Promoting colonisation using rare carbohydrates

Abstract

The present invention relates to bacteria, transmissible elements (eg. conjugative plasmids, phage and phagemid), donor bacteria and recipient bacteria which are engineered to be able to use xylitol and other precursor substrates of xylulose or xylulose-5-phosphate as a food source to enable sustained and higher levels of colonisation of a microbiome of the GI tract in subjects. The bacteria may be engineered to further express desired (e.g. therapeutic) molecules of interest (MOIs). The invention relates to pharmaceutical compositions containing such bacteria and transmissible elements, and their use in the treatment of various diseases.

Description

[0001] PROMOTING COLONISATION USING RARE CARBOHYDRATES

[0002]

[0003] Background to invention

[0004] There have been many attempts to engineer bacteria to produce molecules of interest (MOIs) in situ in local environments, such as the gut microbiome of animals, such as humans, or in soil, water and other natural microbiomes (see for example, Omer et al., Frontiers Bioengineering & Biotechnology, 10, 870675, 2022, doi: 10.3389 / fbioe.2022.870675, and Bai et al., Nat. Rev. Bioeng., 1, 665-679, 2023, doi:https: / / doi.org / 10.1038 / s44222-023-00072-2, for human medical uses). For example, it has been proposed that engineered bacteria can be used to deliver several different categories of molecule for therapeutic uses. For example, Lactococcus lactis has been engineered to deliver anti-tumor necrosis factor (TNF)-a antibody for the treatment of chronic inflammatory bowel disease (IBD), see Vandenbroucke et al., Mucosal Immunol., 3, 2010, 49-56; and Bacteroides ovatus has been engineered to produce human keratinocyte growth factor-2 (KGF-2) to treat IBD, see Hamady et al., Gut, 59, 2010, 461-469. In other fields, Bacillus subtilis has been engineered to express a Sadenosylmethionine methyltransferase gene CmarsM) which converts most of the inorganic arsenic contained in organic manure into dimethylarsenate and tri methylarsine oxide, as a bioremediation strategy, see Huang et al., Appl. Environ. Microbiol., 81 (19), 2015, 6718–6724. In water treatment applications, Synechococcus elongatus has been engineered to produce an oxidative laccase enzyme which can degrade a number of different water contaminants, such as dyes and other chemical pollutants, see Datta et al., Nature Communications, 14, 4742, 2023.

[0005] One ongoing problem which is yet to be addressed is how to effectively introduce such modified bacteria in a way that they reach a sufficient colonisation level to allow them to produce quantities of the MOI to produce the desired effect. In human applications, often the administration of antibiotics precedes the administration of the modified bacteria, killing the existing bacteria in the microbiome to create a niche, into which the modified bacteria can grow. However, there are a number of problems associated with this approach:

[0006] 1. The use of antibiotics tends to remove bacteria indiscriminately, and may remove helpful and useful bacteria from the microbiome of interest;

[0007] 2. The remaining bacteria may comprise a higher amount of antibiotic resistant bacteria, which may subsequently be more difficult to remove;

[0008] 2. The engineered bacteria must compete for colonisation of the niche with the bacteria remaining in the microbiome of interest. This may result in fewer of the engineered bacteria than desired and / or an increase in undesirable (e.g. antimicrobial resistant) bacteria filling the niche if those undesirable bacteria have a selective advantage over the engineered bacteria.

[0009] Even without the administration of antibiotics before administration of the engineered bacteria, colonisation of individual species and strains within a complex microbiome is not well understood, and how well a particular species or strain is able to colonise such a microbiome may bedependent on many interrelated factors, such as competition for macro- and micro-nutrients, avoidance of inhibitory actions from neighbouring cells, phage and host immune systems, and any beneficial actions from neighbouring cells.

[0010] It has been proposed (WO2018 / 112194A1, The Board of Trustees of the Leyland Standford Junior University & Novome Biotechnologies, Inc., and WO2015 / 148680A1, Ginkgo Bioworks, Inc.) to express exogenous genes which use various carbohydrates, such as porphyran, agarose, carrageenan, raffinose and sucrose in various bacteria to support their growth over other bacteria which do not comprise those genes. WO2018 / 112194A1 describes an in vivo mouse colonisation experiment.

[0011] There is a need therefore to be able to control and promote growth of desired or engineered bacteria administered to these complex microbiomes, in particular in the gut. For engineered bacteria which express a MOI from exogenous nucleotide sequences, it has not previously been shown whether the combination of expression of the MOI and enzymes for processing the carbohydrate can function in vivo to provide sustained production of MOI.

[0012] The present invention aims to solve some of the above-mentioned problems.

[0013] Summary

[0014] The present invention addresses some of the potential problems with previously proposed approaches for improving colonisation by administration of carbohydrates.

[0015] For example, WO2015 / 148680 (Gingko Bioworks, Inc) describes using a carbon utilisation operon, the raf operon in Example 7 therein, instead of an antibiotic resistance gene to select for the maintenance and containment of the engineered strains over the parent, wild-type host. The specific genes and reactions of the enzymes in the operon were not elucidated. Other carbon sources suggested in WO2015 / 148680 include sucrose, xylose and arabinose.

[0016] However, it is not envisaged that this approach would be translatable to strains for use in combination with expression of molecules of interest (MOIs) which are useful in treating human or animal subjects. The inventors of the present invention did not select the sugars (carbon sources) described in WO2015 / 148680 because they are either metabolised in the gut microbiome of many subjects, or because they are metabolised by the subject before they reach the desired microbiome (e.g. colon). Raffinose is already metabolised by many bacteria in the human microbiome, see Mao et al., "In vitro fermentation of raffinose by the human gut bacteria", Food Funct., 9(11), 5824-5831, 2018, doi: 10.1039 / c8fo01687a. Sucrose is widely present in the diet and thus is not desirable to use as a carbon source because it is widely used by human cells as a carbon source, so may not be capable of providing the selective advantage that is needed, see Viswanathan & Rao, " Intestinal Disaccharidase Deficiency in Adults: Evaluation and Treatment", Curr. Gastroenterol. Rep., 25(6), 134-139, 2023, doi:10.1007 / s11894-023-00870-z. Xylose also is absorbed and partially metabolised by humans, thus may not be suitable in all patients, see https: / / medlineplus.gov / ency / article / 003606.htm. Further, Xylose can be used as a carbon source by many common human gut bacteria (e.g. E. coli, Bacteroidesetc), so may not be selective enough for the desired engineered target strain to provide the desired colonisation advantages. Arabinose is thought to also be widely metabolised by bacteria, see Abelilla & Stein, "166 Fate of pentoses in the small intestine and hindgut of growing pigs", J. Anim. Sci., 97(Suppl 2), 95, 2019, doi:10.1093 / jas / skz122.171 as well as being linked to the promotion of pathogens, see Cottam et al., " Metabolism of l-arabinose converges with virulence regulation to promote enteric pathogen fitness", Nature Communications, 15, 4462, 2024, doi:https: / / doi.org / 10.1038 / s41467-024-48933-7.

[0017] WO2018 / 112194A1 (The Board of Trustees of the Leyland Standford Junior University & Novome Biotechnologies, Inc.) identifies a particular porphyran utilisation pathway by screening many Bacteroides strains to identify those capable of metabolising porphyran (see Example 1). As explained therein, a minimum of 24 genes (medium PUL) were required to permit growth using porphyran as a carbon source. WO2018 / 112194 confirms there were difficulties in transferring such large pieces of foreign DNA to other species (Example 2). The document describes colonisation in vivo (Examples 2 and 3), either using a naturally occurring strain of Bacteroides which can use porphyran as a carbon source (NB001) or a strain of

[0018]

[0019] a porphyran-utilising plasmid in Example 2. Other carbon sources described include agarose, agaropectin and carrageenan.

[0020] WO2018 / 112194 does not show that the porphyran utilisation pathway could be stably introduced into desired bacteria, nor does it show that it would be compatible with strains for use in combination with expression of molecules of interest (MOIs) which are useful in treating human or animal subjects.

[0021] However, it is not envisaged that this porphyran approach would be translatable to strains for use in combination with expression of molecules of interest (MOIs) which are useful in treating human or animal subjects, because of the size and stability of the various gene modules. Generally, the inventors of the present invention did not select the carbon sources described in WO2018 / 112194 because they are either metabolised in the gut microbiome of many subjects, or because they are metabolised by the subject before they reach the desired microbiome (e.g. colon). For example, carrageenan and agarose are degraded by many native bacterial species in the human gut microbiome, see Li et al., "Degradation of Marine Algae-Derived Carbohydrates by Bacteroidetes Isolated from Human Gut Microbiota", Mar. Drugs, 15(4),92, 2017, doi:10.3390 / md15040092. Agaropectin is not well studied in humans. In addition to being a polymer, it tends to be a relatively heterogeneous molecule with unpredictable sulfate, glucuronate, and pyruvate modifications, increasing the complexities of manufacture and approval for human use. The safe maximum amount of agaropectin has not been determined in humans, but consumption of agar is thought to be 4.5g, of which approximately 39% is agaropectin. Thus, without being bound by theory, the maximum amounts of agaropectin that may be given to a human may not be sufficient to maintain colonisation of an agaropectin-utilising bacterium in vivo.Thus, the present invention aims to solve some of the problems associated with the previously proposed approaches including, but not limited to:

[0022] • a carbon source which is not frequently found in the diet of subjects (e.g. humans), but is safe and non-toxic in the quantities required to provide the selective advantage • a carbon source which is able to reach the lower gut (e.g. colon) of a subject (e.g.

[0023] human) without significant metabolization or degradation

[0024] • a carbon source which is cheap and easy to produce, package and administer to the subject (e.g. human)

[0025] • a pathway which is compatible with transfer to strains by plasmids and by integration into the genome

[0026] • a small size of a pathway, which is expected to reduce the impact on cell fitness / viability as compared to, for example, the large size of the porphyran utilisation pathway

[0027] • a pathway which can be transferred easily to multiple target bacterial strains

[0028] • a pathway which is compatible with MOI production, balancing MOI production and carbon source use vs fitness to the modified bacterial strain

[0029] The present invention therefore provides the following:

[0030] In a first configuration:

[0031] There is provided a bacterium, or transmissible element for transmission to a recipient bacterium, wherein the bacterium or transmissible element comprises: (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, wherein one, more or all of the one or more nucleotide sequences is heterologous to the bacterium or recipient bacterium.

[0032] In a second configuration:

[0033] There is provided a bacterium, or transmissible element for transmission to a recipient bacterium, wherein the bacterium or transmissible element comprises:

[0034] (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, optionally wherein one, more or all of the one or more nucleotide sequences is heterologous to the bacterium or recipient bacterium; and (b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium.

[0035] In a third configuration:

[0036] There is provided a bacterium comprising:(a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate, operably linked to a promoter which is not operably linked to the nucleotide sequence(s) encoding the peptide product(s) in nature, wherein the promoter is not a native promoter of the pUC13 plasmid.

[0037] In a fourth configuration:

[0038] There is provided a pharmaceutical composition comprising (i) a population of bacteria as described herein, (ii) a population of donor bacteria as described herein, or (iii) a plurality of transmissible elements as described herein, and a pharmaceutically acceptable excipient or carrier.

[0039] There is also provided kit comprising (i) a population of bacteria as described herein, (ii) a population of donor bacteria as described herein, (iii) a plurality of transmissible elements as described herein, or (iv) a pharmaceutical composition as described herein; and at least one dose of xylitol for administration to a subject.

[0040] In a fifth configuration:

[0041] There is provided a method of colonising a microbiome of a subject, said method comprising the steps of:

[0042] I. providing xylitol (e.g. D-xylitol) for ingestion by (e.g. for oral administration to) the subject, whereby the subject ingests the xylitol;

[0043] II. administering to the subject (i) a population of bacteria as described herein, (ii) a population of donor bacteria as described herein, (iii) a plurality of transmissible elements as described herein, or (iv) a pharmaceutical composition as described herein.

[0044] There is also provided a (i) population of bacteria as described herein, (ii) population of donor bacteria as described herein, (iii) plurality of transmissible elements as described herein, or (iv) the pharmaceutical composition as described herein, for use in colonising a microbiome of the GI tract of a subject, the use comprising:

[0045] I. providing xylitol (e.g. D-xylitol) for ingestion by (e.g. for oral administration to) the subject, whereby the subject ingests the xylitol;

[0046] II. administering to the subject (i) the population of bacteria, (ii) the population of donor bacteria, (iii) the plurality of transmissible elements, or (iv) the pharmaceutical composition.

[0047] In a sixth configuration:

[0048] There is provided a method of treating or preventing a disease or disorder in a subject, said method comprising carrying out the method as described in the fifth configuration or as described elsewhere herein.

[0049] There is also provided a (i) population of bacteria as described herein, (ii) population of donor bacteria as described herein, (iii) plurality of transmissible elements as described herein, or (iv) the pharmaceutical composition as described herein, for use in treating or preventing a disease or disorderin a subject, said use comprising carrying out the method as described in the fifth configuration or as described elsewhere herein.

[0050] There is also provided the use of a (i) population of bacteria as described herein, (ii) population of donor bacteria as described herein, (iii) plurality of transmissible elements as described herein, or (iv) pharmaceutical composition as described herein, in the manufacture of a medicament for the treatment or prevention of a disease or disorder in a subject, wherein said medicament is administered in combination (sequentially or simultaneously) with xylitol.

[0051] Brief description of the Figures

[0052] Figure 1A: Schematic showing metabolism pathway of D-xylitol and D-xylose. Xylitol dehydrogenase (XOHDH), Sugar isomerase (SI), xylulokinase (XK), Xylitol oxidase (AldO), General metabolism (GM) refers to the pentose phosphate pathway and Krebs cycle.

[0053] Figure 1B: The system for xylitol metabolism and uptake found in M. morganii consists of an ABC transporter (transporter, SEQ ID Nos: 10, 11 and 12), xylulokinase (XK, SEQ ID No:6), xylitol dehydrogenase (XᴏHDH, SEQ ID No:2), sugar isomerase (SI, SEQ ID No:4) and a repressor (R, SEQ ID NO:15)

[0054] Figure 2: Growth of Symbioflor strain carrying the plasmid-borne xylitol pathway from M. morganii in minimal medium containing xylitol as the sole carbon source. Cells were grown in M9 xylitol minimal media, and optical densities (OD) of cultures were recorded at regular intervals. Growth curves of strains b7207, carrying an empty vector and, b7992 containing the xylitol pathway from M. morganii were plotted. The presence of the cloned pathway enabled b7992 to grow using xylitol as the only carbon source.

[0055] Figure 3: Growth of Symbioflor strain carrying the xylitol pathway from M. morganii in the chromosome. Growth curves of Symbioflor strains b7950, carrying the xylitol pathway in the chromosome and, b7659 as control in M9 minimal media. B7950 could grow using xylitol as the only carbon source, unlike the control strain b7659 (left). However, b7659 shows growth when a different carbon source (i.e. arabinose) is added to the media (right).

[0056] Figure 4: Genes used for xylitol metabolism in M. morganii. The system consists of an ABC transporter (transporter, SEQ ID Nos: 10, 11, 12), xylulokinase (XK, SEQ ID No:6), xylitol dehydrogenase (XOHDH, SEQ ID No:2), sugar isomerase (SI, SEQ ID No:4) and a repressor (R, SEQ ID No: 15). Plasmids containing the whole or parts of the system are constructed as shown schematically below. Empty boxes symbolise parts of the system deleted in the respective plasmid.

[0057] Figure 5: Growth of Symbioflor strains containing plasmids with parts of the xylitol pathway from M. morganii. Growth after 6 hours of culture of Symbioflor strains in M9 minimal media with 1% xylitol. Strains carry p2820, containing the full xylitol pathway from M. morganii as a positive control; p2939, lacking the ABC transporter; p2940, lacking xylulokinase (XK); p2987, lacking xylitol dehydrogenase (XOHDH); p2958, lacking sugar isomerase (SI); p2988, carrying only the xylulosedehydrogenase (XOHDH); and the negative control b5652 without any plasmid. All strains carrying the different plasmid versions could grow on xylitol as the only carbon source except the strain carrying p2987 with the XOHDH encoding gene deleted.

[0058] Figure 6: Growth of the engineered Symbioflor strain carrying the full xylitol pathway in the chromosome on different xylitol concentrations compared to the wildtype strain. Growth curves of strain b7950 (SBF xylitol) and b6760 (SBF wt) in M9 media supplemented with Casamino acids and different concentrations of xylitol (2%, 1%, 0.5%, 0.25%, 0.13%, 0.06%, 0.03% and 0%). The engineered Symbioflor strain carrying the xylitol pathway (b7950) shows a positive dose response to the addition of xylitol even at low concentrations. In contrast, the wild-type strain (b6760) is not able to utilize xylitol.

[0059] Figure 7: Dose dependent growth of a strain carrying the full xylitol pathway compared to a control strain in spent LB media at different xylitol concentrations. Growth of strain b8046 with the full xylitol pathway and the control strain b7513, with no pathway, spotted in LB Str plates with 10-fold serial diluted cultures after 0 or 24 hours of the addition of different concentrations of xylitol (0%, 0,02%, 0,2% and 2%) in three types of spent LB media. The different spent media were obtained by culturing either Strain A, Strain B or Strain C for 24 hours, until saturation of the culture.

[0060] Figure 8: Symbioflor G6 / 7 stain carrying the MOI production pathway and the xylitol pathway in the chromosome. Strains b8270 / b8343 carries the MOI production pathway (grey boxes) and the full xylitol pathway (black boxes); XOHDH, xylitol dehydrogenase (SEQ ID No:2); XK, xylulokinase (SEQ ID No:6); SI, sugar isomerase (SEQ ID No:4); R, repressor (Seq ID No: 15) and ABC transporter (Seq ID Nos: 10, 11, 12).

[0061] Figure 9: MOI producing strain grows on xylitol in a dose dependent manner following chromosomal insertion. Strain b8270 (MOI producing strain containing xylitol pathway) grown for 14 hours in M9 media supplemented with different concentrations of xylitol (2%, 1%, 0,5%, 0,25%, 0,13%, 0,06%, 0,03% and 0%). The engineered Symbioflor strain carrying the xylitol pathway (b8270) shows a positive dose response to the addition of xylitol.

[0062] Figure 10: MOI production of Symbioflor strain b8343. MOI production as measured using LC / MS. b8343 contains the full pathway for xylitol degradation integrated into the genome. Strains were grown in M9 minimal media with 1 mM MOI precursor molecule under both anaerobic and aerobic conditions.

[0063] Figure 11: Growth of Klebsiella pneumoniae expressing xylitol dehydrogenase from M. morganii. Growth after 8 hours of culture in M9 minimal media with 2%, 0,5%, 0,12% and 0% xylitol. Klebsiella pneumoniae b2951 with and without carrying xylitol dehydrogenase on p2988; E. coli b8214 carries the xylitol dehydrogenase in the chromosome and acts as a positive control. b2951 carrying p2988 grew on xylitol as the only carbon source in a dose-dependent manner similarto strain b8214. In contrast, b2951 without any plasmid did not grow on xylitol as the only carbon source.

[0064] Figure 12: Group and treatment overview.

[0065] Figure 13: Colonisation of gram-negative bacteria before dosing. No gramnegative bacteria are streptomycin resistant.

[0066] Figure 14: CFU in faeces of groups 1 (no xylitol) and 2 (xylitol). No pre-treatment with streptomycin.

[0067] Figure 15: Faecal CFU counts on selective media for our strain (MacConkey + streptomycin).

[0068] Figure 16. CFU in the tissue and contents of ileum, cecum and colon of mice on Day 6.

[0069] Figure 17: Group and treatment overview.

[0070] Figure 18: CFU / g faeces shown over 16 days of study including no xylitol indicated (day 8 excluded due to lab-related issues).

[0071] Figure 19: Shows CFU counts and MOI levels obtained from b8343 in mice dosed with 0% or 5% xylitol. CFU on left axis (open dots and thin line) and MOI on right axis (black dots and color-filled area under curve). The addition of xylitol increases CFU and measured MOI levels.

[0072] Figure 20: Group and treatment overview.

[0073] Figure 21: CFU / g faeces in groups dosed with 3-5% xylitol through the drinking water.

[0074] Detailed Description

[0075] As previously mentioned, one ongoing problem with the delivery of genetically modified bacterial cells which is yet to be addressed is how to effectively introduce such modified bacteria in a way that they reach a sufficient colonisation level in a native microbiome to allow them to produce quantities of the MOI to produce the desired effect. In human applications, often the administration of antibiotics precedes the administration of the modified bacteria, killing the existing bacteria in the microbiome to create a niche, into which the modified bacteria can grow. However, there are a number of problems associated with this approach:

[0076] 1. The use of antibiotics tends to remove bacteria indiscriminately, and may remove helpful and useful bacteria from the microbiome of interest;

[0077] 2. The remaining bacteria may comprise a higher amount of antibiotic resistant bacteria, which may subsequently be more difficult to remove;

[0078] 2. The engineered bacteria must compete for colonisation of the niche with the bacteria remaining in the microbiome of interest. This may result in fewer of the engineered bacteria thandesired and / or an increase in undesirable (e.g. antimicrobial resistant) bacteria filling the niche if those undesirable bacteria have a selective advantage over the engineered bacteria.

[0079] It has been proposed (WO2018 / 112194A1, The Board of Trustees of the Leyland Standford Junior University & Novome Biotechnologies, Inc., and WO2015 / 148680A1, Ginkgo Bioworks, Inc.) to express exogenous genes which use various carbohydrates, such as porphyran, agarose, carrageenan, raffinose and sucrose in various bacteria to support their growth over other bacteria which do not comprise those genes. WO2018 / 112194A1 describes an in vivo mouse colonisation experiment.

[0080] There is a need therefore to be able to control and promote growth of desired or engineered bacteria administered to these complex microbiomes, in particular in the gut. For engineered bacteria which express a MOI from exogenous nucleotide sequences, it has not previously been shown whether the combination of expression of the MOI and enzymes for processing the carbohydrate can function in vivo provide sustained production of MOI.

[0081] The present invention aims to solve some of the problems associated with the previously proposed approaches including, but not limited to:

[0082] • a carbon source which is not frequently found in the diet of subjects (e.g. humans), but is safe and non-toxic in the quantities required to provide the selective advantage • a carbon source which is able to reach the lower gut (e.g. colon) of a subject (e.g.

[0083] human) without significant metabolization or degradation

[0084] • a carbon source which is cheap and easy to produce, package and administer to the subject (e.g. human)

[0085] • a pathway which is compatible with transfer to strains by plasmids and by integration into the genome

[0086] • a small size of a pathway, which is expected to reduce the impact on cell fitness / viability as compared to, for example, the large size of the porphyran utilisation pathway

[0087] • a pathway which can be transferred easily to multiple target bacterial strains

[0088] • a pathway which is compatible with MOI production, balancing MOI production and carbon source use vs fitness to the modified bacterial strain

[0089] Definitions

[0090] A "beneficial metabolite" refers to a molecule that is beneficial to the local environment of the bacterium, recipient bacterium or donor bacterium, or to the microbiome as a whole, or indeed causes downstream beneficial effects to the organism which may comprise the bacterium or microbiome. Beneficial metabolites may not be present, or may be present in too low a concentration in the bacterium (e.g. engineered / modified, donor or recipient bacterium) which results in a detrimental effect in the bacterium (or microbiome or organism), optionally as compared to other bacteria in amicrobiome. Beneficial metabolites may be molecules which provide a competitive advantage to the bacterium (e.g. engineered, donor or recipient bacterium).

[0091] A "carbon source", as used herein, refers to any organic molecule which can be used by an engineered bacterium, or a donor or a recipient bacterium to synthesise biomass.

[0092] As used herein "colonising", "colonisation" and the like refers to a successful establishment and persistence of an introduced bacterial strain or species in an environment or microbiome. In some cases, the introduced bacteria reaches a 'high level' of colonisation (e.g. a 'high abundance'). In such cases, the introduced bacteria may reach an abundance of 105CFU / µL or more (e.g. 106CFU / µL or more, 107CFU / µL or more). Thus, in some cases, an introduced bacteria reaches an abundance of 107CFU / µL or more. In some cases, the introduced bacteria may reach an abundance such that it attains a population level of 10% or more (e.g. 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more) of total CFUs in the environment or microbiome (e.g. in the target organ). In some examples, the introduced bacteria reaches an abundance such the population attains a population level in a range of from 10%-90% (e.g., from 10%-80%, 10%-70%, 10%-60%, 10%-50%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, or 30%-50%) of total CFUs in the environment or microbiome (e.g. in the target organ). The measurement of CFU may be achieved by day 5 or 6 after introduction into the environment or microbiome (e.g. in the target organ). If a strain or species is not able to colonise, then it is cleared via excretion such that levels decrease and eventually are undetectable in the environment or microbiome into which it was originally introduced. Providing a niche (e.g. by the use of an antibiotic to clear space in the microbiome) may help the newly introduced bacteria to establish. If such a niche is not provided, the newly introduced species must compete directly with the existing, already established, bacteria in the environment or microbiome. Thus in one example, the colonisation is achieved after generation of a niche, e.g. by use of an antibiotic.

[0093] As used herein, a "conjugative plasmid" is a particular transmissible element. A conjugative plasmid is a plasmid which, when comprised within a bacterial cell ("host" or "donor" cell, used interchangeably herein) is able to be transferred to another bacterium ("recipient" cell) through the mechanism of bacterial conjugation. Bacterial conjugation is the unidirectional and horizontal transmission of genetic information from one bacterium to another. Conjugative plasmids generally fall into two classes: mobilizable plasmids and self-transmissible plasmids. Mobilizable plasmids comprise at least an origin of transfer (oriT), a relaxase and other genetic information on the plasmid which is transferred to the recipient cell, and require helper functions provided by e.g. a second plasmid or the chromosome of the donor cell to effect the plasmid transfer. On the other hand, self-transmissible plasmids, in addition to the genetic information on the plasmid which is transferred to the recipient cell, also contain all the molecular machinery needed for self-transfer (e.g. for pilus formation and initiation of gene transfer) on the same plasmid. Both mobilizable plasmids and self-transmissible plasmids comprise relaxase genes which recognises the origin of transfer (oriT) andcatalyses both the initial cleavage of oriT in the donor, to produce the DNA strand from the plasmid that will be transferred, as well as the final ligation of the transported DNA in the recipient cell that reconstitutes the conjugated plasmid. Thus, in one example, the conjugative plasmid is a mobilizable plasmid. In another example, the conjugative plasmid is a self-transmissible plasmid. In another example, the conjugative plasmid includes an origin of transfer (oriT). Plasmid mobility, mechanisms and structures are described in more detail in Smillie et al., Microbiol. Mol. Biol. Rev., 74(3): 434-452, 2010 doi: 10.1128 / MMBR.00020-10, which is incorporated herein in its entirety.

[0094] Bacterial cells possessing a conjugative self-transmissible plasmid contain a surface structure (pilus) encoded by the conjugative machinery on the plasmid that is involved in the coupling of donor and recipient cells, and the transfer of the genetic information contained within the plasmid. Conjugation involves contact between cells, and the transfer of genetic traits can be mediated by many plasmids. Among all natural transfer mechanisms, conjugation is the most efficient. For example, F plasmid of E. coli, pCFIO plasmid of Enterococcus faecalis and pXO16 plasmid of Bacillus thuringiensis employ different mechanisms for the establishment of mating pairs, the sizes of mating aggregates are different, and they have different host ranges within gram-negative (F) as well as gram-positive (pCFIO and pXO16) bacteria. Their plasmid sizes are also different; 54, 100 and 200 kb, respectively. Remarkably, however despite differences in origin and size, those conjugation systems are able to sustain efficient conjugative transfer in liquid medium. The conjugative process permits the protection of plasmid DNA against environmental nucleases, and thus efficient delivery of plasmid DNA into a recipient cell can be obtained. Conjugation functions are naturally plasmid encoded. Numerous conjugative plasmids (and transposons) are known, which can transfer associated genes within one species (narrow host range) or between many species (broad host range). Transmissible plasmids are widespread across the domain of bacteria and similar systems of horizontal gene transfer through pilus structures have recently been described for Archaea. Engineered conjugative plasmids are described in more detail in WO2021 / 037732 (SNIPR Biome ApS), which is incorporated herein in its entirety. The features of such conjugative plasmids and bacterial cells comprising them as described in the claims as filed in WO2021 / 037732 are also incorporated herein by reference.

[0095] " Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked. Constitutive promoters and variants are well known in the art and are described elsewhere herein.

[0096] A "detrimental metabolite" refers to a molecule that is harmful to the local environment of the bacterium, or recipient or donor bacterium, or to the microbiome as a whole, or indeed causes downstream harmful effects to the organism which may comprise the bacterium or microbiome. Detrimental metabolites, whilst needed and / or required to a certain degree, may be present in too high a concentration in the bacterium (e.g. engineered), or donor or recipient bacterium which results in a detrimental effect in the cell (or microbiome or organism), optionally as compared to other bacteria in a microbiome. Detrimental metabolites may be molecules which cause a fitness cost to thebacterium (e.g. engineered), or recipient or donor bacterium. In examples where the second MOI is a detrimental metabolite, the first MOI is a molecule which either has a neutral effect on the bacterium, or recipient or donor bacterium (or microbiome or organism), or is easily excreted by the cell and / or organism, or is a beneficial metabolite.

[0097] " Exogenous" or "heterologous" are used interchangeably and refer to a nucleotide sequence that is not normally found in a given cell in nature. The given cell may the bacterium (e.g. engineered), or donor or recipient bacterium (e.g. comprising a transmissible element), in particular the recipient bacterium, or the parental bacterium before any genome engineering has occurred. Thus, an exogenous or heterologous nucleotide sequence may be one which is not naturally found in the parental, donor or recipient bacterium. As used herein, a heterologous sequence encompasses a nucleotide sequence (or amino acid sequence) that is exogenously introduced into a given cell. A heterologous gene includes a native gene, or fragment thereof, that has been introduced into the cell. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native cell. Thus, a heterologous gene may be foreign or native to the bacterium, or donor or recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and / or the polypeptide which it encodes; and / or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. For plasmids, such as conjugative plasmid, a heterologous gene is one which has been engineered to be included on such plasmid (e.g. conjugative plasmid). However, in alternative examples relating to plasmids, such as conjugative plasmids, a heterologous or exogenous gene is heterologous to the recipient bacterium.

[0098] As used herein an "exporter" refers to a membrane-integrated protein which is capable of exporting a molecule (such as a naturally occurring cellular substrate or metabolite, an MOI, or any other peptide, protein or small molecule) out of the bacterial cell. As discussed elsewhere herein, in some bacteria there are no endogenous exporters of MOIs, and thus the cell must rely on diffusion of the MOIs to remove the MOIs from the cell. Without being bound by theory, the export of MOIs in gram-negative bacteria may be particularly problematic, due to the presence of both an inner and outer membrane. Thus, the addition of a heterologous exporter capable of exporting the MOI in the transmissible element may provide further benefits in this type of bacterial cell. In other bacteria, there may be endogenous exporters which export substrates which are used in the production of the second MOI. When these substrates are exported from the cell, they are no longer available to be used in the production of the second MOI, thus potentially decreasing the amount of second MOI available in the cell to be converted into the first MOI. It may be desirable to deactivate or reduce the ability of these exporters to export the endogenous substrates from the cell. The ability of any given protein to act as an exporter can be easily measured by those skilled in the art. Identification ofputative exporters can be achieved through literature searches as well as by using protein and genetic databases, such as GenBank, which are well-known to those skilled in the art.

[0099] As used herein an "importer" refers to a membrane-integrated protein which is capable of importing a molecule (such as a naturally occurring cellular substrate or metabolite, a precursor molecule such as xylitol, a second MOI or any other peptide, protein or small molecule) into the bacterial cell. As discussed elsewhere herein, in some bacteria there are no endogenous importers of precursor molecules such as xylitol, of second MOIs and / or of substrates which are used in the production of the second MOI, and thus the cell must rely on diffusion of the precursor molecules (e.g. xylitol), second MOI and / or such substrates to produce second MOIs to import them into the cell. Thus, the addition of a heterologous importer capable of importing xylitol, the second MOI and / or substrates which are used in the production of the second MOI may provide further benefits in the bacterium or recipient bacterium. The ability of any given protein to act as an importer can be easily measured by those skilled in the art. Identification of putative importers can be achieved through literature searches as well as by using protein and genetic databases, such as GenBank, which are well-known to those skilled in the art.

[0100] An "inducible promoter" refers to a promoter which transcribes a coding sequence or gene under its control and / or to which it is operably linked in the presence of an inducer of said promoter. The inducer may be one or more environmental condition(s) and / or one or more inducing molecule(s).

[0101] A "kill switch" refers to a biocontainment system which is included in the transmissible element (e.g. the conjugative plasmid) or engineered bacterium and is designed to destroy the engineered bacterium or the recipient bacterium, or in the case of a plasmid (e.g. a conjugative plasmid), either the plasmid itself only, or the plasmid and the bacterium comprising the plasmid together, when no longer contained within its desired environment (e.g. within a microbiome, such as a gut microbiome, within a subject). Such means are well-known in the art, and are regulatable, for example by the addition of non-naturally occurring substances (e.g. synthetic amino acids), temperature and the like.

[0102] A "microbiome," as used herein, refers to the totality of microbes in a particular environment (e.g. in / on an organism, in a marine environment (e.g. ocean), and / or in a terrestrial environment (e.g. soil)). In some examples, a microbiome may refer to the totality of microbes that reside, or are stably maintained, for example, on the surface and in deep layers of the skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tracts of an organism. The microbiome may exist within any of the organs described elsewhere herein.

[0103] " Peptide" and "protein" are used interchangeably herein.

[0104] " Phage" or "bacteriophage" are used interchangeably and refer to obligate intracellular parasites that multiply inside bacteria by co-opting some or all of the host biosynthetic machinery. Phage genomes come in a variety of sizes and shapes. Most phages range in size from 24-200 nm in diameter. Phages contain nucleic acid (i.e. genome) and proteins, and may be enveloped by a lipid membrane. Depending upon the phage, the genome can be either DNA or RNA, and can exist in eithercircular or linear forms. The size of the phage genome varies depending upon the phage. The simplest phages have genomes that are only a few thousand nucleotides in size, while the more complex phages may contain more than 100,000 nucleotides in their genome, and in rare instances more than 1,000,000. The number and amount of individual types of protein in phage particles will vary depending upon the phage.

[0105] A "phagemid" refers to a bacteriophage-derived vector containing the replication origin of a plasmid and the packaging site of a bacteriophage. Examples of phagemids that may be used in accordance with the present disclosure include, without limitation, M13-derived phagemids containing the fl origin for filamentous bacteriophage packaging such as, for example, pBluescript II SK (+ / -) and KS (+ / -) phagemids, pBC SK and KS phagemids, pADL and Pl-based phagemids (see, e.g. Westwater CA et al., Microbiology 148, 943-50 (2002); Kittleson JT et al., ACS Synthetic Biology 1, 583-89 (2012); Mead DA et al., Biotechnology 10, 85-102 (1988)). Other phagemids may be used and, for example, can be made to work with packaging systems from natural, engineered or evolved bacteriophage.

[0106] " Preventing" as used herein in relation to transcription or expression of a gene or protein refers to a complete ablation of transcription or expression.

[0107] As used herein, "promoting viability" and like terms refers to increasing the fitness of a cell (e.g. bacterial cell) in a given environment or microbiome. This will make the given cell (e.g. bacterial cell) more able to use existing or exogenously provided resources or to more easily withstand environmental conditions (such as pH, presence or absence of oxygen and the like). In the context of the present invention, the engineered bacteria are able to use an exogenously-added carbon source, which promotes the fitness of that cell compared to other species and strains which cannot use that carbon source in the environment or microbiome.

[0108] As used herein "providing a selective advantage" and like terms refers to providing a cell (e.g. bacterial cell) means to process a nutrient or carbon source more effectively than the majority (e.g. more than 80%, 90% 95% or more than all) of the other cells in the environment or microbiome. Thus, the engineered bacteria herein are selected over and above other bacteria in the environment or microbiome due to their ability to use the rare carbon source (e.g. xylitol). In one example the selective advantage is not by the provision or existence of an antibiotic resistance gene or mechanism in the cell (e.g. bacterial cell).

[0109] " Reducing" as used herein in relation to transcription or expression of a gene or protein refers to a reduction in gene transcription or expression, such as at least a 50% reduction of transcription or expression. In one example, the reduction is at least a 60%, at least a 70%, at least an 80% reduction. In one example, the reduction is at least an 85% reduction. In one example, the reduction is at least a 90% reduction. In one example, the reduction is at least a 95% reduction. In one example, the reduction is at least a 97% reduction. In one example, the reduction is at least a 99% reduction. In one example, the reduction is a 100% reduction.As used herein, with respect to treatment methods, "prevention" includes a reducing of the risk of contracting the disease. The "treatment or prevention" may be complete or partial treatment or prevention, i.e. a reduction, but not complete reduction of the disease / condition or symptoms thereof; or a reducing of the risk but not total prevention of the disease / condition or a symptom thereof. Similarly, the methods treat or prevent (i.e. reduces the risk of) an undesirable symptom of the disease or condition or the therapy.

[0110] A "therapeutic metabolite" refers to a molecule that is able to act in the local environment of the bacterium, recipient bacterium or donor bacterium to provide a therapeutic effect. This may be by, for example, counteracting the presence of another detrimental molecule or causing a downstream reaction which provides the therapeutic effect.

[0111] A "transmissible element" as used herein refers to any means which can be transferred to a recipient bacterium, including, but not limited to a plasmid, a conjugative plasmid, a phage or a phagemid. In some examples, a transmissible element may include other mobile genetic elements, such as transposons and the like. In a particular example, the transmissible element is selected from a conjugative plasmid, and phage and a phagemid. In another particular example, the transmissible element is a conjugative plasmid.

[0112] Any percentage identity herein may be at least (about) 70%. For example, any percentage identity herein may be at least (about) 80%. For example, any percentage identity herein may be at least (about) 90%. For example, any percentage identity herein may be at least (about) 95%. For example, any percentage identity herein may be at least (about) 96%. For example, any percentage identity herein may be at least (about) 97%. For example, any percentage identity may be at least (about) 98%. For example, any percentage identity herein may be at least (about) 99%. Percent identity for amino acid sequences are determined using the blastP algorithm with the following parameters: - The default parameters are adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 6. Regions of low compositional complexity are masked. The employed scoring matrix is 'BLOSUM62', with scoring costs to create and extend a gap being 11 and 1 respectively. Conditional compositional score matrix adjustment is employed to compensate for amino acid composition of compared sequences.

[0113] Percent identity for nucleotide sequences are determined using the blastn algorithm with the following parameters: - The default parameters are automatically adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 28. Regions of low compositional complexity are masked. The query sequence is masked while producing seed sequences used to scan databases, but not masked for extensions. Matches are scored as +1 and mismatches are scored as -2.For example, an amino acid sequence described herein is identical to the reference SEQ ID No, except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change. For example, a nucleic acid sequence described herein is identical to the reference SEQ ID No, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide changes.

[0114] For example, an amino acid sequence described herein is identical to the reference SEQ ID No except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in the reference sequence. For example, a nucleotide sequence described herein is identical to the reference SEQ ID No except for a total number of nucleotide changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of nucleotides in the reference sequence.

[0115] Heterologous nucleotide sequences

[0116] The nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates, or nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes) which are comprised by the engineered bacteria, transmissible elements, donor bacteria and / or recipient bacteria (described elsewhere herein) may be from various sources, e.g. plant, mammal, yeast, bacterial. In one example, any of the nucleotide sequences described herein may be from a prokaryotic origin. In one example, any of the nucleotide sequences described herein may be from a bacterial origin where such gene exists (e.g. from any of the bacterial strains described elsewhere herein, for example any of the strains listed in Table 4).

[0117] Particular sources of nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides, importers of any second MOI or further substrates, or nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes) are described herein. However, as will be apparent to those skilled in the art, the nucleotide sequences described may not encompass all known genes which encodes peptide products that catalyse the stated reaction, or import / export the stated substrate molecule. Further genes and protein products may be identified, for example, through literature searching, using known databases (such as GenBank, Kegg, EMBL or protein databases such as Uniprot and the like), either through keyword searching, putative annotations and / or sequence homology to a known enzyme (protein and / or DNA sequence). A list of databases can be found here: https: / / en.wikipedia.org / wiki / List_of_biological_databases.Peptide products for the conversion of a precursor substrate to xylulose or xylulose-5-phosphate The engineered bacteria, transmissible elements, donor bacteria and / or recipient bacteria comprise in part (a), one or more nucleotide sequences for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate. In some examples, one, more or all of the one or more nucleotide sequences or part (a) is heterologous to the bacterium or recipient bacterium. In other examples, one, more or all of the one or more nucleotide sequences or part (a) is endogenous to the bacterium or recipient bacterium. In yet another example, the one or more nucleotide sequences or part (a) are a combination of nucleotide sequences which are heterologous and endogenous to the bacterium or recipient bacterium.

[0118] In one example, there is provided a bacterium, or transmissible element for transmission to a recipient bacterium, wherein the bacterium or transmissible element comprises: (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, wherein one, more or all of the one or more nucleotide sequences is heterologous to the bacterium or recipient bacterium. The bacterium or transmissible element may further comprise: (b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium. The first MOI is as described elsewhere herein.

[0119] In another example, there is provided a bacterium, or transmissible element for transmission to a recipient bacterium, wherein the bacterium or transmissible element comprises: (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate; and (b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium. In this example, one, more or all of the one or more nucleotide sequences of part (a) may be heterologous to the bacterium or recipient bacterium. In a particular example, all of the one or more nucleotide sequences of part (a) may be heterologous to the bacterium or recipient bacterium. Alternatively, one, more or all of the one or more nucleotide sequences of part (a) may be endogenous to the bacterium. This example is useful for bacteria which are naturally able to use a precursor substrate such as xylitol as a food source, but are further engineered to be able to produce the first MOI.

[0120] The inventors showed that the addition of XOHDH, which converts xylitol to xylulose, alone provided increased growth in the presence of xylitol, thus, it is expected that an increase in production of xylulose in some bacteria may provide a selective advantage and increased colonisation and / or growth, without the need for the addition of a further enzyme to convert the xylulose to xylulose-5-phosphate. Thus in some examples, the bacterium, recipient bacterium or transmissible elements provide peptide products for the conversion of a precursor substrate (e.g. xylitol) to xylulose. In other examples, the bacterium, recipient bacterium or transmissible elements provide peptide products for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phophate. In other examples, thebacterium, recipient bacterium or transmissible elements may require peptide products for the conversion of a precursor substrate which is not xylitol to xylulose or xylulose-5-phosphate, depending on the intrinsic ability of the bacterium to process xylitol. All of these potential configurations are useful in producing an improved bacterium which can successfully use xylitol as a carbon source to improve colonisation and provide a selective advantage.

[0121] There is also provided a transmissible element for transmission to a recipient bacterium, wherein: (a) the recipient bacterium comprises one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, and the transmissible element comprises (b) at least one heterologous nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the recipient bacterium and the transmissible element. In this example, the recipient bacterium may already be part of the microbiome (e.g. microbiome of the GI tract of a subject). The recipient bacteria may have been previously administered to the subject, and may have already colonised the microbiome. Alternatively, the recipient bacterium may be a naturally occurring bacterium in the microbiome, and comprises endogenous nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate.

[0122] In other examples, a bacterium which comprises endogenous nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate may be further engineered to increase expression of the peptide product(s). This could be by increasing the number of copies of the endogenous nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, such that the bacterium comprises both endogenous nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, and additional copies of the nucleotide sequences which have been introduced by genetic engineering techniques (for example, engineered into the chromosome of the bacterium) or by a transmissible element. In this latter example, the transmissible element comprises nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, where the nucleotide sequences are endogenous to the recipient bacterium and the recipient bacterium comprises endogenous nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate.

[0123] Additionally or alternatively, increasing expression of the peptide products encoded by a bacterium which comprises endogenous nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate may be achieved by increasing the strength of the promoter (e.g. one, more than one or all of the promoters) controlling expression of the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate. Suitable promoters are described elsewhere herein. Thus, there is provided a bacterium comprising (a) one or more nucleotidesequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate, operably linked to a promoter which is not operably linked to the nucleotide sequence(s) encoding the peptide product(s) in nature, wherein the promoter is not a native promoter of the pUC13 plasmid. The bacterium may be further engineered to be able to produce a first MOI. Thus, the bacterium may further comprise (b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium.

[0124] Figure 1 shows various pathways for the conversion of xylitol to xylitol-5-phosphate which can be further metabolised by the cell to provide both energy and a source of carbon which is capable of being used to make biomass. Xylitol can be directly converted to xylulose using a xylitol dehydrogenase (XOHDH), or alternatively can be converted first to xylose using a xylitol oxidase (AldO), and then to xylulose using a sugar isomerase (SI). Xylulose can then be converted to the xylulose-5-phosphate using a xylulose kinase (XK). Further details on each of these reactions and enzymes are provided herein.

[0125] A particular peptide product for the conversion of xylitol to xylulose includes, but is not limited to xylitol dehydrogenase (XOHDH). Thus, in a particular example, the one or more nucleotide sequences of (a) encodes a xylitol dehydrogenase (XOHDH). XOHDH may be from an Aspergillus species, for example from Aspergillus flavus, for example the sequence described as " AFLA_097930" in Figure 1 of Kumar et al. " Characterization of a xylitol dehydrogenase from Aspergillus flavus and its application in l-xylulose production", Front. Bioeng. Biotechnol., 10, 1001726, 2022, doi: 10.3389 / fbioe.2022.1001726, also see Genbank accession number: XP_041148561 which is incorporated herein by reference and intended to form part of the disclosure of the present invention. XOHDH may be from a Gluconobacter species, for example from Gluconobacter oxydans, see for example, Qi et al., " Enhanced xylitol production: Expression of xylitol dehydrogenase from Gluconobacter oxydans and mixed culture of resting cell", J. Biosci. & Bioeng., 122(3), 257-262, 2016, doi:https: / / doi.org / 10.1016 / j.jbiosc.2016.02.009, and the sequence described in Genbank entry: AKH80557.1 (https: / / www.ncbi.nlm.nih.gov / protein / AKH80557.1, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. XOHDH may be from a Saccharomyces species, for example from Saccharomyces Cerevisiae, for example, Richard, et al., " Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase", FEBS Letters, 457(1), 135-138, 1999, doi:https: / / doi.org / 10.1016 / S0014-5793(99)01016-9, and the sequence described in Genbank entry: EDV09150.1 (see https: / / www.ncbi.nlm.nih.gov / protein / EDV09150.1), each of which is incorporated herein by reference and intended to form part of the disclosure of the present invention. XOHDH may be from a Candida species, for example from Candida tropicalis (see for example, Ko et al., " Production of Xylitol from d-Xylose by a Xylitol Dehydrogenase Gene-Disrupted Mutant of Candida tropicalis', Appl. Environ. Microbiol., 72(6), 4207-4213, 2006, doi:10.1128 / AEM.02699-05), and the sequence described in Genbank entry: ABG49459.1 (seehttps: / / www.ncbi.nlm.nih.gov / protein / ABG49459.1), which is incorporated herein by reference and intended to form part of the disclosure of the present invention. XOHDH may be from a Pichia species, for example from Pichia (Scheffersomyces) stipites, see, for example, Metzger & Hollenberg, " Amino Acid Substitutions in the Yeast Pichia Stipitis Xylitol Dehydrogenase Coenzyme-Binding Domain Affect the Coenzyme Specificity", Eur. J. Biochem., 228, 50-54, 1995, doi:https: / / doi.org / 10.1111 / j.1432-1033.1995.0050o.x, and for example, the sequence described in Genbank entry: A16166 (see https: / / www.ncbi.nlm.nih.gov / nuccore / A16166), each of which is incorporated herein by reference and intended to form part of the disclosure of the present invention. XOHDH may be from a Providencia species, for example from Providencia stuartii, for example Doten & Mortlock, infra, and the sequence described in Genbank entry: EOF5044640.1, see https: / / www.ncbi.nlm.nih.gov / protein / EOF5044640.1, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. In particular, XOHDH may be from a Morganeiia species. Most particularly, the XOHDH is from Morganella morganii In one particular example, the nucleotide sequence encoding the XOHDH comprises a nucleotide sequence of Seq ID No:2. In one example, the nucleotide sequence encoding the XOHDH has at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to Seq ID No:2, and the XOHDH converts xylitol to xylulose. In one particular example, the XOHDH comprises an amino acid sequence of SEQ ID No:l. In one example, the XOHDH comprises an amino acid sequence having at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to Seq ID No:l, and converts xylitol to xylulose. In an example, the XOHDH is heterologous to the cell (e.g. engineered bacterium or recipient bacterium). Alternatively, the XOHDH is endogenous to the cell (e.g. engineered bacterium or recipient bacterium). In another particular example, the XOHDH is capable of converting D-xylitol to D-xylulose. The XOHDH, when isolated, may have a KM of less than 100 mM in an in vitro assay. For example, the KM is less than 50 mM or less than 20 mM. In another example, the XOHDH, when isolated, may have a KM of approximately 16 mM (e.g. when the XOHDH is from M. morganii, see Doten et ai., infra) or approximately 13 nM (e.g. when the XOHDH is from P. stuartii, see Doten etai., infra) in an in vitro assay. The in vitro assay may be as described elsewhere herein.

[0126] A peptide product for the conversion of xylose to xylulose includes, but is not limited to a sugar isomerase (SI), see for example Tucker et al., " Properties of genetically overproduced E. coli xylose isomerase", Biotechnol. Letters, 10(2), 79-84, 1988, doi: https: / / doi.org / 10.1007 / BF01024630. Thus, in a particular example, the one or more nucleotide sequences of (a) encodes a sugar isomerase (SI). SI may be from a species or strain listed in Table 1, whose sequences are described in the UniProt database (https: / / www.uniprot.org / ) under the " SI identifier" provided in Table 1 for each species, and each UniProt sequence for the species in Table 1 is incorporated herein by reference and intended to form part of the disclosure of the present invention. The SI may be from a Escherichia species, for example from E. coli, for example Lawlis et ai., infra, and the sequence described in Genbank entry: P00944 (see https: / / www.ncbi.nlm.nih.gov / protein / P00944), which is incorporated herein by reference and intended to form part of the disclosure of the present invention. In particular,the SI may be from a Morganella species. Most particularly, the SI is from Morganella morganii. In one particular example, the nucleotide sequence encoding the SI comprises a nucleotide sequence of Seq ID No:4. In one example, the nucleotide sequence encoding the SI has at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to Seq ID No:4, and the SI converts xylose to xylulose. In one particular example, the SI comprises an amino acid sequence of SEQ ID No:3. In one example, the SI comprises an amino acid sequence having at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to Seq ID No: 3, and converts xylose to xylulose. In an example, the SI is heterologous to the cell (e.g. engineered bacterium or recipient bacterium). Alternatively, the SI is endogenous to the cell (e.g. engineered bacterium or recipient bacterium). In another particular example, the SI is capable of converting D-xylose to D-xylulose. The SI, when isolated, may have a KM of less than 10 mM in an in vitro assay. The in vitro assay may be as described elsewhere herein.

[0127] Table 1: Species and strains comprising SI proteins

[0128]

[0129]

[0130] A peptide product for the conversion of xylulose to xylulose-5-phosphate includes, but is not limited to xylulokinase (XK). Thus, in a particular example, the one or more nucleotide sequences of (a) encodes a xylulokinase (XK). XK may be from an Escherichia species, for example from E. coli, for example, see Lawlis etai., " Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coil', Appl. Environ. Microbiol., 47, 1984, doi:https: / / doi.org / 10.1128 / aem.47.1.15-21.1984, and the sequence described in Genbank entry: P09099 (see https: / / www.ncbi.nlm.nih.gov / protein / P09099), which is incorporated herein by reference and intended to form part of the disclosure of the present invention. XK may be from a Bacteroides species. XK may be from a Bifidobacterium species. Other XK enzymes that may be used in the present applications are described in https: / / www.ncbi.nlm.nih.gov / gene / ?term=2.7.1.17%5BEC%5D, including enzymes from mammalian origins such as Homo sapiens (Uniprot ID: 075191.3), Canis lupus familiaris (Uniprot ID: A0A8I3Q4W2), Rattus norvegicus (Uniprot ID: Q3MIF4.1), Macaca mulatta (Uniprot ID: A0A1D5R7B) etc, as well as enzymes from bacterial origins, such as Escherichia coli str. K-12 substr. MG1655 (Uniprot ID: P09099), Bifidobacterium adolescentis ATCC 15703 (Uniprot ID: A1A0I0), Bacillus pumiiusSAFR-032 (Uniprot ID: A8FE32), Bacteroides cellulosilyticus (Uniprot ID: A0A0N7IES2), Bacteroides ovatus (Uniprot ID: A0A6N2VBY6) and Bacteroides xylanisolvens Uniprot ID: A0A415KMN6), whose sequences are described in the UniProt database (https: / / www.uniprot.org), which is incorporated herein by reference and intended to form part of the disclosure of the present invention. In particular, SI may be from a Morganella species. Most particularly, the XK is from Morganella morganii. In one particular example, the nucleotide sequence encoding the XK comprises a nucleotide sequence of Seq ID No:6. In one example, the nucleotide sequence encoding the XK has at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to Seq ID No:6, and the XK converts xylulose to xylulose-5-phosphate. In one particular example, the XK comprises an amino acid sequence of SEQ ID No: 5. In one example, the XK comprises an amino acid sequence having at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to Seq ID No: 5, and converts xylulose to xylulose-5-phosphate. In an example, the XK is heterologous to the cell (e.g. engineered bacterium or recipient bacterium). Alternatively, the XK is endogenous to the cell (e.g. engineered bacterium or recipient bacterium). In another particular example, the XK is capable of converting D-xylulose to D-xylulose-5-phosphate. The XK, when isolated, may have a KM of less than 400 pM (e.g. less than 300 pM) in an in vitro assay. The KM may be approximately 290 pM. The in vitro assay may be as described elsewhere herein. See for example, Luccio et al., " Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coif, J. Mol. Biol., 365(3), 783-98, 2007, doi: 10.1016 / j.jmb.2006.10.068, which is incorporated herein by reference in its entirety.

[0131] A peptide product for the conversion of xylitol to xylose includes, but is not limited to xylitol oxidase (AldO), see for example https: / / www.genome.jP / dbget-bin / www_bget7enzyme-i-l.l.3.41, and https: / / www.uniprot.org / uniprotkb / Q9ZBUl / entry. Thus, in a particular example, the one or more nucleotide sequences of (a) encodes a xylitol oxidase (AldO). AldO may be from an Streptomyces species, for example from Streptomyces coelicolor, see Heuts et al., infra, and whose sequences are described in Uniprot entry: Q9ZBU1 (see https: / / www.uniprot.org / uniprotkb / Q9ZBUl / entry), which is incorporated herein by reference and intended to form part of the disclosure of the present invention. In another particular example, the AldO is capable of converting D-xylitol to D-xylose. In an example, the AldO is heterologous to the cell (e.g. engineered bacterium or recipient bacterium). Alternatively, the AldO is endogenous to the cell (e.g. engineered bacterium or recipient bacterium). The AldO, when isolated, may have a KM of less than 1 mM (e.g. less than 0.5 mM, or less than 0.4 mM) in an in vitro assay. The KM may be approximately 0.32 mM, see Heuts et al., " Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor', J. Biol Chem., 282(28), 20283-91, 2007, doi:10.1074 / jbc. M610849200. The in vitro assay may be as described elsewhere herein. AldO may be more advantageous than xylose reductase, because it is able to catalyse the reaction of xylitol to xylose in a single direction, whereas xylose reductase catalyses a reversible reaction. Thus, xylose reductase, in addition to converting xylitol to xylose it can convert xylose back to xylitol, which is expected to be less efficient than the non-reversible conversion of xylitol to xylose by AldO.Another peptide product for the conversion of xylitol to xylose includes, but is not limited to xylose reductase, see for example, Neuhauser, efa / .," NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis, Biochem. J., 326, 683-692, 1997, which is incorporated herein by reference, and https: / / www.brenda-enzymes.org / literature. php?e=l.1.1.307&r=286260. Thus, in an example, the one or more nucleotide sequences of (a) encodes a xylose reductase. The xylose reductase may be from a Neurospora species, for example from Neurospora crassa, whose sequences are described in Supplementary Figure 5 of Woodyer et ai., " Heterologous expression, purification, and characterization of a highly active xylose reductase from Neurospora crassd’, Appl. Environ. Microbiol., 71(3), 1642-7, 2005, doi: 10.1128 / AEM.71.3.1642-1647.2005, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. The xylose reductase may be from a Yamadazyma species, for example from Yamadazyma tenuis, whose sequences are described in the UniProt database, entry: O74237, see https: / / www.uniprot.org / uniprotkb / O74237 / entry, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. The xylose reductase may be from a Scheffersomyces species, for examples from Scheffersomyces stipites, whose sequences are described in Figure 1 of Son et ai. " Structural insight into D-xylose utilization by xylose reductase from Scheffersomyces stipitis', Sci. Rep., 8, 17442, 2018, doi:https: / / doi.org / 10.1038 / s41598-018-35703-x, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. The xylose reductase may be from a Chaetomium species, for examples from Chaetomium thermophiium, whose sequences are described in Figure 8 of Quehenberger et ai., " Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum', Int. J. Mol. Sci., 20(1), 185, 2019, doi:10.3390 / ijms20010185, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. In an example, the xylose reductase is heterologous to the cell (e.g. engineered bacterium or recipient bacterium). Alternatively, the xylose reductase is endogenous to the cell (e.g. engineered bacterium or recipient bacterium). In another particular example, the xylose reductase is capable of converting D-xylitol to D-xylose. The xylose reductase, when isolated, may have a KM (for xylose) of less than 300 mM (for example, less than 260mM) in an in vitro assay. The KM may be approximately 257 mM The in vitro assay may be as described elsewhere herein.

[0132] The in vitro assay for determining KM values may be substantially as described in Doten & Mortlock, J. Bacteriol., " Directed evolution of a second xylitol catabolic pathway in Klebsiella pneumoniad', 159(2), 730-735, 1984, doi:10.1128 / jb.159.2.730-735.1984, which methods are incorporated herein by reference in their entirety. Alternatively, the in vitro assay for determining KM values may be substantially as described in Ehrensberger et ai., Structure, " Structure-Guided Engineering of Xylitol Dehydrogenase Cosubstrate Specificity", 14(3), 567-575, 2006, doi:https: / / doi.org / 10.1016 / j.str.2005.11.016, which methods are incorporated herein by reference in their entirety. In an example, the assay comprises the steps of providing a buffer with substrate and NAD(P)+, adding enzyme at various concentrations and observing the increase in absorbance at 340nm, and fitting the values to a Michaelis-Menten equation. In an example, the buffer Tris (pH 7.0), optionally at (approximately) 100 mM. In an example the substrate concentration, e.g. xylitol is at a saturating concentration, e.g.1.5 M. In an example the NAD(P)+is at a saturating concentration, e.g. between 2 and lOmM, depending on the enzyme concentration. In an example, the enzyme is added a concentrations between 28 and 280 nM. In an example, the absorbance is measured on a spectrophotometer, e.g. a Shimadzu UV-160U spectrophotometer. The Michaelis-Menten equation may be fitted using a program, e.g. DeltaGraph (SPSS, Inc).

[0133] In particular examples, there is provided a bacterium (e.g. engineered or donor), recipient bacterium, or transmissible element which comprises:

[0134] (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of xylitol to xylulose-5-phosphate, wherein the peptide products comprise i. a xylitol dehydrogenase (XOHDH) (as described elsewhere herein), ii. a sugar isomerase (SI) (as described elsewhere herein), and iii. a xylulokinase (XK) (as described elsewhere herein); and

[0135] (b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium (as described elsewhere herein); and

[0136] (c) a further nucleotide sequence encoding an ABC transporter which is capable of importing xylitol into the bacterium or recipient bacterium (optionally from a species selected from a Morganella species, for example from Morganella morganii, or as described elsewhere herein).

[0137] In particular examples, there is provided a bacterium (e.g. engineered or donor), recipient bacterium, or transmissible element comprises:

[0138] (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of. xylitol to xylulose-5-phosphate, wherein the peptide products comprise i. a xylitol dehydrogenase (XOHDH) from Morganella morganii, ii. a sugar isomerase (SI) from Morganella morganii, and iii. a xylulokinase (XK) from Morganella morganii, optionally wherein the peptide products are under the control of the native Morganella morganii promoter; and

[0139] (b) at least one heterologous nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium (as described elsewhere herein); and

[0140] (c) a further nucleotide sequence encoding an ABC transporter which is capable of importing xylitol into the bacterium or recipient bacterium from Morganella morganii, optionally wherein the transporter is under the control of the native Morganella morganii promoter.

[0141] In a most particular example, there is provided a bacterium (e.g. engineered or donor), recipient bacterium, or transmissible element comprises:

[0142] (a) one or more nucleotide sequences encoding peptide product(s) for the conversion of. xylitol to xylulose-5-phosphate, wherein the peptide products comprise i. a xylitol dehydrogenase (XOHDH), comprising an amino acid sequence of SEQ ID No:l, or wherein the nucleotide sequenceencoding the XOHDH comprises a nucleotide sequence of SEQ ID No:2, ii. a sugar isomerase (SI) comprising an amino acid sequence of SEQ ID No:3, or wherein the nucleotide sequence encoding the SI comprises a nucleotide sequence of SEQ ID No:4, and iii. a xylulokinase (XK) comprising an amino acid sequence of SEQ ID No:5, or wherein the nucleotide sequence encoding the XK comprises a nucleotide sequence of SEQ ID No: 6, optionally wherein the peptide products are under the control of the native Morganella morganii promoters (e.g. the promoters of SEQ ID No: 13); and

[0143] (b) at least one heterologous nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium (as described elsewhere herein); and

[0144] (c) a further nucleotide sequence encoding an ABC transporter comprising the amino acid sequences of SEQ ID No:7, SEQ ID No:8 and SEQ ID No:9, or wherein the nucleotide sequence encoding the transporter comprises nucleotide sequences of SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, optionally wherein the transporter is under the control of the native Morganella morganii promoters (e.g. the promoters of SEQ ID No: 13).

[0145] Optionally, and in a particular example, the native Morganella morganii promoter comprises a nucleotide sequence of SEQ ID No: 13, and the promoters control expression of a repressor encoded by a nucleotide sequence of SEQ ID No: 14, or control expression of a nucleotide sequence encoding a repressor having an amino acid sequence of SEQ ID No: 15, and wherein the nucleotide sequence encoding the repressor is downstream (3') of the promoter sequence of SEQ ID No: 13. The inclusion of the repressor has the effect of making the promoter inducible, see the discussion elsewhere herein, and may be particularly beneficial. More particularly, the sequences of part (a) and (c) are under the control of a promoter of SEQ ID No: 13, and the bacterium, recipient bacterium, or transmissible element further comprises a nucleotide sequence of SEQ ID No: 14 which encodes a repressor of the promoter of SEQ ID No: 13, wherein the nucleotide sequence encoding the repressor is downstream (3') of the promoter sequence of SEQ ID No: 13 (and optionally wherein the nucleotide sequences encoding the SI is downstream (3') of the promoter sequence of SEQ ID No: 13 and upstream of the sequence encoding the SI, and further optionally wherein the nucleotide sequences encoding the transporter of part (c), the XK and the XOHDH is upstream (5') of the promoter sequence of SEQ ID No: 13 (e.g. wherein the sequence encoding the transporter is upstream of the sequence encoding the XK; and the sequence encoding the XK is upstream of the sequence encoding the XOHDH.

[0146] Importers of precursor substrates, in particular xylitol

[0147] While some bacteria are capable of importing precursor substrates, such as xylitol, e.g. by diffusion, the Examples below show that the conversion on xylitol and subsequent growth of cell can be increased if the bacteria comprise a transporter which is capable of importing the xylitol into the cell (e.g. engineered bacterium or recipient bacterium). Thus, the bacterium, recipient bacterium or transmissible element may further comprise: (c) a further nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium or recipient bacterium.Other bacteria may already express such a transporter, thus, the transporter which is capable of importing the xylitol into the cell may be endogenous to the cell (e.g. engineered bacterium or recipient bacterium). Thus, in other examples, a bacterium which comprises (c) a further nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium may be further engineered to increase expression of the transporter. This could be by increasing the number of copies of the nucleotide sequence encoding the transporter, such that the bacterium comprises both endogenous nucleotide sequences encoding the transporter, and additional copies of the nucleotide sequences which have been introduced by genetic engineering techniques (for example, engineered into the chromosome of the bacterium) or by a transmissible element. In this latter example, the transmissible element comprises nucleotide sequences encoding a transporter, where the nucleotide sequences are endogenous to the recipient bacterium and the recipient bacterium comprises endogenous nucleotide sequences encoding the transporter.

[0148] Additionally or alternatively, increasing expression of the transporter encoded by a bacterium which comprises endogenous nucleotide sequences encoding the transporter may be achieved by increasing the strength of the promoter (e.g. one, more than one or all of the promoters) controlling expression of the transporter. Suitable promoters are described elsewhere herein. Thus, there is provided a bacterium comprising (c) a nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium, operably linked to a promoter which is not operably linked to the nucleotide sequence encoding the transporter in nature.

[0149] However, in a particular example, the bacterium (e.g. engineered or donor), recipient bacterium or transmissible element may further comprise: (c) a further nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium or recipient bacterium, wherein the nucleotide sequence is heterologous to the bacterium or recipient bacterium.

[0150] Any transporter herein may be from a species selected from a Morganella species, for example from Morganella morganii.

[0151] In an example, the transporter is a xylose transporter. The xylose transporter may be xylFGI. The xylose transporter may be from an Escherichia species, e.g. E. coli, for example the sequence described in Genbank entry: P37387 (see https: / / www.ncbi.nlm.nih.gov / protein / P37387 for xylF), Genbank entry: P37388 (see https: / / www.ncbi.nlm.nih.gov / protein / P37388 for xylG), and Genbank entry: P0AGI4 (see https: / / www.ncbi.nlm.nih.gov / protein / P0AGI4 for xyll), each of which is incorporated herein by reference and intended to form part of the disclosure of the present invention.

[0152] In another example, the transporter is a phosphotransferase transporter. The phosphotransferase transporter may be fruI. The phosphotransferase transporter may be from a Streptococcus species, e.g. Streptococcus mutans, for example Wen et al., " Characterization of two operons that encode components of fructose-specific enzyme II of the sugar: phosphotransferase system of Streptococcus mutant, FEMS Microbiol. Lett., 205(2), 337-42, 2001, doi: 10.1111 / j.l574-6968.2001.tbl0969.x. and the sequence described in Genbank entry: (see ), which is incorporatedherein by reference and intended to form part of the disclosure of the present invention. These phosphotransferases phosphorylate xylitol as it crosses the bacterial membrane. In some bacteria, the xylitol-phosphate is converted back into xylitol through hydrolysis, which can then be subsequently used by the cells herein to produce a carbon source as described. In other bacteria the xylitol- phosphate accumulates inside the cell, and becomes toxic, which is thought to be dues to the xylitol phosphate inhibiting glucose uptake and glycolytic enzymes in the bacteria, see Trahan et al., " Transport and Phosphorylation of Xylitol by a Fructose Phosphotransferase System in Streptococcus mutant, Caries Res., 19(1), 53-63, 1985, doi: https: / / doi.org / 10.1159 / 000260829, and Trahan et a / ., " Intracellular xylitol-phosphate hydrolysis and efflux of xylitol in Streptococcus sobrinus', Oral Microbiol. & Immunol., 6(1), 41-50, 1991, doi:https: / / doi.org / 10.1111 / j.l399-302X.1991.tb00450.x, and Kõljalg et al., " Effect of erythritol and xylitol on Streptococcus pyogenes causing peritonsillar abscesses", Sci. Rep., 11, 15855, 2021, doi:https: / / doi.org / 10.1038 / s41598-021-95367-y, and https: / / www.genome.jp / entry / R02136, each of which are incorporated herein by reference in its entirety. A skilled person is able to screen and test for potential toxicity of xylitol which is taken up through a phosphotransferase transporter, for example using the methods described in Koljalg et al.

[0153] In another example, the transporter is hxt11. The hxtll transporter may be from a Saccharomyces species, e.g. Saccharomyces cerevisiae, for example the sequence described in the UniProt database, entry: P54862, see https: / / www.uniprot.org / uniprotkb / P54862 / entry, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. See additionally, Jordan et al., " Hxtl3, Hxtl5, Hxtl6 and Hxtl7 from Saccharomyces cerevisiae represent a novel type of polyol transporters", Sci Rep., 6, 23502, 2016, doi:https: / / doi.org / 10.1038 / srep23502, and Shin et al., " An engineered cryptic Hxtll sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae', Biotechnol. Biofuels, 8, 176, 2015, doi:10.1186 / sl3068-015-0360-6, each of which is incorporated herein by reference in its entirety.

[0154] In another example, the transporter is hxt15. The hxt15 transporter may be from a Saccharomyces species, e.g. Saccharomyces cerevisiae, for example the sequence described in the UniProt database, entry: P54854, see https: / / www.uniprot.org / uniprotkb / P54854 / entry, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. See for example, Jordan et al., supra.

[0155] In another example, the transporter is plt4. The plt4 transporter may from a Lotus species, e.g. Lotus Japonicus, for example the sequence described in Figure 1 of Kalliampakou et al., " Cloning and functional characterization of LjPLT4, a plasma membrane xylitol H+-symporter from Lotus japonicus', Molecular Membrane Biology, 28(1), 1-13, 2011, doi:https: / / doi.org / 10.3109 / 09687688.2010.500626, which is incorporated herein by reference and intended to form part of the disclosure of the present invention. See for example, Kalliampakou et a!., " Cloning and functional characterization of ZyPLT4, a plasma membrane xylitol H+-symporter from Lotus japonicus" MolecularMembrane Biology, 28(1), 1-13, 2011, doi:https: / / doi.org / 10.3109 / 09687688.2010.500626, which is incorporated herein by reference in its entirety.

[0156] As is shown below in Example 3, a particularly useful transporter is an ABC transporter. Thus, in a particular example, the bacterium (e.g. engineered or donor), recipient bacterium or transmissible element further comprises (c) a further nucleotide sequence encoding an ABC transporter which is capable of importing xylitol into the bacterium or recipient bacterium. In one particular example, the nucleotide sequence encoding the transporter comprises a nucleotide sequences of each of SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12. In one example, the nucleotide sequences encoding the transporter have at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to each of SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, and the transporter is capable of importing xylitol into the bacterium or recipient bacterium. In one particular example, the transporter comprises an amino acid sequence of each of SEQ ID No:7, SEQ ID No:8 and SEQ ID No:9. In one example, the transporter comprises an amino acid sequence having at least 80% identity (such as 85%, 90% or 95%, in particular 90% identity) to each of SEQ ID No:7, SEQ ID No:8 and SEQ ID No:9, and is capable of importing xylitol into the bacterium or recipient bacterium. In an example, the ABC transporter is heterologous to the cell (e.g. engineered bacterium or recipient bacterium). Alternatively, the ABC transporter is endogenous to the cell (e.g. engineered bacterium or recipient bacterium).

[0157] The ABC transporter may be controlled by a native promoter from Morganella morganii, for example the promoters of SEQ ID No: 13. The ABC transporter may be located upstream (5') of the promoter sequence of SEQ ID No: 13. Optionally any XK and XOHDH sequences may also be located upstream (5') of the promoter sequence of SEQ ID No: 13 (e.g. wherein the sequence encoding the transporter is upstream of the sequence encoding the XK; and the sequence encoding the XK is upstream of the sequence encoding the XOHDH). Additionally, any nucleotide sequence encoding the SI may be downstream (3') of the promoter sequence of SEQ ID No: 13. Optionally, any nucleotide sequence encoding a repressor may also be located downstream (3') of the promoter sequence of SEQ ID No: 13 (e.g. also upstream of the sequence encoding the SI).

[0158] Administration of precursor substrates such as xylitol

[0159] When the subject is administered a precursor substrate, such as xylitol, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) act on the precursor substrate to produce a carbon source which can be used by the bacterium (e.g. engineered bacterium) or recipient bacterium to provide a selective advantage and maintain or increase the colonisation levels of the bacterium or recipient bacterium. The colonisation may be in a desired microbiome, for example in the gut of a subject.

[0160] In the most particular example, the precursor substrate is xylitol. The xylitol may particularly be D-xylitol.In an example, wherein the precursor substrate is a D precursor substrate (e.g. D-xylitol), and wherein all products (e.g. xylose, xylulose and / or xylulose-5-phosphate) derived from said precursor substrate (e.g. D-xylitol) are D products (e.g. D-xylose, D-xylulose and / or D-xylulose-5-phosphate).

[0161] In a particular example, the xylitol is provided in the form of a composition for oral administration. The xylitol may be food grade xylitol. The xylitol may be a lyophilised formulation, in particular a lyophilised formulation for oral administration. The xylitol may be an encapsulated formulation to be released in the lower gut of the subject, in particular an encapsulated formulation for oral administration. The xylitol may be in the form of a powder for reconstitution in water, for example where the reconstituted xylitol is orally administered. The xylitol may be in the form of a solution for reconstitution in water, for example where the reconstituted xylitol is orally administered. The xylitol may be in the form of a powder for reconstitution in an ingestible liquid, for example where the reconstituted xylitol is orally administered. The xylitol may be in the form of a solution for reconstitution in an ingestible liquid, for example where the reconstituted xylitol is orally administered. The formulation may be comprised by a sachet. The xylitol may be in the form of a tablet for oral administration. The formulation may be comprised by a blister pack.

[0162] In one example, the xylitol may be polymerised or oligomerised with another molecule which would be non-toxic if ingested by a subject, e.g. a human. This would be useful for allowing the polymerised xylitol to more easily reach the colon instead of being taken up earlier in the digestive tract.

[0163] Doses of xylitol may be determined by those skilled in the art. In one example, the xylitol is provided in a dosage form comprising 5g of xylitol. The xylitol may be provided in a dosage form comprising 10g of xylitol. The xylitol may be provided in a dosage form comprising 15g of xylitol. The xylitol may be provided in a dosage form comprising 20g of xylitol. The dosage form may be a sachet of xylitol. The dosage form may be comprised by a sachet.

[0164] Each dose of xylitol may be administered (at least) twice a day. Each dose of xylitol may be administered (at least) three times a day. Each dose of xylitol may be administered (at least) four times a day. Each dose of xylitol may be ingested by the subject (at least) twice a day. Each dose of xylitol may be ingested by the subject (at least) three times a day. Each dose of xylitol may be ingested by the subject (at least) four times a day. The dosing regimen of xylitol may be of the same duration as the dosing regimen of the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition. In a particular example, the xylitol is not administered at the same time (for example within one hour) as the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition.

[0165] In particular, 5g of xylitol is administered to the subject twice a day. Alternatively, 10g of xylitol is administered to the subject twice a day. In an example, the subject ingests up to 50g of xylitol per day. Without being bound by theory, it is thought that, even if used by the bacteriadescribed herein, that excess xylitol may accumulate in the colon, which may have undesirable side effects, such as excess gas, and diarrhoea. That said, the more that is metabolised by the bacteria described herein, the more xylitol can be tolerated before such side effects may be seen.

[0166] The total dose of xylitol per day may be between 0.1 and 0.6g / kg / day, such as between 0.4 and 0.5 g / kg / day or between 0.15 and 0.25 g / kg / day. In particular, the total dose may be 0.18 g / kg / day or may be 0.44 g / kg / day.

[0167] For example, if the subject receives the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition over two months, then xylitol may also be administered over two months. The dosing regimen of the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition may not be identical to that of the xylitol.

[0168] Exogenous Genes for the production of a first MOI

[0169] In any example herein, the at least one nucleotide sequence of (b) may be comprised by a transmissible element. Alternatively, the at least one nucleotide sequence of (b) may be comprised by a recipient bacterium. Further alternatively, the at least one nucleotide sequence of (b) may be comprised by a bacterium, e.g. an engineered bacterium.

[0170] In a particular example, all of the proteins encoded by the at least one nucleotide sequence of (b) are heterologous to the bacterium or recipient bacterium.

[0171] The one or more exogenous nucleotide sequences may be for the production of a first molecule of interest (MOI) in the bacteria (e.g. engineered bacteria) or recipient bacterium. The at least one nucleotide sequence of (b) may encode the first MOI. The at least one nucleotide sequence of (b) may comprise or consist of a heterologous gene for the production of the first MOI. The production of the first MOI may be direct (i.e. the heterologous nucleotide sequence expresses the first MOI from the sequence).

[0172] In an example, the first MOI is selected from a therapeutic molecule (e.g. a peptide molecule) and a beneficial or therapeutic metabolite. In particular, the first MOI is a beneficial or therapeutic metabolite.

[0173] The first MOI may be a therapeutic molecule, such as a peptide molecule (e.g. an antibody fragment, a hormone such as GLP-1, an interleukin, a cytokine, a chemokine, an eukaryotic growth factor, or an enzyme) or a small molecule (e.g. a metabolite such as L-DOPA, indole-3-acetic acid, butyrate etc).

[0174] The at least one nucleotide sequence for the production of a first MOI of (b) may encode an MOI which is a therapeutic molecule.

[0175] In one example, the first MOI is a therapeutic peptide molecule, e.g. a therapeutic protein. Therapeutic proteins may be used to, for example, replace a protein that is deficient or abnormal, augment an existing biological pathway, provide a novel function or activity, interfere with a moleculeor organism, and / or deliver other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins.

[0176] Therapeutic proteins include antibody fragments, hormones, interleukins, cytokines, chemokines, eukaryotic growth factors and / or enzymes.

[0177] When the first MOI is a protein molecule (e.g. a therapeutic protein molecule), a bacterial transport signal peptide (SP) may be needed so that the bacteria can secrete the active substances out of the bacteria. SPs are short peptides with 16-30 amino acids and are N-terminal extensions of secreted proteins. They act as a target and recognition signal for signal peptidases which remove SPs from the translocated protein across the cytoplasmic membrane and cell wall. This process will result in the extracellular release of the mature protein or peptide.

[0178] Thus, a signal peptide may also be required to aid secretion of the first MOI (e.g. therapeutic protein molecule) to the periplasm of the bacterium or recipient bacterium, to the cell surface of the bacterium or recipient bacterium, or to the extracellular space outside of the bacterium or recipient bacterium. In any example herein, the bacterium, recipient bacterium or transmissible element may further comprise (e) a further nucleotide sequence encoding a signal peptide for the secretion of the first MOI (e.g. therapeutic protein molecule) to the periplasm of the bacterium or recipient bacterium. In any example herein, the bacterium, recipient bacterium or transmissible element may further comprise (e) a further nucleotide sequence encoding a signal peptide for secretion of the first MOI (e.g. therapeutic protein molecule) to the cell surface of the bacterium or recipient bacterium. In any example herein, the bacterium, recipient bacterium or transmissible element may further comprise (e) a further nucleotide sequence encoding a signal peptide for secretion of the first MOI (e.g. therapeutic protein molecule) to the extracellular space outside of the bacterium or recipient bacterium.

[0179] Further, as is known in the art, diffusion of molecules through bacterial membrane(s) (both gram-negative and gram-positive bacteria) can limit the amount of a particular molecule which diffuses to the local environment. Without being bound by theory, in general, bacterial membrane(s) are hydrophobic, so the more hydrophilic the first MOI is, the less likely it is to be able to cross the membrane. However, certain molecules which are detrimental to the cell (for example toxins, bacteriocins, etc) are exported more often, along with certain molecules which provide specific extracellular functions (such as molecules associated with quorum sensing, iron acquisition, etc). In addition, some molecules are actively transported through the membrane(s) by dedicated exporters (for example, excess amino acids may be exported from the cell to maintain homeostasis, such as the alaE exporter of alanine, and the leuE exporter of leucine both found in E. coli), or exporters which export a certain class of molecule (for example, the setA transporter found in E. coli exports various sugar molecules). Thus, in many bacteria, secretion of MOIs may be limited by the rate of diffusion. The inventors have engineered bacteria to express heterologous exporters which are capable of exporting an first MOI across the membrane(s) of the bacteria. Unexpectedly, heterologous exporters are able to form and function within the bacterial membrane (G4 in Figure 8 is an exporter of the firstMOI). This leads to an increased secretion of the first MOI into the local environment, and contributes to the reduced fitness disadvantage of the expression of the first MOI within the bacteria, because (without being bound by theory) the first MOI is removed from the cytoplasm and / or periplasm into the local environment.

[0180] Thus, in any example herein, the bacterium (e.g. engineered or donor), recipient bacterium or transmissible element may further comprise (d) a further nucleotide sequence encoding an exporter which is capable of exporting the first MOI (e.g. the therapeutic protein molecule or metabolite) out of the bacterium or recipient bacterium. In any example herein, the transmissible element may further comprise an exogenous nucleotide sequence encoding one or more exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite) from the bacterium or recipient bacterium. In a particular example, the nucleotide sequence encodes an exporter of the first MOI.

[0181] In one example, the bacterium, recipient bacterium or transmissible element comprises a nucleotide sequence which encodes, in 5' to 3' direction, a promoter, a sequence encoding a signal peptide, at least one nucleotide sequence for the expression of the first MOI and optionally a further nucleotide sequence encoding one or more (e.g. one) exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite) from the bacterium or recipient bacterium. In a particular example, the nucleotide sequence encodes an exporter of the first MOI.

[0182] The therapeutic molecule may be an antibody therapy. In particular, the therapeutic molecule is an antibody fragment. The therapeutic molecule may comprise an antibody (or in particular an antibody fragment) comprising the binding domains of any of the specific antibody molecules described herein. The therapeutic molecule may comprise an antibody fragment which binds to any of the targets of any of the antibody molecules described herein. Fc fusion proteins are also contemplated.

[0183] The antibody therapy may be an immune checkpoint inhibitor antibody or fragment thereof. The antibody or fragment thereof may be selected from an anti-PD-Ll, anti-PD-1, anti-CTLA4, anti-TIM3, anti-TNFa superfamily member (such as an anti-TNFa, TNFR1 or BAFF), anti-IL6R, anti-IL-4Ra, or anti-PCSK9.

[0184] Examples of antibodies, antibody fragments, and / or Fc fusion proteins include, without limitation, Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (or tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, Cedelizumab, Certolizumab, pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Conatumumab, Concizumab, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizu mab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN, Ticilimumab (or tremelimumab), Tildrakizumab, Tigatuzumab, TNX-, Tocilizumab (or atlizumab), Toralizumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab,Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab and Zolimomab aritox.

[0185] Therapeutic hormones include glucagon-like peptide-1 (GLP-1) or GLP-t and insulin. Other examples of peptide hormones include, without limitation, Amylin (or Islet Amyloid Polypeptide), Antimullerian hormone (or Müllerian inhibiting factor or hormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin), Angiotensi nogen and angiotensin, Antidiuretic hormone (or vasopressin, arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin), Brain natriuretic peptide, Calcitonin, Cholecystokinin, Corticotropin-releasing hormone, Enkephalin, Endothelin, Erythropoietin, Follicle-stimulating hormone, Galanin, Gastrin, Ghrelin, Glucagon, Gonadotropin-releasing hormone, Growth hormone-releasing hormone, Human chorionic gonadotropin, Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-like growth factor (or somatomedin), Leptin, Lipotropin, Luteinizing hormone, Melanocyte stimulating hormone, Motilin, Orexin, Oxytocin, Pancreatic polypeptide, Parathyroid hormone, Prolactin, Prolactin releasing hormone, Relaxin, Renin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulating hormone (or thyrotropin), and Thyrotropin-releasing hormone.

[0186] Examples of interleukins include, without limitation, interleukin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. The interleukin may be interleukin-2, interleukin-4, interleukin-6, interleukin-7, interleukin-10, interleukin-11 or nterleukin-13. The interleukin may in particular be interleukin 1-17.

[0187] Examples of cytokines include, without limitation pegfilgratim, anakinra, emfilermin, denileukin diftitox, interferon-a, erythropoietin, granulocyte-macrophage colony stimulating factor, interleukin-1 receptor antagonist, granulocyte colony stimulating factor, Cintredekin besudotox, Romiplostim, Regramostim, Albinterferon Alfa-2B, Maxy-G34, Thrombopoietin, stem cell factors, erythropoiesis stimulating agents, Avotermin, Nagrestipen, Binetrakin, Dulanermin, Muplestim, Molgramostim, Balugrastim, Lipegfilgrastim, CD40-ligand, Leridistim, Bempegaldesleukin, Pegilodecakin, ALT-801, Teceleukin, Lorukafusp-a, edodekin-a, viral macrophage inflammatory protein-II (vMIP), interferon-K, Efineptakin-a and Tengonermin. Examples of interferons (IFNs) include, without limitation, IFN-a, IFN-p, IFN-co and IFN-y.

[0188] Examples of chemokines include, without limitation, CC chemokines (e.g. CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10), CXC chemokines (e.g. CXCR1, XCR2, CXCR3, CXCR4, CXCR5, CXC6 and CXCR7), C chemokines or CX3C chemokines (e.g. CX3CR1).

[0189] Examples of growth factors include, without limitation, Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and otherneurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-a), Transforming growth factor beta (TGF-β), Tumor necrosis factor-alpha (TNF-a), Vascular endothelial growth factor (VEGF), placental growth factor (PIGF), Foetal Bovine Somatotrophin (FBS) and IL-1-IL7.

[0190] Examples of enzymes include, without limitation, any of the enzymes assigned an Enzyme Commission Number (EC) number (e.g. EC1-EC6) by the International Union of Biochemistry and Molecular Biology (IUBMB) (Webb, Edwin C. Enzyme nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. San Diego: Published for the International Union of Biochemistry and Molecular Biology by Academic Press. ISBN 0-12-227164-5 (1992), incorporated herein by reference). Other examples include: styrene monooxygenase (StyAB), toluene dioxygenase (TODC1C2AB), luciferase and lactase. In some examples, the enzyme is toluene dioxygenase. In some examples, the enzyme is styrene monooxygenase.

[0191] In any example, the first MOI is a therapeutic molecule described in Table 2 below.

[0192] Other therapeutic molecules include, without limitation, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, and thrombolytics. Other examples include those that bind non-covalently to target (e.g. monoclonal antibodies or fragments thereof), those that affect covalent bonds (e.g. enzymes), and those that exert activity without specific interactions (e.g. serum albumin).

[0193] Therapeutic molecules include recombinant therapeutic proteins, used to treat, for example, cancers, immune disorders, infections and / or other diseases.

[0194] In one example, the therapeutic protein is selected from Etanercept, Bevacizumab, Rituximab, Adalimumab, Infliximab, Trastuzumab, Insulin glargine, Epoetin alfa, Pegfilgrastim, Ranibizumab, Darbepoetin alfa, Interferon beta-la, Insulin aspart, Rhu insulin, Octocog alfa, Insulin lispro, Cetuximab, Peginterferon alfa-2a, Interferon beta-lb, Eptacog alfa, Insulin aspart, OnabotulinumtoxinA, Epoetin beta, Rec antihemophilic factor, Filgrastin, Insulin detemir, Natalizumab, Insulin (humulin) and Palivizumab.

[0195] Examples of Fc fusion proteins include, without limitation, Etanercept, Alefacept, Abatacept, Rilonacept, Romiplostim, Belatacept and Aflibercept.

[0196] Examples of anticoagulants and / or blood factors include, without limitation, Protein C, Protein S, and antithrombin, Factors I-VIII, prothrombinase, prothrombin, thrombin von Willebrand Factor (vWF), fibrinogen, fibrin and fibrinopeptides.

[0197] Examples of bone morphogenetic proteins (BMPs) include, without limitation, BMP1-BMP7, BMP8a, BMP8b, BMP10, and BMP15.Table 2: MOIs produced bv genetically modified bacteria

[0198]

[0199]

[0200] Table produced from Omer eta / ., supra and Zhou eta / ., Engineering Microbiol., 2(3), 100034, 2022, doi:https: / / doi.org / 10.1016 / j.engmic.2022 100034

[0201] Zeng etal., Appl. Microbiol. Biotechnol., 101, 7177-7186, 2017, doi:10.1007 / s00253-017-8410-6

[0202] 5 Carvalho etal., Microb. Cel. Fact., 16, 27-0624, 2017, doi:10.1186 / sl2934-017-0624-x

[0203] An etal J. Microbiol., 59, 202-21, 2021, doi:10.1007 / sl2275-021-0562-5

[0204] Ciacma etal., Microb. Cel. Fact., 17, 018-1028, 2018, doi:10.1186 / sl2934-018-1028-2

[0205] Yue etal., Microb. Cel. Fact., 19, 213, 2020, doi:10.1186 / sl2934-020-01466-2

[0206] Gusmao-Silva etal., Front. Immunol., 11, 562905, 2020, doi: 10.3389 / fimmu.2020.562905

[0207] 0 Zhang etal., PLoS One, 12, e0188960, 2017, doi:10.1371 / journal. pone.0188960

[0208] Peng etal., Sci. Rep., 8, 6435, 2018, doi:10.1038 / s41598-018-24879-x

[0209] Mao etal., Sci. Transl. Med., 10, eaao2586, 2018, doi:10.1126 / scitranslmed.aao2586Kikuchi eta!., Biochem. Biophysical Res. Commun., 493(1), 306-312, 2017, doi:10.1016 / j.bbrc.2017.09.026 Guo et al., J. Pharm. Biomed. Sci., 7(6), 2017, doi:10.20936 / JPBMS / 170602

[0210] Liu eta / ., Int. Immunopharmacology, 57, 25-32, 2018, doi:10.1016 / j.intimp.2018.02.004

[0211] Geldart eta / ., Bioeng. Translational Med., 3, 197-208, 2018, doi:10.1002 / btm2.10107

[0212] 5 Praveschotinunt eta / ., Nat. Commun., 10, 5580, 2019, doi:10.1038 / s41467-019-13336-6

[0213] Yoon eta / ., Eur. J. Cancer, 70, 48-61, 2017, doi: 10.1016 / j.ejca.2016.10.010

[0214] Puurunen eta!., Nat. Metab., 3, 1125-1132, 2021, https: / / doi.org / 10.1038 / s42255-021-00430-7 Chowdhury eta / ., Nat. Med., 25, 1057-1063, 2019, https: / / doi.org / 10.1038 / s41591-019-0498-z

[0215] Zeng eta / ., Appl. Microbiol. Biotechnol., 101, 7177-7186, 2017, https: / / doi.org / 10.1007 / s00253-017-8410-6 10 Chiabai eta / ., BMC Biotechnol., 19, 38, 2019, https: / / doi.org / 10.1186 / sl2896-019-0518-6Other examples of therapeutic proteins include, without limitation, Insulin (blood glucose regulator), Pramlintide acetate (glucose control), Growth hormone GH (growth failure), Pegvisoman (growth hormone receptor antagonist), Mecasermin (IGF1, growth failure), Factor VIII (coagulation factor), Factor IX (coagulation factor, Protein C concentrate (anti-coagulation), al-proteinase inhibitor (anti-trypsin inhibitor), Erythropoietin (stimulates erythropoiesis), Filgrastim (granulocyte colonystimulating factor, G-CSF; stimulates neutrophil proliferation), Sargramostim 36, 37 (granulocytemacrophage colony-stimulating factor, GM-CSF), Oprelvekin (interleukin-11, IL-11), Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-a (human luteinizing hormone), Interleukin 2 (IL-2), Interleukin-1 Receptor Agonist, Denileukin diftitox (fusion of IL-2 and Diphtheria toxin), Interferon alfacon 1 (consensus interferon), Interferon-a2a (IFN-a2a), Interferon-a2b (IFN-a2b), Interferon-an3 (IFN-an3), Interferon-pia (rIFN-p), Interferon-plb (rIFN-p), Interferon-ylb (IFNy), Salmon calcitonin (32-amino acid linear polypeptide hormone), Teriparatide (part of human parathyroid hormone 1-34 residues), Exenatide (Incretin mimetic with actions similar to glucagon-like peptide 1), Octreotide (octapeptide that mimics natural somatostatin), Dibotermin-a (recombinant human bone morphogenic protein 2), Recombinant human bone morphogenic protein 7, Histrelin acetate (gonadotropin-releasing hormone; GnRH), Palifermin (Keratinocyte growth factor, KGF), Becaplermin (platelet-derived growth factor, PDGF), Nesiritide (recombinant human B-type natriuretic peptide), Lepirudin (recombinant variant of hirudin, another variant is Bivalirudin), Anakinra (interleukin 1 (IL-1) receptor antagonist), Enfuviritide (an HIV-1 gp41-derived peptide), £-Glucocerebrosidase (hydrolyzes to glucose and ceramide), Alglucosidase-a (degrades glycogen), Laronidase (digests glycosaminoglycans within lysosomes), Idursulfase (cleaves O-sulfate preventing GAGs accumulation), Galsulfase (cleave terminal sulphage from GAGs), Agalsidase-0 (human a-galactosidase A, hydrolyzes glycosphingolipids), Lactase (digest lactose), Pancreatic enzymes (lipase, amylase, protease; digest food), Adenosine deaminase (metabolizes adenosine), Tissue plasminogen activator (tPA, serine protease involved in the breakdown of blood clots), Factor Vila (serine protease, causes blood to clot), Drotrecogin-a (serine protease, human activated protein C), Trypsin (serine protease, hydrolyzes proteins), Botulinum toxin type A (protease, inactivates SNAP-25 which is involved in synaptic vesicle fusion), Botulinum toxin type B (protease that inactivates SNAP-25 which is involved in synaptic vesicle fusion), Collagenase (endopeptidase, digest native collagen), Human deoxyribonuclease I (endonuclease, DNase I, cleaves DNA), Hyaluronidase (hydrolyzes hyaluronan), Papain (cysteine protease, hydrolyzes proteins), L-Asparaginase (catalyzes the conversion of L-asparagine to aspartic acid and ammonia), Rasburicase (urate oxidase, catalyzes the conversion of uric acid to allantoin), Streptokinase (Anistreplase is anisoylated plasminogen streptokinase activator complex (APSAC)), and Antithrombin III (serine protease inhibitor).

[0216] Examples of small molecule therapeutics include beneficial metabolites. Therapeutics include GLP1, GLP2, IL10, IL22, TNF-a, a STING agonist such as ci-di-AMP and human epidermal growth factor. Beneficial metabolites include, without limitation, L-DOPA, butyrate, propionate, acetate, GLP1,GLP2, IL10, IL22, indole, tryptophan, indole-3-acetic acid (IAA), arginine, tryptophan, kynurenine and kynurenic acid.

[0217] Production of TNF-a (first MOI) is described in WO2022 / 072636A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety.

[0218] Production of the STING agonist, ci-di-AMP (first MOI), using the dacA gene from Listeria monocytogenes is disclosed in WO2022 / 150779A1 and W02020 / 097424A1 (both Synlogic Operating Company, Inc.), each of which is incorporated herein by reference in its entirety. This MOI is useful in treating cancers.

[0219] Production of human epidermal growth factor (hEGF, first MOI), which uses the human EGF cDNA sequence (NCBI accession number:gq214314, codon-optimised for expression in E. coli), with N-terminal sec secretion signal sequences of either PhoA (21 amino acids), PcIB (22 amuio acids), or OmpA (21 amino acids) is disclosed in WO2022 / 221273A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety. Other expression constructs which could be used in the present disclosure for expression of a first MOI are disclosed in Example 1 therein.

[0220] In one example, the at least one nucleotide sequence of (b) does not comprise any reporter genes. In one example, the at least one nucleotide sequence of (b) does not comprise any antibiotic resistance genes.

[0221] Exogenous genes for the conversion of a second MOI to a first MOI

[0222] In an example, the at least one nucleotide sequence of (b) encodes one or more protein(s) for the conversion of a second MOI to the first MOI.

[0223] In a particular example, all of the proteins encoded by the at least one nucleotide sequence of (b) are heterologous to the bacterium or recipient bacterium.

[0224] Where a second MOI is converted to a first MOI, the first MOI may be selected from a therapeutic molecule (e.g. as described elsewhere herein) and a beneficial or therapeutic metabolite. Alternatively, the second MOI is a detrimental metabolite

[0225] For example, the therapeutic molecule may be a prodrug, and the protein encoded by the at least one nucleotide sequence of (b) is an enzyme which converts the prodrug to the active therapeutic drug. In another example, the therapeutic molecule is a peptide or fusion protein, and the protein encoded by the at least one exogenous nucleotide sequence cleaves the peptide or fusion protein to release a desired sub-unit of the original peptide or fusion protein.

[0226] Examples of small molecule therapeutics include beneficial metabolites. Beneficial metabolites include, without limitation, L-3,4-dihydroxyphenylalanine (L-DOPA), butyrate, propionate, acetate, GLP1, GLP2, IL10, IL22, indole, tryptophan, indole-3-acetic acid (IAA), arginine, tryptophan, kynurenine and kynurenic acid.

[0227] Alternatively, the second MOI may be a detrimental metabolite. In this example, the first MOI is either neutral or does not cause the detrimental effect of the second MOI to the cell, organism ormicrobiome. Thus, reducing levels of detrimental metabolites may be achieved by the transmissible elements herein.

[0228] Such detrimental metabolites include leucine, isoleucine, valine, chenodeoxycholic acid (CDCA), oxalate, uric acid, propionate and methionine.

[0229] In a particular example, the first MOI is a beneficial metabolite or a therapeutic metabolite. L-DOPA (a first MOI), for example, can be made from tyrosine (second MOI) using tyrosine 3-hydroxylase (EC 1.14.16.2); or from L-phenylalanine (second MOI) using two enzymes, phenylalanine 4-hydroxylase (EC 1.14.16.1) and tyrosine 3-hydroxylase. Methods for gene insertion into bacteria are provided in W02022 / 013407A1 (Danmarks Tekniske Universitet), which is incorporated herein by reference in its entirety.

[0230] Genes for producing butyrate (first MOI) from crotonyl-CoA (second MOI) are described for example in WO2017 / 136792A2 and WO2017 / 074566A1 (Synlogic Operating Company, Inc.), which are both incorporated herein by reference in their entirety. Genes may include one or more (for example all) of the genes from butyrate production pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk); or trans-2-enoynl-CoA reductase (ter) from Treponema denticoia, in combination with thiAi, hbd, crt2, pbt, and buk ovn Peptoclostridium difficile 630; or a thioesterase (tesB) from E. Coli in combination with bcd2, etfB3, etfA3, thiAl, hbd, crt2 from Peptoclostridium difficile 630; or ter from Treponema denticoia, in combination with thiAi, hbd, crt2, pbt, and buk from Peptoclostridium difficile 630 and tesB from E. coli. Any of the intermediate substrates of these reactions could be the second MOI, and the number of exogenous genes could be reduced if the bacterium or recipient bacterium is able to produce the intermediate substrate itself.

[0231] WO2017 / 074566A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the production of gut barrier enhancer molecules, such as propionate, acetate, GLP2, IL-22, IL-10, indole and tryptophan (each first MOIs).

[0232] WO2017 / 123418A1, WO2021 / 242897A1 and WO2023 / 225667A2 (all Synlogic Operating Company, Inc.), each of which is incorporated herein by reference in its entirety, disclose genes for the production of IAA (first MOI) from tryptophan (second MOI), and deletions in trpR and tnaA genes to increase the production of IAA, also see other examples disclosed herein for further genes and configurations for the production of IAA from various molecules.

[0233] W02016 / 090343A1 and W02020 / 232063A1 (both Synlogic Operating Company, Inc), each of which is incorporated herein by reference in its entirety, disclose methods of converting glutamate (second MOI) to arginine (first MOI) in an eight-step enzymatic process involving N-acetylglutamate synthetase (ArgA), N-acetylglutamate kinase (ArgB), N-acetylglutamate phosphate reductase (ArgC), acetylornithine aminotransferase (ArgD), N-acetylornithinase (ArgE), carbamoyl phosphate synthase (encoded by carA and carB), ornithine transcarbamylase (ArgF and ArgI), argininosuccinate synthase (ArgG), and argininosuccinate lyase (ArgH). Ornithine acetyltransferase (ArgJ) is bifunctional and can be used instead of both N-acetylglutamate synthetase (ArgA) and N-acetylornithinase (ArgE). All ofthe genes encoding these enzymes are subject to repression by arginine via its interaction with ArgR to form a complex that binds to the regulatory region of each gene and inhibits transcription. Thus, bacterium or recipient bacterium may comprise a modification, or the transmissible element may comprise a nucleic acid modifier, which reduces or prevents arginine-mediated repression, for example by preventing binding of arginine (first MOI) to ArgR or by preventing ArgR binding to the regulatory region of each gene (known as an " ARG box"), or by arginine binding to N -acetylglutamate synthetase.

[0234] WO2016 / 210384A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the production of various metabolic or satiety effector molecules, including bile salt hydrolase, n-acyl-phophatidylethanolamine (NAPE), n-acyl-ethanolamines (NAE), ghrelin receptor antagonist, peptide YY3-36, acholecystokinin (CCK), CCK58, CCK33, CCK22, CCK8, bombesin, gastrin releasing peptides (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptides, shortchain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotides (NAD), nicotinamide mononucleotides (NMN), nucleotide riboside (NR), nicotinamide, nicotinic acid (NA), but in particular examples tryptophan, kynurenine, kynurenic acid, indole and IAA, as well as transporters for importing these molecules into the cell.

[0235] In an example, the second MOI may be a detrimental metabolite.

[0236] W02016 / 201380A1, WO2021 / 188618A1 and WO2021 / 146394A1 (all Synlogic Operating Company, Inc.), each of which is incorporated herein by reference in its entirety, disclose genes for the reduction of branched chain amino acids, such as leucine, isoleucine, valine (second MOIs), as well as various mutations in exporters to reduce export of the second MOIs, and importers to increase import of the second MOIs into the cell for increased degradation.

[0237] Further, W02020 / 257610A1 and W02020 / 257707A1 (both Synlogic Operating Company, Inc. and Ginkgo Bioworks, Inc.) each of which is incorporated herein by reference in its entirety, disclose genes for the reduction of leucine (second MOI), converting it into isopentanol (first MOI) and including importers to increase import of the second MOI into the cell for increased degradation.

[0238] WO2022 / 169909A2 (Novome Biotechnologies, Inc.), incorporated herein by reference in its entirety, describes Bacteroides strains which have been engineered to convert CDCA (second MOI) to ursodeoxycholic acid, UDCA (first MOI). Chenodeoxycholic acid, CDCA is detrimental metabolite which is elevated in diseases such as IBS, whereas UDCA is generally considered tolerable for humans. Two enzymes 7a-HSDH from E. co / / Nissle 1917 and 70-HSDH from Colinsella aerofaciens ATCC 25986 are used to convert CDCA into UDCA.

[0239] Oxalate accumulation in urine causes hyperoxaluria. Elevated urinary oxalate has been linked to recurrent calcium oxalate kidney stones, kidney damage, and eventually end-stage renal disease. WO2020 / 123483A1 (Novome Biotechnologies, Inc.), incorporated herein by reference in its entirety, describes various oxalate-metabolising enzymes and proteins (e.g. oxalate decarboxylase (OXDC), EC 4.1.1.2, e.g. from O. formigenes, oxalate decarboxylase; oxalate oxidase (OXO), EC 1.2.3.4, e.g. fromHordeum vulgare, oxalate oxidoreductase (00R), EC 1.2.7.10, e.g. from Moorella thermoacetica, ATCC 39073; oxalate-CoA ligase / oxalyl-CoA synthetase (OXS), EC 6.2.1.8, e.g. from A. thaliana or S. cerevisiae, formyl-CoA:oxalate CoAtransferase (FCOCT), EC 2.8.3.16, e.g. from E. coli, acetyl-CoA:oxalate CoA-transferase (ACOCT), EC 2.8.3.19, e.g. from E. coli, succinyl-CoA:oxalate CoAtransferase (SCOCT), EC 2.8.3.2, e.g. from Cupriavidus oxalaticus, oxalyl-CoA decarboxylase (OXC), EC 4.1.1.8, e.g. from A. thaliana or S. cerevisiae, and oxalyl-CoA reductase / glyoxylate: NADP+ oxidoreductase (OXR), EC 1.2.1.17, e.g. from Methylobacterium extorquens) for the degradation of oxalate (second MOI) into various products such as formate (first MOI), oxalyl-CoA (first MOI), formyl-CoA (first MOI), or glyoxylate (first MOI).

[0240] Importers of oxalate (e.g. oxalate:formate antiporter (OxIT)) and exporters of formate may further be included in the transmissible elements.

[0241] W02017 / 040719A1, WO2021 / 146397A1 and W02022 / 204406A1 (all Synlogic Operating Company, Inc.), and each incorporated herein by reference in their entirety, disclose various genes, such as frc, ScAAE3, YfdE, Oxc the degradation of oxalate (second MOI), optionally in combination with OxIT importer of oxalate.

[0242] WO2021 / 173808A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes such as uricase, rasburicase, aegA or ygfT for the degradation of uric acid (second MOI), optionally in combination with an importer of uric acid, such as ygfY, uacT or ygfU.

[0243] WO2017 / 023818A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the degradation of propionate (second MOI) and genes which are importers of propionate.

[0244] Dietary methionine (second MOI) is converted to cysteine using several different enzymes, and mutations in one or more of these enzymes leads to homocystinuria. WO2021 / 163421A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the degradation of methionine using methionine gamma lyase (MGL) or methionine decarboxylase (MDC) from various sources, along with increasing expression of the methionine importer, metNIQ, and decreasing or preventing expression of the methionine exporter yjeH.

[0245] In the case of first MOIs which are peptides or proteins, the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element may further comprise a nucleotide sequence encoding a signal peptide as described herein.

[0246] For all first MOIs, the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element may further comprise a nucleotide sequence encoding one or more exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite). In any example herein, the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element may further comprise (d) a further nucleotide sequence encoding an exporter which is capable of exporting the first MOI (e.g. the therapeutic protein molecule or metabolite) out of the bacterium or recipient bacterium. In a particular example, the nucleotide sequence encodes an exporter of the first MOI.For all first MOIs, the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element may further comprise a nucleotide sequence encoding one or more importer(s) of the second MOI (e.g. a protein molecule or substrate for the production of the first MOI). In any example herein, the at least one nucleotide sequence of (b) further encodes one or more (e.g. one) importer(s) of the second MOI (e.g. a protein molecule or substrate for the production of the first MOI). In a particular example, the nucleotide sequence encodes an importer of the second MOI.

[0247] Where the first MOI is a metabolite, the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element may further comprise a nucleotide sequence encoding one or more importer(s) of any further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI (e.g. a substrate used in the pathway for the production of the first MOI). In any example herein, the at least one nucleotide sequence of (b) further encodes one or more (e.g. one) importer(s) of any further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI (e.g. a substrate used in the pathway for the production of the first MOI). In a particular example, the nucleotide sequence encodes an importer of the any further substrate.

[0248] In one particular example, the at least one nucleotide sequence of (b) encodes one or more protein(s) for the conversion of a second MOI to the first MOI, and an exporter which is capable of exporting the first MOI out of the bacterium or recipient bacterium. The nucleotide sequence of (b) may further encode an importer of a further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

[0249] In one example, the at least one nucleotide sequence of (b) encodes one or more protein(s) for the conversion of a second MOI to the first MOI, and an importer of the second MOI. The nucleotide sequence of (b) may further encode an importer of a further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

[0250] In one example, the at least one nucleotide sequence of (b) encodes one or more protein(s) for the conversion of a second MOI to the first MOI, and an exporter of the first MOI and an importer of the second MOI. The nucleotide sequence of (b) may further encode an importer of a further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

[0251] In one example, the at least one nucleotide sequence of (b) does not comprise any reporter genes. In one example, the at least one nucleotide sequence of (b) does not comprise any antibiotic resistance genes.

[0252] MOIs or nucleotide sequences encoding agents designed to target neighbouring cells in the microbiome

[0253] In some examples, the bacteria (e.g. engineered or recipient bacteria) or transmissible elements may comprise nucleotide sequences encoding products which are able to act on cells nearby in the microbiome, for example to kill, inhibit the growth of, or otherwise modify or alter suchneighbouring cells. For example, if an engineered bacterium or recipient bacterium could kill neighbouring cells (for example by introducing a cut in the chromosome of the cell which cannot be repaired), then an expanded niche could be provided, into which the engineered or recipient bacteria would be able to expand, in particular due to the selective advantage conferred by the one or more nucleotide sequences of (a) encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate. Alternatively, the engineered or recipient bacteria could modify antibiotic resistance genes comprised by neighbouring cells (e.g. comprised by a plasmid or episome) to sensitise the neighbouring cell to future antibiotic treatment, which again, could provide an expanded niche into which the engineered or recipient bacteria would be able to expand, upon treatment with antibiotics. Further alternatively, the engineered or recipient bacteria could modify genes comprised by neighbouring cells to either promote expression of metabolites which are beneficial to the engineered or recipient bacteria or to reduce expression of metabolites which are harmful to the engineered or recipient bacteria.

[0254] In other examples, the nucleic acid modifiers described herein may be comprised by the bacterium, recipient bacterium or transmissible element as a kill switch (as described elsewhere herein).

[0255] Base Editors

[0256] In an example, the first MOI comprises a nucleic acid modifier which is a base editor. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is a base editor.

[0257] Base editors that may be used as a nucleic acid modifier are well known in the art. The base editor may be a modified nuclease that is modified to be unable to perform DNA double strand breaks, while retaining its DNA binding capacity and that also is fused to a domain to perform base editing. Base editors are described in general, for example, in Rees eta / ., Nature Genetics Reviews, 19, 2018, 770-788, which is incorporated herein by reference in its entirety.

[0258] Thus, base editors include systems where CRISPR / Cas protein(s) (such as dead-Cas9 (dCas9) or nickase Cas9 (nCas9)) is / are fused to a cytosine or adenosine deaminase domain and directed to the target sequence to make the desired modification. Thus, in one example, the base editor encodes a dCas9 or nCas9 fused to a cytosine or adenosine deaminase domain and a guide RNA (or crRNA).

[0259] In one example, the base editor is selected from:

[0260] A. Cytosine Base Editors (CBEs) that convert C: G into T: A (Komor eta / ., Nature, 533:420-4, 2016, incorporated herein by reference in its entirety)

[0261] CBEs rely on ssDNA cytidine deaminase among which: APOBEC1, rAPOBECl, APOBEC1 mutant or evolved version (evoAPOBECl), and APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine deaminase 1 (CDA 1), evoCDA 1, FERNY, evoFERNY.

[0262] B. Adenine Base Editors (ABEs) that convert A: T into G: C (Gaudelli et al., Nature, 551 (7681), 464-471, 2017, incorporated herein by reference in its entirety)ABEs rely on deoxyadenosine deaminase activity of a tandem fusion TadA-TadA* where TadA* is an evolved version of TadA, an E. coli tRNA adenosine deaminase enzyme, able to convert adenosine into inosine on ssDNA. TadA* include TadA-8a-e and TadA-7.10.

[0263] C. Cytosine Guanine Base Editors (CGBEs) that convert C: G into G: C (Chen et a!., Biorxiv, 2020; Kurt eta / ., Nature Biotechnology, 2020, both incorporated herein by reference in its entirety) CGBEs generally consist of a nickase CRISPR / Cas protein fused to a cytosine deaminase (rAPOBEC) and base excision repair proteins, such as rXRCCl, or of a nickase CRISPR / Cas protein fused to a rat APOBEC1 variant (R33A) protein and an E. coli-derived uracil DNA N-glycosylase (eUNG).

[0264] D. Cytosine Adenine Base Editors (CABEs) that convert C: G into A: T (Zhao et a!., Nature Biotechnology, 2020, incorporated herein by reference in its entirety)

[0265] CABEs generally consist of a nickase CRISPR / Cas protein (e.g. a Cas9 nickase), a cytidine deaminase (e.g. AID), and a uracil-DNA glycosylase (Ung).

[0266] E. Adenine Cytosine Base Editors (ACBEs) that convert A: T into C: G (W02020 / 181180, incorporated herein by reference in its entirety)

[0267] ACBEs generally include a nucleic acid programmable DNA-binding protein and an adenine oxidase.

[0268] F. Adenine Thymine Base Editors (ATBEs) that convert A: T into T: A (W02020 / 181202, incorporated herein by reference in its entirety)

[0269] ATBEs generally consist of a CRISPR / Cas nickase protein (e.g. a Cas9 nickase) and one or more adenosine deaminase or an oxidase domain.

[0270] G. Thymine Adenine Base Editor (TABE) that convert T: A into A: T (W02020 / 181193; W02020 / 181178; W02020 / 181195, each of which is incorporated herein by reference in its entirety).

[0271] TABEs generally consist of a CRISPR / Cas nickase protein (e.g. a Cas9 nickase) and an adenosine methyltransferase, a thymine alkyltransferase, or an adenosine deaminase domain.

[0272] Further additional modules can be added to base editors to increase precision, modularity and editing efficiency. These include the addition of one or two uracil DNA glycosylase inhibitor domain(s) (UGI) to prevent base excision repair mechanism to revert base edition. Another module that can be added is Mu-GAM that decreases the insertion-deletion rate by inhibiting DNA repair by the non-homologous end joining mechanism (NHEJ) in the cell. For CRISPR / Cas based base editing systems, the CRISPR / Cas system may rely on nickase activity (for example by mutating Cas9 to form the well-known nCas9 D10A). The use of a nickase cuts the non-edited strand, and favours its repair and thus the fixing of the edited base.

[0273] In an example, the base editor is selected from the group consisting of: POBEC1, rAPOBECl, APOBEC1 mutant or evolved version (evoAPOBECl), APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine deaminase 1 (CDA1), evoCDA 1, FERNY, evoFERNY, BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3,SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Casl2a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRYCBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4, enAsBEl.l, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBEl.l, AsBE1.4, CRISPR-Abest, CRISPR-Cbest, eA3A-BE3 and AncBE4. Examples of DNA-based editor proteins include, but are not limited to BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Casl2a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRYCBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4, enAsBEl.l, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBEl.l, AsBEl.4, CRISPR-Abest, CRISPR-Cbest, eA3A-BE3 and AncBE4.

[0274] A particular example of a base editor is shown in WO2024 / 047151A1 (SNIPR Biome, Aps), see for example claims 26 to 30, Example 5, and associated disclosure (incorporated herein by reference in its entirety).

[0275] Prime Editors

[0276] In an example, the first MOI comprises a nucleic acid modifier which is a prime editor. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is a prime editor.

[0277] Prime editors that may be used as a nucleic acid modifier are well known in the art. Prime editors include systems where Cas9 (dead-Cas9) or nCas9 (nickase Cas9) is fused to a reverse transcriptase domain and directed to the target sequence to make the desired modification with the help of a pegRNA (prime editing guide RNA, which is a guide RNA that includes a template region for reverse transcription). Thus, in one example, the prime editor may encode a Cas9 (dead-Cas9) or nCas9 (nickase Cas9) fused to a reverse transcriptase domain and a pegRNA (prime editing guide RNA) which directs the prime editor to the target endogenous nucleic acid to modify said target nucleic acid.

[0278] Prime Editing allows introduction of insertions, deletions (indels), and 12 base-to-base conversions. Prime editing relies on the ability of a reverse transcriptase, fused to a Cas nickase variant, to convert RNA sequence brought by a pegRNA into DNA at the nick site generated by the Cas protein. The DNA flap generated from this process is then included or not in the targeted DNA sequence.

[0279] Prime editors, as described in Anzalone et a!., Nature, 576, 149-157, 2019, which is hereby incorporated by reference in its entirety, generally consist of a nickase CRISPR / Cas protein (e.g. nCas9) fused to a reverse transcriptase used in combination with a pegRNA. These systems include systems such as a Cas9-H840A (as Cas9 nickase variant) fused to a reverse transcriptase domain such as M-MLV RT or its mutant version (for example selected from M-MLV RT(D200N), M-MLVRT(D200N / L603W) and M-MLV RT(D200N / L603W / T330P / T306K / W313F)) in combination with a pegRNA.

[0280] Examples of prime editing systems include, but are not limited to PE1, PE1-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3 and PE3b.

[0281] RNA interference systems

[0282] In an example, the first MOI comprises a nucleic acid modifier which is an RNAi system. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is an RNAi system.

[0283] For bacterial uses, the RNAi system comprises small regulatory RNAs (sRNAs). These may be naturally occurring, or synthetic. In a particular example, the sRNA is an artificial sRNA. These sRNAs are sometimes referred to as non-coding RNAs. sRNAs function by base pairing between an sRNA and a messenger RNA (mRNA), which can regulate gene expression by changing the accessibility of the ribosome-binding site or altering the RNA-turnover rate. Such systems are described for example, in Nora et al., RNA Biol. 2017; 14(2), 206-218, 2016, doi:10.1080 / 15476286.2016.1270001, which is incorporated herein by reference in its entirety.

[0284] RNAi systems are also commercially available (e.g. from ThermoFischer etc).

[0285] CRISPR interference systems (CRISPRi)

[0286] In an example, the first MOI comprises a nucleic acid modifier which is a CRISPRi system. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is a CRISPRi system.

[0287] CRISPRi systems that may be used as a nucleic acid modifier are well-known in the art, and are commercially available (e.g. from VectorBuilder, Horizon Discovery, etc). CRISPRi systems include systems where dCas9 (dead-Cas9) is fused to one or more transcriptional repressor(s). When coupled with a gRNA (or crRNA) which is complementary to the target sequence, dCas9 targets a specific DNA location and the repressor reduces or prevents expression of the target gene. Usually, the gRNA (or crRNA) targets a region downstream of (e.g. 0-300 base pairs) the target gene's transcriptional start site (TSS) to result in interference. Thus, in one example, the nucleic acid modifier is a CRISPRi system which encodes a dCas9 (dead-Cas9) fused to one or more transcriptional repressor(s) and a gRNA (or crRNA) which directs the CRISPRi system to the target endogenous nucleic acid to modify a target nucleic acid.

[0288] Examples of fusions include dCas9-KRAB (see, for example, Gilbert, eta / ., Cell, 154(2), 442-51, 2013; Moghadam, et al., Nat. Cell Biol., 22(9), 1143-1154, 2020; and Alerasool, et al., Nat. Methods, 17(11), 1093-1096, 2020; each of which is incorporated herein by reference in its entirety), dCas9-KRAB-MeCP2, dCas9-SALL1-SDS3 (see for example Alland, eta / ., Mol. & Cell Bio., 22(8), 2743-2750, 2002, incorporated herein by reference in its entirety). Thus, the CRISPRi system encodes a system selected from: dCas9-KRAB, dCas9-KRAB-MeCP2 and dCas9-SALL1-SDS3.In a particular example, the CRISPRi system is a CasΦ system, as described in WO2024 / 047151A1 (SNIPR Biome, Aps), see for example claims 7 to 10 and 30, Example 4, and associated disclosure (incorporated herein by reference in its entirety).

[0289] Nuclease systems

[0290] In an example, the first MOI comprises a nucleic acid modifier which is a nuclease. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is a nuclease.

[0291] In an example, the first MOI comprises a nucleic acid modifier which is a nickase. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is a nickase.

[0292] In an example, the nucleic acid modifier is a nuclease in combination with a gRNA or crRNA. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a nucleic acid modifier which is a nuclease or nickase and a gRNA or crRNA.

[0293] Nuclease systems that may be used as a nucleic acid modifier are well known in the art. A particular nucleic acid modifier is a CRISPR / Cas nuclease or nickase. The CRISPR / Cas nucleic acid modifier may in particular be a CRISPR / Cas nuclease.

[0294] In some examples, nucleases which are mutated such that they are no longer able to cleave DNA can be used to silence genes. Suitable nuclease systems include, but are not limited to CRISPR / Cas systems, such as dCas9, type IV CRIPSR / Cas systems or type I CRISPR / Cas systems in which the nuclease (Cas3) has been removed, a TALEN or a zinc finger protein. Thus, in one example, the nucleic acid modifier is a nuclease which cannot introduce double stranded breaks in the target endogenous nucleic acid, and the gRNA (or crRNA) directs the nuclease to the target endogenous nucleic acid to modify said target nucleic acid.

[0295] In one example, the nuclease is selected from a CRISPR / Cas system, a meganuclease, a zinc finger, a TALEN or a restriction enzyme which has been modified to prevent cutting of DNA or RNA. In a particular example, the nucleic acid modifier is a CRISPR / Cas nuclease or nickase (e.g. nuclease) which does not cut (e.g. has been modified to not cut) DNA or RNA.

[0296] In some examples, nucleases which cleave DNA (or RNA) can be used to cut the genome of a cell (e.g. chromosome, plasmid or episome). CRISPR / Cas-based guided nucleases are particularly attractive for killing neighbouring cells, or removing (curing) plasmids from the genome of neighbouring cells, because they can precisely cut DNA. Their ease of design and specificity to their target sequences also make them attractive, particularly as they are able to distinguish between very closely related sequences, cutting the target and leaving intact those DNA / RNA without the correct sequence. In a particular example, the nucleic acid modifier is a CRISPR / Cas nuclease. In an example, the nuclease is selected from a Type I, Type II, Type III or Type V nuclease.In an example, the nucleic acid modifier is a CRISPR / Cas nickase, such as a Type I, Type II, Type III or Type V nickase.

[0297] Such CRISPR / Cas nucleases are used in the present applications in combination with a nucleotide sequence which expresses a gRNA or crRNA. gRNAs are generally a single RNA sequence, whereas a crRNA is processed after transcription of a pre-crRNA, which optionally complexes with the tracrRNA. Both gRNA and crRNA comprise one or more repeat sequences and a spacer sequence. The spacer sequence is complementary to a protospacer sequence in the target sequence, and provides the nuclease with the precise location to bind and cut (or nick) the target sequence. Sequences of specific CRISPR / Cas proteins from various source strains can be found in various databases, such as https: / / crisprcas.i2bc.paris-saclay.fr / .

[0298] In any of the descriptions of gRNA or crRNAs relating to CRISPR / Cas systems as peptide products, in an example, the nuclease (or nickase) forms a complex with the gRNA or crRNA which comprises a first spacer that is cognate to a first protospacer in the target sequence. In one example, the nuclease (or nickase) forms a ribonucleoprotein complex with a gRNA or crRNA which comprises a first spacer that is cognate to a first protospacer in the target sequence. In one example, the gRNA or crRNA comprises two repeat sequences (e.g. two repeat sequences).

[0299] The gRNA or crRNA may comprise a first spacer that is cognate to a first protospacer in the target sequence, wherein the protospacer is immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence.

[0300] In another example, the nuclease is selected from a meganuclease, a zinc finger, a TALEN or a restriction enzyme which cuts DNA or RNA. In a particular example, the nucleic acid modifier is a CRISPR / Cas nuclease or nickase (e.g. nuclease) which cuts DNA or RNA.

[0301] Target sequences

[0302] The neighbouring cells may comprise a target sequence which is targeted, and optionally modified, by the nucleic acid modifier. Alternatively, the recipient cell or bacterial cell (e.g. engineered bacterial cell) may comprise a target sequence which is targeted and modified by the nucleic acid modifier (in particular when the nucleic acid modifier is being used as a kill switch).

[0303] In an example, the target sequence is in an essential gene of the neighbouring target cell, of the bacterial cell (e.g. engineered bacterial cell) or of the recipient bacterial cell. Blocking or preventing transcription of an essential gene in a target cell (e.g. bacteria) can be used to kill the target cell whilst leaving other, non-target cells unaffected, in particular clearing a niche in a microbiome into which the engineered or recipient cell can grow with the assistance of xylitol as a food source. When being used as a kill switch, blocking or preventing transcription of an essential gene in the bacterial cell (e.g. engineered bacterial cell or recipient bacterial cell) can be used to kill the target bacterial cell (reducing or preventing production of the first MOI) whilst leaving other, non-target cells unaffected.

[0304] In one example, the nucleic acid modifier may cut the target sequence, and result in death of the target (neighbouring, engineered bacterial or recipient) cell. Thus, in one example, the targetsequence is comprised by the chromosome of the target (neighbouring, engineered bacterial or recipient) cell, and the nucleic acid modifier cuts the DNA and results in the death of the cell. The cutting may be double-stranded cutting of the chromosome.

[0305] In another example, the nucleic acid modifier may modify the target sequence which is comprised by a gene essential to growth or maintenance of the target (neighbouring) cell, and results in a reduction of growth of the target cell, allowing the engineered cell or recipient cell to more effectively compete with the neighbouring cells for nutrients and space in the microbiome. When being used as a kill switch, the nucleic acid modifier may modify the target sequence which is comprised by a gene essential to growth or maintenance of the bacterial cell (e.g. engineered bacterial cell or recipient bacterial cell), and results in a reduction of growth of the cell, reducing production of the first MOI.

[0306] In other examples, the target sequence is comprised by a plasmid within the target (neighbouring, engineered bacterial or recipient) cell.

[0307] This alternative example may be useful to cut a target sequence in a plasmid or vector comprised by the target (neighbouring) cell, and result in destruction of the plasmid or vector. This may re-sensitise the target (neighbouring) cell to antibiotics if the plasmid or vector comprises an antibiotic resistance gene. It may also be used to remove genes of concern that are comprised within the plasmid or vector (e.g. virulence genes, toxin-producing genes, conjugative genes or mobile genetic elements (MGEs), and the like).

[0308] Thus, in one example, the target sequence is comprised by a plasmid within the target cell (e.g. neighbouring), and the nucleic acid modifier modifies the target sequence by double-stranded cutting of the plasmid which prevents expression of an antibiotic resistance gene by destroying the plasmid. In this example, the method may further comprise administration of the antibiotic to which the resistance gene conferred protection, thereby resulting in death of the target (neighbouring) cell which comprised the antibiotic resistant plasmid.

[0309] When being used as a kill switch and the target sequence is comprised by a plasmid within the target cell (e.g. engineered bacterial or recipient), the nucleic acid modifier cuts a target sequence in a plasmid or vector (e.g. which produces the first MOI) comprised by the cell (e.g. engineered bacterial or recipient) and results in destruction of the plasmid or vector (reducing or preventing production of the first MOI).

[0310] Thus, in one example, the target sequence is comprised by a plasmid within the target cell (e.g. engineered bacterial or recipient), and the nucleic acid modifier modifies the target sequence by double-stranded cutting of the plasmid which prevents expression of the first MOI by destroying the plasmid.

[0311] In any example, the nucleic acid modifier modifies the target sequence by cutting the nucleic acid. In another example, the modification of the target sequence is double-stranded cutting of the target sequence.In some examples, the cutting is of single stranded DNA or RNA, wherein the nucleic acid modifier comprises a nickase or RNA nuclease. The nickase, RNA nuclease and single stranded DNA or RNA cutting may be as described elsewhere herein.

[0312] In other particular examples, the modification is cutting of a double-stranded DNA, and the peptide product comprises a nuclease, such as an I-TevI nuclease. The nuclease and double-stranded DNA cutting may as described elsewhere herein.

[0313] In some example, the modification results in death of the cell.

[0314] Toxic peptide products

[0315] In an example herein, the first MOI is a peptide toxin. In another example, the bacterium, recipient bacterium or transmissible element comprises, or further comprises, (f) a further nucleotide sequence encoding a peptide toxin.

[0316] The peptide toxin may be a bacteriocin. The peptide toxin may be a lysin. The peptide toxin may be a toxic peptide of a toxin-antitoxin system. The toxin is toxic to at least the target (neighbouring) cell. The peptide toxin may be a molecule that causes cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms.

[0317] Examples of toxin-antitoxins include but are not limited to peml-pemK genes of plasmid R100, the phd-doc genes of phage Pl, the ccdA-ccdB genes of plasmid F, mazE-mazF (also known as chpAI-chpAK) of E. co / ZK-12, sof-gefc E. coli K-12, kicA-kicBc E. co / / K-12, re / B-relEc E. co / i\\-12, and chpBI-chpBK of E. coli.-12 (toxin in bold). See Westwater eta / ., Antimicrobial agents and chemotherapy, 47, 1301-1307, 2003; doi:10.1128 / AAC.47.4.1301-1307.2003.

[0318] Other examples of toxins and lysis proteins are described in in Knudsen, eta / ., Appl. Environ. Microbiol., 57, 85-92, 1991; and Callura, et al., Proc. Natl Acad. Sci. USA, 107, 15898-15903, 2010, each of which is incorporated herein by reference in its entirety. Molecules which are toxic because they are capable of the degradation of essential proteins are discussed in Chan, et a / ., Nat. Chem. Biol., 12, 82-86, 2016 which is incorporated herein by reference in its entirety.

[0319] The peptide toxin may be an antimicrobial peptide, which include natural antimicrobial peptides, for example from frog's skin, human sweat or ant's venom. Classes of antimicrobial peptides include: anionic, linear cationic a-helical, cationic peptides enriched for specific amino acids, anionic and cationic peptides that contain cysteine and form disulphide bonds, and anionic and cationic peptide fragments of larger proteins. For a discussion of these classes, see the review paper Brogden, Nat Rev Microbiol, 3, 238-250, 2005, doi:10.1038 / nrmicrol098, which is incorporated herein by reference in its entirety. In particular, see Table 1 of Brogen for specific examples of antimicrobial peptides, which peptides are intended to form part of the subject-matter of this disclosure.

[0320] In one example, the antimicrobial peptide is selected from a-defensin, p-defensin, plectasin, Protegrin-1, LL-37, nisin, mersacidin, polymyxin B. Other peptide antimicrobials are described in Figure 2 of Hancock & Sahl, Nat. Biotechnol., 24(12), 1551, 2006, doi:10.1038 / nbtl267, which is incorporated herein by reference in its entirety. In particular, see Table 1 of Hancock & Sahl for specificexamples of antimicrobial peptides, which peptides are intended to form part of the subject-matter of this disclosure.

[0321] W02010 / 141135A2 (Trustees of Boston University & Massachusetts Institute of Technology), which is incorporated herein by reference in its entirety, describes a number of toxic peptide molecules. Thus, in an example, the peptide product is selected from Indolicidin (SEQ ID No:6 of W02010 / 141135A2), Cecropin PI (SEQ ID No: 11 of W02010 / 141135A2), Dermaseptin (SEQ ID No: 14 of W02010 / 141135A2), Ponericin WI (SEQ ID No:44 of W02010 / 141135A2), Ponericin W3 (SEQ ID No:40 of W02010 / 141135A2), Ponericin W4 (SEQ ID No: 18 of W02010 / 141135A2), Ponericin W5 (SEQ ID No:42 of W02010 / 141135A2) and Ponericin W6 (SEQ ID No:22 of W02010 / 141135A2) or variants thereof. In another example, the peptide product comprises any of the amino acid sequences of SEQ ID No: 10 or SEQ ID Nos:36-45 of W02010 / 141135A2. Each of the sequences of W02010 / 141135A2 which are referred to herein are incorporated by reference and intended to form part of the subject-matter of this disclosure.

[0322] Other examples of antimicrobial peptides include, but are not limited to, mefloquine, venturicidin A, antimycin, myxothiazol, stigmatellin, diuron, iodoacetamide, potassium tellurite hydrate, aDL-vinylglycine, N-ethylmaleimide, L-a I lyg lycine, diaryquinoline, betaine aldehyde chloride, acivcin, psicofuraine, buthionine sulfoximine, diaminopemelic acid, 4-phospho-D-erythronhydroxamic acid, motexafin gadolinium and / or xycitrin or modified versions or analogues thereof.

[0323] W02010 / 141135A2 also described a lytic enzyme, LysK. Thus, in one example, the peptide product is LysK or a functional fragment thereof, e.g. the functional fragment of LysK is CHAP165 (SEQ ID NO: 71 of W02010 / 141135A2 which sequence is incorporated by reference and intended to form part of the subject-matter of this disclosure).

[0324] A particular peptide toxin is a RaIR peptide toxin.

[0325] Promoters

[0326] The nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates; nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes) which are comprised by the engineered bacteria, transmissible elements and / or recipient bacteria (each as described elsewhere herein) may use promoter(s) which is / are endogenous to the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element. Alternatively, the nucleotide sequences described herein which are comprised by the engineered bacteria, transmissible elements and / or recipient bacteria (described elsewhere herein) may use promoter(s) which is / are heterologous to the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element. Such promoters may be inducible or constitutive.Where the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate of (a), or the at least one nucleotide sequence for the production of a first molecule of interest (MOI) of (b) comprises more than one expressed product (for example more than one of a protein, peptide product, enzyme, importer and / or exporter), each element of the nucleotide sequence encoding said expressed products may be under the control of a different promoter. Thus, when the one or more nucleotide sequences encodes multiple peptide products for the conversion of the precursor substrate (e.g. xylitol) to xylulose-5-phosphate of (a), each encoded peptide product may be under the control of a different promoter. Additionally or alternatively, when the at least one nucleotide sequence for the production of a first molecule of interest (MOI) of (b), (d), (e) and / or (f) encodes multiple peptide products, each encoded peptide product may be under the control of a different promoter.

[0327] In other examples, all the expressed peptide products from the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate of (a) are comprised by a single operon under the control of a single promoter. Additionally or alternatively, all the expressed peptide products from the at least one nucleotide sequence for the production of a first molecule of interest (MOI) of (b), (d), (e) and / or (f) are comprised by a single operon under the control of a single promoter.

[0328] In other particular examples, all the expressed peptide products from the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate of (a) and the further nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium or recipient bacterium of (c) are comprised by two operons each under the control of a single promoter. Additionally or alternatively, all the expressed peptide products from the at least one nucleotide sequence for the production of a first molecule of interest (MOI) of (b), (d), (e) and / or (f) are comprised by a single operon under the control of a single promoter. The promoters may be any of the promoters described elsewhere herein.

[0329] In other examples, all the expressed peptide products from the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate of (a) and the further nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium or recipient bacterium of (c) are comprised by a single operon under the control of a single promoter. Additionally or alternatively, all the expressed peptide products from the at least one nucleotide sequence for the production of a first molecule of interest (MOI) of (b), (d), (e) and / or (f) are comprised by a single operon under the control of a single promoter. The promoters may be any of the promoters described elsewhere herein.

[0330] In an example, the at least one nucleotide sequence of (b) encoding one or more protein(s) may be comprised by one or more operons (e.g. by one operon). The promoters may be any of the promoters described elsewhere herein. In particular, the one or more operons are each under the control of a constitutive promoter. In a most particular example, the at least one nucleotide sequenceof (b) encoding one or more protein(s) may be comprised by one operon under the control of a single constitutive promoter.

[0331] In an example,

[0332] i) the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) are each or all (e.g. all) under the control of a promoter (e.g. an inducible promoter, for example a native inducible promoter); and / or

[0333] ii) the at least one nucleotide sequence for the production of the first MOI of (b) are each or all (e.g. all) under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); and

[0334] iii) optionally, the further nucleotide sequence encoding the transporter of (c) is under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); and

[0335] iv) further optionally the further nucleotide sequence encoding the exporter of (d) is under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); and

[0336] v) further optionally the further nucleotide sequence encoding the signal peptide of (e) is under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter), and

[0337] vi) further optionally, the further nucleotide sequence encoding a nucleic acid modifier of (f) is under the control of a promoter (e.g. an inducible promoter).

[0338] In an example, the promoter of any of parts i) to vi) is an inducible promoter. In another example, parts i) and iii) are controlled by one or two (e.g. one, and in particular 2 [as in the promoter of SEQ ID No: 13]) inducible promoter(s). In another example, the nucleotide sequences of i) and iii) are comprised by a single operon under the control of an inducible promoter (e.g. the dual promoter of SEQ ID No: 13). In another example, the nucleotide sequences of i) and iii) are comprised by two operons, each under the control of an inducible promoter (e.g. as in Figure IB). In another example, all of the nucleotide sequences of part i) to vi) are controlled by one or more inducible promoters. For example, all of the nucleotide sequences of part i) to vi) are controlled by a single inducible promoter. This configuration may be useful to drive both the production of the MOI and the machinery for promoting colonisation and providing a selective advantage through the consumption of xylitol at the same time, particularly when using the inducible promoter which comprises a nucleotide sequence of SEQ ID No: 13, wherein the promoter controls expression of repressor encoded by a nucleotide sequence of SEQ ID No: 14, or controls expression of a nucleotide sequence encoding a repressor having an amino acid sequence of SEQ ID No: 15, and wherein the nucleotide sequence encoding the repressor is downstream (3') of the promoter sequence of SEQ ID No: 13.In another example, the promoter of any of parts i) to vi) is a constitutive promoter. In another example, parts ii), iv), v) and vi) are controlled by one or two (e.g. one) constitutive promoter(s). In another example, the nucleotide sequences of parts ii), iv), v) and vi) are comprised by a single operon under the control of a constitutive promoter. In another example, all of the nucleotide sequences of part i) to vi) are controlled by one or more constitutive promoters. For example, all of the nucleotide sequences of part i) to vi) are controlled by a single constitutive promoter.

[0339] In a particular example, the nucleotide sequences of parts ii), iv), v) and vi) are comprised by a single operon under the control of a constitutive promoter and the nucleotide sequences of i) and iii) are comprised by a single operon under the control of an inducible promoter. In another example, the nucleotide sequences of parts ii), iv), v) and vi) are comprised by a single operon under the control of a constitutive promoter and the nucleotide sequences of i) and iii) are comprised by two operons, each under the control of an inducible promoter.

[0340] In one example, the promoter is a promoter which is based on the sequence of a tac promoter. Tac promoters are based on a combination of promoters from the trp and lac operons, see de Boer, eta / ., PNAS, 80(1), 21-25, 1983. doi:10.1073 / pnas.80.1.21, which is incorporated herein by reference in its entirety. Several tac-based promoters have been reported in the art, see e.g. Zhang et al., Microb. Cell Fact, 16:84, 2017, doi: 10.1186 / S12934-017-0700-2, which is incorporated herein by reference in its entirety. In one example, the promoter is a Pc-tga promoter. In one example, the promoter has a nucleotide sequence of Seq ID No: 16.

[0341] Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli cs promoter (e.g. an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli o2promoter (e.g. htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli o70promoter (e.g. lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001), M13K07 gene I promoter (BBa_M13101), M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis oApromoter (e.g. promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PiepA(BBa_K823002), Pveg(BBa_K823003)), a constitutive Bacillus subtilis oBpromoter (e.g. promoter etc (BBa_K143010) or promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g. Pspv2 from Salmonella (BBa_K112706), Pspv from Salmonella (BBa_K112707)), a bacteriophage T7 promoter (e.g. T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g. SP6 promoter (BBa_J64998)).In another example, the promoter is a constitutive Pc promoter having the nucleic acid sequence of SEQ ID No: 17.

[0342] One way for measuring the strength of activity is by measuring the Anderson score of any given promoter. The activity of the reporters is measured by the relative fluorescence of the promoter when used in the control plasmid EX-Ptet-S-rbsRFP-P " RFP reporter" (see http: / / parts.igem. Org / Part: BBa_J61002) in strain TGI grown in LB media to saturation. BBa_J23119 is the "consensus" promoter sequence and the strongest member of the family. The Nhel and Avril restriction sites present within these promoter parts make them a scaffold for further modification. For more information, see http: / / parts.igem. Org / Part: BBa_J23114.

[0343] Thus, in one example, the constitutive promoter is a strong constitutive promoter (for example a promoter having an Anderson Score (AS) of AS >0.4, such as >0.5). In another example, the promoter has an Anderson score of between 0.1 and 0.4 or between 0.1 and 0.5.

[0344] Table 3: Anderson Promoter Collection

[0345]

[0346] a: also shown in the Anderson Catalog, see http: / / parts.igem.org / Promoters / Catalog / Anderson

[0347] b: Strength is the Anderson Score (AS), e.g. a strength of 1 is a AS of 1. Reported activities of the promoters are given as the relative fluorescence of plasmids in strain TGI grown in LB media to saturation. A suitable plasmid is EX-Ptet-S-rbsRFP-P " RFP reporter" as described at http: / / parts.igem. Org / Part: BBa_J61002; insertion of a promoter element between Xbal and Spel sites results in a RFP reporter.

[0348] In an example, a promoter herein is an inducible promoter.

[0349] In some examples, it may be desirable to include a promoter which is inducible to produce the first MOI (and components of that system) and / or the xylulose or xylulose-5-phosphate under only certain conditions. For example, the production of the first MOI could be under the control of a promoter which is active only under certain environmental conditions. Alternatively or additionally, the expression of the peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate and / or importers of xylitol could be under the control of a promoter which is active only under certain environmental conditions. For example, the inducible promoter is active under environmental conditions which are specific to the gut of a subject. In one example, the inducible promoter is active under environmental conditions which are specific to the upper gastrointestinal tract of a subject (e.g. bile acids). In one example, the inducible promoter is active under environmental conditions which are specific to the lower gastrointestinal tract of a subject (e.g. anaerobic conditions). In one example, the inducible promoter is active under the low oxygen or anaerobic conditions which are specific to gut (e.g. the upper gastrointestinal tract and / or the lower gastrointestinal tract) of a subject.

[0350] The inducible promoter may be a native inducible promoter of the transporter of (c) or a native inducible promoter of any of the peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a).

[0351] In a particular example, the inducible promoter comprises a nucleotide sequence of SEQ ID No: 13, and the promoter controls expression of repressor encoded by a nucleotide sequence of SEQ ID No: 14, or controls expression of a nucleotide sequence encoding a repressor having an amino acid sequence of SEQ ID No: 15, and wherein the nucleotide sequence encoding the repressor is downstream (3') of the promoter sequence of SEQ ID No: 13. This native promoter and repressor sequences from Morganella morganii are inducible by the presence of xylitol and / or xylulose. It comprises a repressor sequence and a promoter. The presence of xylitol and / or xylulose allows expression of the components under control of this promoter. Without being bound by theory, it is thought that the repressor binds to the promotor sequence, preventing transcription of the components under its control. However, when a sufficient concentration of xylitol and / or xylulose is present, it is thought that the xylitol and / or xylulose (or an immediate derivative thereof) binds to the repressor, thereby preventing the repressor from binding to the promoter, turning on transcription ofthe components under its control. Small variations to the repressor sequences (SEQ ID Nos: 14 and 15) may be tolerated and still produce this inducible effect. Thus, the bacterium, recipient bacterium or transmissible elements may comprise a nucleotide sequence having at least 80% (in particular at least 90%) identity to Seq ID No: 14 encoding a repressor, or comprise a nucleotide sequence encoding a repressor having an amino acid sequence at least 80% (in particular at least 90%) identity to SEQ ID No: 15.

[0352] In another example, the inducible promoter is a temperature sensitive promoter, such as one which is active under physiological temperatures (e.g. approximately 35 to 39 °C, for example approximately 36 to 38 °C, such as approximately 37 °C). For a discussion on the use of this type of promoter in a kill switch, see https: / / wyss.harvard.edu / news / kill-switches-for-engineered-microbes-gone-rogue / and the "cryodeath" system which is described in more detail in Stirling eta!., Mol. Cell, 68, 686-697. e683, 2017, which is incorporated herein in its entirety.

[0353] In another example, the promoter is active only in the presence of certain molecules present in the local physiological environment (such as molecules present only in the gut). These inducible promoters may therefore turn on and off production and secretion of the first MOI and / or the peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate and / or importers of xylitol when in the desired location (such as the gut). Such inducible promoters are described herein.

[0354] Other promoters of interest are ones which have been designed to be active when the bacterial cell is in a certain state, for example when it is a "stress-phase active". Such stress-phase active promoters (SPAs) are described in GB2303409.3, which is incorporated herein in its entirety and for its disclosure relating to SPAs, in particular for any of promoters of SEQ ID Nos: 1 to 10 disclosed therein (Seq ID Nos:38 to 47 in Table 9 hereinbelow) or any promoters in claims 77 to 86 therein.

[0355] Thus, in one example the promoter is a promoter selected from a Re / B, BoiA, Hya, YiaG and a RpoH promoter. The promoter may be a promoter selected from a Re / B promoter sequence, o70; a BolA promoter sequence, oS, o70; a Hya promoter sequence, oS, o70; a YiaG promoter sequence, oS; a RpoH promoter sequence Pl, o70; a RpoH promoter sequence P2, aS; a RpoH promoter sequence P3, o24; a RpoH promoter sequence P4, o70; a RpoH promoter sequence P5, o70; and a RpoH promoter sequence P6, o54. The promoter may be a promoter having a nucleotide sequence selected from any one of Seq ID Nos:38 to 47, or a nucleotides sequence having 90% (or 95%) homology thereto.

[0356] Kill Switches

[0357] In one example, the bacterium (e.g. engineered bacterium), recipient bacterium or transmissible element further comprises a kill switch.

[0358] In some examples, the kill switch may comprise a nucleic acid modifier as described elsewhere herein. The nucleic acid modifier may silence an essential gene of the bacterium or recipientbacterium. Alternatively, the nucleic acid modifier may cut the chromosome of the bacterium or recipient bacterium. In this example, the cutting of the chromosome results in death or reduction in growth of the bacterium or recipient bacterium.

[0359] In some examples, it may be desirable to include a kill switch in the bacterium or transmissible element. A kill switch is a biocontainment system which is included in the bacterium or transmissible element and is designed to destroy the bacterium or transmissible element (e.g. the conjugative plasmid or plasmid) itself only, or the transmissible element and the bacterium comprising the transmissible element (e.g. the recipient bacterium) together, when no longer contained within its desired environment (e.g. within a microbiome, such as a gut microbiome, within a subject). Such means are well-known in the art, and are regulatable, for example by the addition of non-naturally occurring substances (e.g. synthetic amino acids), temperature and the like. Specific examples of promoters and kill switches (in particular, for removal of plasmids from bacteria) are provided in W02023 / 012109A2 (SNIPR Biome, ApS), which is incorporated herein by reference in its entirety.

[0360] Bacteria comprising kill switches have been engineered for in vitro research purposes, e.g. to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease may also be programmed to die at a specific time after the expression and delivery of the first MOI, or after the subject has experienced the therapeutic effect. For example, in some examples, the kill switch is activated to remove the transmissible element (e.g. the conjugative plasmid or plasmid) from the bacterium comprising the transmissible element (e.g. the recipient bacterium), or to kill the bacterium comprising the transmissible element (e.g. the recipient bacterium), after a period of time following oxygen leveldependent expression of the first MOI. In some examples, the kill switch is activated in a delayed fashion following oxygen level dependent expression of the first MOI. Alternatively, the bacterium comprising the transmissible element (e.g. the recipient bacterium) may be engineered to die after the bacterium has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the bacterium comprising the transmissible element (e.g. the recipient bacterium), spread of the bacterium comprising the transmissible element (e.g. the recipient bacterium) outside the area of interest (for example, outside the gut) within the subject, or spread of the bacterium comprising the transmissible element (e.g. the recipient bacterium) outside of the subject into the environment (for example, spread to the environment through the stool of the subject).

[0361] Kill-switches can be designed such that toxin(s) is / are produced in response to an environmental condition or external signal (e.g. the recipient bacterium is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased. Examples of such promoters are also described elsewhere herein. The toxin(s) is / are toxic to the bacterium which produces and secretes the first MOI.The switches that control production of the toxin(s) can be based on, for example, transcriptional activation, translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis, reactive oxygen species, temperature, bile acids, pH, lactate, caffeine or other biosensors. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl P-D- I-thiogaiactopyra noside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death.

[0362] Thus, in some examples, the bacterium or recipient bacterium is further programmed to die or specifically degrade the transmissible element (e.g. the conjugative plasmid or plasmid) after sensing an exogenous environmental signal, for example, in a low-oxygen environment.

[0363] Kill switches can be permissive, such that the bacterium or recipient bacterium will continue to thrive in its environment, unless a defined condition changes. Cell survival or proliferation may be impeded by the expression of toxins and lysis proteins (e.g. as discussed in Knudsen, et al., Appl. Environ. Microbiol., 57, 85-92, 1991; Callura, et al., Proc. Natl Acad. Sci. USA, 107, 15898-15903, 2010, each of which is incorporated herein by reference in its entirety), or the degradation of essential proteins (e.g. as discussed in Chan, et al., Nat. Chem. Biol., 12, 82-86, 2016 which is incorporated herein by reference in its entirety).

[0364] Thus, in one example, the kill switch comprises a toxin gene, expression of which is induced in response to an environmental condition(s) and / or signal(s).

[0365] Expression of the toxin gene may be provided by an inducible promoter. Such promoters are well-known to those skilled in the art, and may include oxygen level-dependent promoters (e.g. an FNR [fumarate and nitrate reductase regulator]-inducible promoter, the ANR [anaerobic arginine deiminiase and nitrate reductase regulator]-inducible promoter, and the DNR [dissimilatory nitrate respiration regulator]-inducible promoter).

[0366] The inducible promoter may be a promoter which is induced by inflammation or an inflammatory response (e.g. an RNS and / or ROS-inducible promoter).

[0367] The inducible promoter may be a temperature-sensitive promoters (e.g. as described elsewhere herein). Temperature sensitive promoters include TIpA, Tel, TetR (and A89D and I193N mutants thereof), Lad (and A241T and G265D mutants thereof), GrpE, HtpG, Lon, RpoH, Clp and DnaK (which are described in more detail in Piraner eta / ., Nature Chemical Biology, 13, 75-80, 2016, doi:10.1038 / nchembio.2233; which is incorporated herein in its entirety).

[0368] In one example, the kill switch comprises a toxin gene expression of which is induced by a change in temperature. In one example, the kill switch comprises a toxin gene expression of which is induced by an increase in oxygen levels. In one example, the kill switch comprises a toxin geneexpression of which is induced by the addition of a molecule which is not usually present in the local environment, such as the gut (e.g. arabinose, sugar alcohol (e.g. sorbitol), tetracycline, IPTG, rhamnose, and non-naturally occurring amino acids).

[0369] The inducible promoter may be a promoter induced by a substance that may or may not be naturally present in the local environment (i.e. is exogenously added to the local environment). Examples include, but are not limited to an arabinose-inducible promoter (e.g. a pBAD promoter), tetracycline-inducible promoters, IPTG-inducible promoters, rhamnose-inducible promoters, xylitolinducible promoters, sorbitol-inducible promoters, and nutritional-inducible promoters.

[0370] Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination.

[0371] Transmissible elements

[0372] In particular examples, the transmissible element is a conjugative plasmid. A conjugative plasmid is a plasmid which, when comprised within a bacterial cell ("donor" or "host" cell) is able to be transferred to another bacterium ("recipient" cell) through the mechanism of bacterial conjugation. Bacterial conjugation is the unidirectional and horizontal transmission of genetic information ("horizontal gene transfer") from one bacterium to another. Conjugative plasmids generally fall into two classes: mobilizable plasmids and self-transmissible plasmids.

[0373] In any example, the conjugative plasmid is capable of being transferred to a recipient bacterial cell. In any example, the conjugative plasmid is transferred to a bacterial cell (i.e. a recipient cell). The recipient cell may be any bacterial cell described elsewhere herein (e.g. a gram-negative bacterial cell).

[0374] There is provided a transmissible element as described herein which is a conjugative plasmid. The conjugative plasmids described herein and donor (host) cells comprising such conjugative plasmids are useful as medicaments.

[0375] There is provided a donor (host) bacterium comprising a conjugative plasmid as described herein. The donor (host) bacterium may be any bacterium described elsewhere herein (e.g. a gramnegative bacterium).

[0376] There is provided a recipient bacterium comprising a conjugative plasmid as described herein. The recipient bacterium may be any bacterium described elsewhere herein (e.g. a gram-negative bacterium).

[0377] There is also provided a donor (host) bacterium comprising a transmissible element as described herein. The donor (host) bacterium may be any bacterium described elsewhere herein (e.g. a gram-negative bacterium).There is also provided a recipient bacterium comprising a transmissible element as described herein. The recipient bacterium may be any bacterium described elsewhere herein (e.g. a gramnegative bacterium).

[0378] Mobilizable plasmids comprise at least an origin of transfer (oriT), a relaxase and other genetic information on the plasmid which is transferred to the recipient cell. They require helper functions provided by e.g. a second plasmid or the chromosome of the donor cell to effect the plasmid transfer. In one example, the conjugative plasmid is a mobilizable plasmid. In one example, the conjugative plasmid is a mobilizable plasmid comprising an origin of transfer (oriT) and a relaxase.

[0379] A self-transmissible plasmid, in addition to the genetic information on the plasmid which is transferred to the recipient cell, also contain all the molecular machinery needed for self-transfer (e.g. for pilus formation and initiation of gene transfer) on the same plasmid. In one example, the conjugative plasmid is a self-transmissible plasmid. In one example, the conjugative plasmid is a self-transmissible plasmid which comprises all of the molecular machinery necessary for self-transfer. In one example, the conjugative plasmid comprises an or / and encodes all proteins required to mobilise the plasmid for conjugative transfer between cells.

[0380] Engineered conjugative plasmids are described in more detail in WO2021 / 037732 (SNIPR Biome ApS), which is incorporated herein in its entirety. The features of such conjugative plasmids and bacterial cells comprising them as described in the claims as filed in WO2021 / 037732 are also incorporated herein by reference.

[0381] Thus, in one example, the conjugative plasmid is devoid of a hypC2 nucleotide sequence, or a homologue thereof, for example a hypC2 nucleotide sequence of Seq ID No:48. The conjugative plasmid may comprise an OriT of an IncX plasmid. The conjugative plasmid may be an IncX plasmid. The conjugative plasmid may be a β10 plasmid.

[0382] The conjugative plasmid (or any other plasmid described herein) may be a plasmid based on any plasmid found in a bacterium disclosed herein. For example, the plasmid may be an Enterobacteriaceae plasmid. In one example, the plasmid is an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter plasmid. In one example, the plasmid may be from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one example, the plasmid may be from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one example, the genera is a Bacteroides genus. In one example, the genera is a Bifidobacterium genus. In one example, the plasmid may be from a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus sa / ivarius, Limosilactobacillus reuteri and Lactococcus iactis. In one example, the plasmid may be from a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroidesuniformis, Bacteroides vu / gatus and Bacteroides thetaiotaomicron. In one example, the plasmid may be from a genus or species disclosed in Table 4.

[0383] In an example, the conjugative plasmid is capable of replicating in a bacterial cell from any bacterial genus or species described herein. For example, the conjugative plasmid is capable of replicating in an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter host (donor) cell. In one example, the conjugative plasmid is capable of replicating in a bacterial cell from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one example, the conjugative plasmid is capable of replicating in a bacterial cell from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one example, the genera is a Bacteroides genus. In one example, the genera is a Bifidobacterium genus. In one example, the conjugative plasmid is capable of replicating in a bacterial cell from a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus sa / ivarius, Limosilactobacillus reuteri and Lactococcus iactis. In one example, the conjugative plasmid is capable of replicating in a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron. In one example, the conjugative plasmid is capable of replicating in a bacterial cell from a genus or species disclosed in Table 4.

[0384] In one example the conjugative plasmid (or any other plasmid described herein) is capable of being hosted in an Enterobacteriaceae cell. In one example, the plasmid is capable of being hosted in an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter cell. In one example, the plasmid is capable of being hosted in a bacterial cell from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one example, the plasmid is capable of being hosted in a bacterial cell from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one example, the genera is a Bacteroides genus. In one example, the genera is a Bifidobacterium genus. In one example, the plasmid is capable of replicating in a bacterial cell from a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus sa / ivarius, Limosilactobacillus reuteri and Lactococcus iactis. In one example, the plasmid is capable of being hosted in a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron. In one example, the plasmid is capable of being hosted in a bacterial cell from a genus or species disclosed in Table 4.In one example, the conjugative plasmid is capable of being conjugatively transferred to an Enterobacteriaceae cell. In one example, the plasmid is capable of being conjugatively transferred to an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter cell. In one example, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one example, the conjugative plasmid is capable of being conjugatively transferred to in a bacterial cell from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one example, the genera is a Bacteroides genus. In one example, the genera is a Bifidobacterium genus. In one example, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus, Bacteroides theta iotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus sa / ivarius, Limosilactobacillus reuteri and Lactococcus iactis. In one example, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron. In one example, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a genus or species disclosed in Table 4.

[0385] The conjugative plasmid may be based on a bacterial conjugative plasmid selected from one of the following bacterial conjugative plasmid families: IncA, IncB / O (InclO), IncC, IncD, IncE, IncFl, IncF2, IncG, IncHIl, IncHI2, Incll, Incl2, IncJ, 10 IncK, IncL / M, IncN, IncP, IncQl, IncQ2, IncR, IncS, IncT, IncU, IncV, IncW, IncXl, IncX2, IncY, IncZ, ColEl, ColE2, ColE3, pl5A, pSClOl, IncP-2, IncP-5, IncP-7, IncP-8, IncP-9, Incl, Inc4, Inc7, Inc8, Inc9, Incll, Incl3, Incl4 and Incl8.

[0386] In one example, the conjugative plasmid is a conjugative plasmid isolated from E. coli. In another example, the conjugative plasmid is a 010 conjugative plasmid from E. coli.

[0387] In one example, the donor (host) cell comprising a conjugative plasmid as described herein is an E. co / / (such as a symbioflor E. coli, e.g. G6 / 7) host cell. In another example, the donor (host) cell comprising a conjugative plasmid as described herein is an E. hormaechei host cell.

[0388] In one example, the recipient cell is an E. co / / recipient strain comprising a conjugative plasmid as described herein. The plasmid is introduced via conjugation from a donor (host) cell described herein. The E. coli recipient strain may be comprised by a native microbiome, e.g. in the gut of a subject. In another example, the recipient cell is a kiebsieiia recipient strain comprising a conjugative plasmid as described herein. The plasmid is introduced via conjugation from a donor (host) cell described herein. The kiebsieiia recipient strain may be comprised by a native microbiome, e.g. in the gut (GI tract) of a subject.In any example described herein, the transmissible element may be capable of being introduced into the recipient bacterium without heat stock. In any example described herein, the transmissible element may be capable of being introduced into a recipient bacterium without electroporation. In any example described herein, the transmissible element may be capable of being introduced into a recipient bacterium without in vitro transformation techniques, including chemical induction (e.g. using calcium).

[0389] As an alternative, the transmissible element is delivered by a phage particle. In one example, the transmissible element is a phage or a phagemid (e.g. a phagemid which is packaged in a phage particle). Thus, there is provided a phage particle comprising a transmissible element as described elsewhere herein. In one example, the phage chromosome comprises a transmissible element as described elsewhere herein. In another example, the phage particle comprises a plasmid (e.g. a phagemid) which comprises a transmissible element as described elsewhere herein.

[0390] The various nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates; nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes) may be delivered by a phage particle.

[0391] There is provided a recipient bacterium which comprises a transmissible element which is a plasmid which has been introduced by a phage. There is provided a recipient bacterium which comprises a transmissible element which is a plasmid which has been introduced by a packaged phagemid (i.e. a phagemid packaged within a phage particle). There is also provided a host bacterium comprising a transmissible element which is a phagemid and is used for the production of a packaged phagemid (i.e. a phagemid packaged within a phage particle).

[0392] There is also provided a plasmid comprised within a phage particle for transmission to a recipient bacterium, wherein the plasmid comprises various nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates; nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes). There is provided a recipient bacterium which comprises a transmissible element which is a plasmid which has been introduced by a phage. There is also provided a host bacterium comprising a transmissible element which is a plasmid comprised within a phage particle which is used for the production of said phage. There is also provided a host bacterium comprising a transmissible element which is a phage or phagemid which is used for the production of said phage or of a packaged phagemid.In another example, the a various nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates; nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes) may be comprised in the chromosome of the phage particle. Thus, there is provided a phage for transmission to a recipient bacterium, wherein the phage chromosome comprises various nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates; nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes). There is provided a recipient bacterium which comprises a transmissible element (as described elsewhere herein) which is a prophage which has been introduced by a phage. There is also provided a host bacterium comprising a transmissible element (as described elsewhere herein) comprised by the chromosome of a phage which is used for the production of said phage.

[0393] Phages that may be used as transmissible elements herein may be as described below. The phage may be a tail phage.

[0394] The tail phage may be a Bacteroides X.a\\ phage. Examples of Bacteroides tail phages include Bacteroides phage crAss002, Bacteroides phage crAss001, Bacteroides phage DAC15 and Azobacteroides phage ProJPt-Bp1.

[0395] The tail phage may be a Bifidobacterium tail phage. Examples of Bifidobacterium \\ phages include Bifidobacterium phage BadAargau2 and Bifidobacterium phage BadAztecl.

[0396] The tail phage may be a Clostridium tail phage. Examples of Clostridium tail phages include Clostridium phage CpVl, Clostridium phage CPD2, Clostridium phage CPS1, Clostridium phage phi24R, Clostridium phage phiCP7R, Clostridium phage CPS2, Clostridium phage phiCPV4, Clostridium phage phiZP2, Clostridium phage susfortuna, Clostridium phage phiCD505, Clostridium phage CDKM9, Clostridium phage CDKM15, Clostridium phage phiMMP02, Clostridium phage phiCDlll, Clostridium phage phiCD146, Clostridium phage phiCD38-2, Clostridium phage phi CD119, Clostridium phage phiCDHM19, Clostridium phage phiCD506, Clostridium phage phiCD481-l, Clostridium phage phiCDHMll, Clostridium phage phiCDHM13, Clostridium phage phiCDHM14, Clostridium phage phiMMP04, Clostridium phage CDMH1, Clostridium phage phiMMPOl, and Clostridium phage phiMMP03.

[0397] The tail phage may be an Enterococcus tail phage. Examples of Enterococcus tail phages include Enterococcus phage ECP3, Enterococcus phage EF24C, Enterococcus phage phiEF24C-P2, Enterococcus phage EFLK1, Enterococcus phage EFDG1, Enterococcus phage EFP01, Enterococcus phage EfV12-phil, Enterococcus phage AE4_17, Enterococcus phage vB_EfaP_Ef6.2, Enterococcusphage vB_EfaP_Ef6.3, Enterococcus phage vB_EfaP_Ef7.2, Enterococcus phage vB_EfaP_Ef7.3, Enterococcus phage vB_EfaP_Ef7.4, Enterococcus phage vB_Efae230P-4, Enterococcus phage vB_EfaP_Efmusl, Enterococcus phage vB_EfaP_Efmus3, Enterococcus phage vB_EfaP_Efmus4, Enterococcus phage Idefix, Enterococcus phage vB_EfaP_IME195, Enterococcus phage vB_EfaP_IME199, Enterococcus phage vB_EfaP_Zip, Enterococcus phage 9183, Enterococcus phage nattely, Enterococus phage vipetofem, Enterococcus phage vB_EfaS_140, Enterococcus phage vB_EfaS_AL2, Enterococcus phage vB_EfaS_AL3, Enterococcus phage AUEF3, Enterococcus phage Ec-ZZ2, Enterococcus phage IME_EF3, Enterococcus phage IME-EF4, Enterococcus phage EfaCPTl, Enterococcus phage vB_EfaS_IME196, Enterococcus phage LY0322, Enterococcus phage PMBT2, Enterococcus phage SANTORI, Enterococcus phage phiSHEF2, Enterococcus phage phiSHEF4, Enterococcus phage phiSHEF5, Enterococcus phage phiFLIA, Enterococcus phage phiFL2A, Enterococcus phage phiFL3A, Enterococcus phage BC611, Enterococcus phage IME-EF1 and Enterococcus phage SAP6.

[0398] The tail phage may be a Klebsiella tail phage. Examples of Klebsiella tail phages include Klebsiella phage 0507-KN2-1, Klebsiella phage 13, Klebsiella phage 1513, Klebsiella phage 2044-307w, Klebsiella phage 3LV2017, Klebsiella phage 4LV2017, Klebsiella phage AltoGao, Klebsiella phage F19, Klebsiella phage GH-K3, Klebsiella phage GML-KpColl, Klebsiella phage Henul, Klebsiella phage JD001, Klebsiella phage JD18, Klebsiella phage JY917, Klebsiella phage Kll, Klebsiella phage K5, Klebsiella phage K5-2, Klebsiella phage K5-4, Klebsiella phage K64-1, Klebsiella phage KL, Klebsiella phage KLPN1, Klebsiella phage KN1-1, Klebsiella phage KN3-1, Klebsiella phage KN4-1, Klebsiella phage KNP2, Klebsiella phage KOX1, Klebsiella phage KP-Rio / 2015, Klebsiella phage KP15, Klebsiella phage KP179, Klebsiella phage KP1801, Klebsiella phage Kp2, Klebsiella phage KP27, Klebsiella phage KP32, Klebsiella phage KP32Jsolate 192, Klebsiella phage KP32Jsolate 194, Klebsiella phage KP32Jsolate 195, Klebsiella phage KP32Jsolate 196, Klebsiella phage KP34, Klebsiella phage KP36, Klebsiella phage KP8, Klebsiella phage KpCHEMY26, Klebsiella phage KpKT21phil, Klebsiella phage KPN N137, Klebsiella phage KPN N141, Klebsiella phage KpS8, Klebsiella phage kpssk3, Klebsiella phage KPV15, Klebsiella phage KpV41, Klebsiella phage KpV475, Klebsiella phage KpV71, Klebsiella phage KPV811, Klebsiella phage LASTA, Klebsiella phage Magnus, Klebsiella phage Marfa, Klebsiella phage Matisse, Klebsiella phage May, Klebsiella phage Menlow, Klebsiella phage MezzoGao, Klebsiella phage Mineola, Klebsiella phage myPSH1235, Klebsiella phage NJS1, Klebsiella phage NTUH-K2044-Kl-1, Klebsiella phage Pharr, Klebsiella phage phiBOlE, Klebsiella phage PhiKpNIH-2, Klebsiella phage phiKpS2, Klebsiella phage PKO111, Klebsiella phage PKP126, Klebsiella phage Pylas, Klebsiella phage Seifer, Klebsiella phage SH-Kp 152234, Klebsiella phage SH-Kp 152410, Klebsiella phage Shelby, Klebsiella phage Sin4, Klebsiella phage Skenny, Klebsiella phage Soft, Klebsiella phage SopranoGao, Klebsiella phage ST13-OXA48phil2.1, Klebsiella phage ST147-VIMlphi7.1, Klebsiella phage ST15-OXA48phil4.1, Klebsiella phage ST16-OXA48phi5.4, Klebsiella phage ST437-OXA245phi4.1, Klebsiella phage ST437-OXA245phi4.2, Klebsiella phage ST512-KPC3phil3.2, Klebsiella phage Sugarland, Klebsiella phage Sushi, Klebsiella phage Sweeny, Klebsiella phage TAH8, Klebsiella phage TSK1,Klebsiella phage UPM 2146, Klebsiella phage vB_KleM_RaK2, Klebsiella phage vB_Kpl, Klebsiella phage vB_Kpn_F48, Klebsiella phage vB_Kpn_IME260, Klebsiella phage vB_KpnM_BIS47, Klebsiella phage vB_KpnM_KB57, Klebsiella phage vB_KpnM_KpS110, Klebsiella phage vB_KpnM_KpV477, Klebsiella phage vB_KpnM_KpV52, Klebsiella phage vB_KpnM_KpV79, Klebsiella phage vB_KpnP_BIS33, Klebsiella phage vB_KpnP_IL33, Klebsiella phage vB_KpnP_IME205, Klebsiella phage vB_KpnP_KpV289, Klebsiella phage vB_KpnP_KpV48, Klebsiella phage vB_KpnP_KpV74, Klebsiella phage vB_KpnP_KpV763, Klebsiella phage vB_KpnP_KpV766, Klebsiella phage vB_KpnP_KpV767, Klebsiella phage vB_KpnP_P184, Klebsiella phage vB_KpnP_PRA33, Klebsiella phage vB_KpnP_SU503, Klebsiella phage vB_KpnP_SU552A, Klebsiella phage vB_KpnS_15-38_KLPPOU149, Klebsiella phage vB_KpnS_Alina, Klebsiella phage vB_KpnS_Call, Klebsiella phage vB_KpnS_Domnhall, Klebsiella phage vB_KpnS_FZ10, Klebsiella phage vB_KpnS_IME279, Klebsiella phage vB_KpnS_IMGroot, Klebsiella phage vB_KpnS_KpV522, Klebsiella phage vB_KpnS_SegesCirculi, Klebsiella phage vB_KpP_FBKp27, Klebsiella phage YMC16 / 01 / N133_KPN_BP, Klebsiella phage ZCKP1, Klebsiella phage_vB_KpnP_IME321 and Klebsiella pneumoniae strain E16KP0115 plasmid unnamed.

[0399] The tail phage may be a Escherichia tail phage. Examples of Escherichia tail phages include Escherichia coli 0157 typing phage 3, Escherichia coli strain 13P477T plasmid pl3P477T-2, Escherichia phage 121Q, Escherichia phage 13a, Escherichia phage 172-1, Escherichia phage 186, Escherichia phage 1H12, Escherichia phage 285P, Escherichia phage 2B8, Escherichia phage 2G7b, Escherichia phage 2H10, Escherichia phage 4A7, Escherichia phage 4MG, Escherichia phage 500465-1, Escherichia phage 500465-2, Escherichia phage 503458, Escherichia phage 520873, Escherichia phage 64795_ecl, Escherichia phage 933W, Escherichia phage aalborv, Escherichia phage AAPEc6, Escherichia phage aaroes, Escherichia phage ADB-2, Escherichia phage alia, Escherichia phage alpha3, Escherichia phage anhysbys, Escherichia phage AnYang, Escherichia phage APCEcOl, Escherichia phage APECcO2, Escherichia phage ARI, Escherichia phage ArgO145, Escherichia phage atuna, Escherichia phage Av-05, Escherichia phage BA14, Escherichia phage BF23, Escherichia phage Bp4, Escherichia phage Bp7, Escherichia phage Cl, Escherichia phage Cl 19, Escherichia phage C130_2, Escherichia phage C5, Escherichia phage CAjan, Escherichia phage CBA120, Escherichia phage CF2, Escherichia phage chee24, Escherichia phage CICC 80001, Escherichia phage damhaus, Escherichia phage DE3, Escherichia phage DT571 / 2, Escherichia phage DT57C, Escherichia phage DTL, Escherichia phage E21, Escherichia phage e4 / lc, Escherichia phage Ebrios, Escherichia phage EC1-UPM, Escherichia phage EC121, Escherichia phage EC6, Escherichia phage EC6098, Escherichia phage ECA2, Escherichia phage ECBP1, Escherichia phage ECBP2, Escherichia phage ECBP5, Escherichia phage ECD7, Escherichia phage ECML-117, Escherichia phage ECML-134, Escherichia phage ECML-4, Escherichia phage EcNPl, Escherichia phage Eco_BIFF, Escherichia phage ECO4, Escherichia phage EcoDSl, Escherichia phage ECPI, Escherichia phage EcSl, Escherichia phage EG1, Escherichia phage egaa, Escherichia phage EK99P-1, Escherichia phage EP75, Escherichia phage EPS7, Escherichia phage ESCO13, Escherichia phage ESCO5, Escherichia phage ESSI2_evO15, Escherichia phage ESSI2_ev040, Escherichia phage ESSI2_evl29, Escherichia phage ESSI2_ev239, Escherichia phageev017, Escherichia phage ev099, Escherichia phage ev207, Escherichia phage ev243, Escherichia phage F2, Escherichia phage FEC14, Escherichia phage FEC19, Escherichia phage FFH2, Escherichia phage flopper, Escherichia phage fpOl, Escherichia phage FV3, Escherichia phage G4, Escherichia phage GA2A, Escherichia phage Gostya9, Escherichia phage grams, Escherichia phage H8, Escherichia phage haarsle, Escherichia phage Henu7, Escherichia phage Henu8, Escherichia phage herni, Escherichia phage HK022, Escherichia phage HK106, Escherichia phage HK446, Escherichia phage HK542, Escherichia phage HK544, Escherichia phage HK578, Escherichia phage HK629, Escherichia phage HK630, Escherichia phage HK633, Escherichia phage HK75, Escherichia phage HK97, Escherichia phage HP3, Escherichia phage HX01, Escherichia phage HY01, Escherichia phage HY02, Escherichia phage HY03, Escherichia phage HZ2R8, Escherichia phage HZP2, Escherichia phage 122, Escherichia phage ID2 Moscow / ID / 2001, Escherichia phage ID21, Escherichia phage ID32, Escherichia phage ID52, Escherichia phage ID62, Escherichia phage Ifl, Escherichia phage IME08, Escherichia phage ime09, Escherichia phage IME11, Escherichia phage IMM-002, Escherichia phage J8-65, Escherichia phage JES2013, Escherichia phage JH2, Escherichia phage JK06, Escherichia phage JL1, Escherichia phage JLK-2012, Escherichia phage JMPW1, Escherichia phage JMPW2, Escherichia phage JS10, Escherichia phage JS98, Escherichia phage JSE, Escherichia phage Kl-5, Escherichia phage K1F, Escherichia phage K1G, Escherichia phage K1H, Escherichia phage Klindl, Escherichia phage Klind2, Escherichia phage K30, Escherichia phage KBNP1711, Escherichia phage KIT03, Escherichia phage Lambda, Escherichia phage LL11, Escherichia phage LL2, Escherichia phage LL5, Escherichia phage LM33_P1, Escherichia phage Lwl, Escherichia phage Lyzl2581Vzw, Escherichia phage M13, Escherichia phage Mangalitsa, Escherichia phage mEp234, Escherichia phage mEpXl, Escherichia phage mEpX2, Escherichia phage Min27, Escherichia phage Minorna, Escherichia phage MLF4, Escherichia phage MtlBl_P17, Escherichia phage Mu, Escherichia phage Murica, Escherichia phage mutPKlA2, Escherichia phage muut, Escherichia phage MX01, Escherichia phage N15, Escherichia phage N30, Escherichia phage N4, Escherichia phage NC-A, Escherichia phage NC28, Escherichia phage NC29, Escherichia phage NC35, Escherichia phage nepoznato, Escherichia phage nieznany, Escherichia phage NJ01, Escherichia phage OSYSP, Escherichia phage pOOOv, Escherichia phage Pl, Escherichia phage P2, Escherichia phage P2_2H1, Escherichia phage P2_2H4, Escherichia phage P2_4A7b, Escherichia phage P2_4B2, Escherichia phage P2_4C9, Escherichia phage P2_4E6b, Escherichia phage P483, Escherichia phage P694, Escherichia phage P88, Escherichia phage P88_4B11, Escherichia phage PA2, Escherichia phage PA28, Escherichia phage PBECO4, Escherichia phage PE3-1, Escherichia phage PE37, Escherichia phage PGN590, Escherichia phage PGT2, Escherichia phage phAPEC8, Escherichia phage PhaxI, Escherichia phage Phil, Escherichia phage phil91, Escherichia phage phi92, Escherichia phage phiAPCEcO3, Escherichia phage phiC120, Escherichia phage phiE142, Escherichia phage phiEB49, Escherichia phage phiEco32, Escherichia phage phiEcoM-GJl, Escherichia phage phiK, Escherichia phage phiKP26, Escherichia phage phiKT, Escherichia phage phiLLS, Escherichia phage phiSUSPl, Escherichia phage phiSUSP2, Escherichia phage phiVIO, Escherichia phage phiX174, Escherichia phage phT4A, Escherichia phage Pollock,Escherichia phage PP01, Escherichia phage prol47, Escherichia phage pro483, Escherichia phage PTXU04, Escherichia phage Qbeta, Escherichia phage QL01, Escherichia phage RB14, Escherichia phage RB16, Escherichia phage RB3, Escherichia phage RB32, Escherichia phage RB43, Escherichia phage RB49, Escherichia phage RB69, Escherichia phage RCS47, Escherichia phage Ro45lw, Escherichia phage Roguel, Escherichia phage Rtp, Escherichia phage sausl32, Escherichia phage SECphi27, Escherichia phage Seurat, Escherichia phage SF, Escherichia phage SH2026Stxl, Escherichia phage Skarpretter, Escherichia phage slur02, Escherichia phage slur03, Escherichia phage slur04, Escherichia phage slur07, Escherichia phage slur09, Escherichia phage slurl6, Escherichia phage Sortsne, Escherichia phage SP15, Escherichia phage SRT7, Escherichia phage SRT8, Escherichia phage SSL-2009a, Escherichia phage St-1, Escherichia phage ST31, Escherichia phage ST32, Escherichia phage Stx2 II, Escherichia phage Tl, Escherichia phage T2, Escherichia phage T4, Escherichia phage T5, Escherichia phage T7, Escherichia phage teqdroes, Escherichia phage teqhad, Escherichia phage teqskov, Escherichia phage tiwna, Escherichia phage TL-2011C, Escherichia phage TIs, Escherichia phage tonijn, Escherichia phage tonn, Escherichia phage tonnikala, Escherichia phage tuntematon, Escherichia phage tunus, Escherichia phage UFV-AREG1, Escherichia phage ukendt, Escherichia phage V18, Escherichia phage V5, Escherichia phage vB_Eco_mar001Jl, Escherichia phage vB_Eco_mar003J3, Escherichia phage vB_Eco_mar004NP2, Escherichia phage vB_Eco_SLUR29, Escherichia phage vB_Eco_swan01, Escherichia phage vB_EcoM_005, Escherichia phage vB_EcoM_112, Escherichia phage vB_EcoM_ACG-C40, Escherichia phage vB_EcoM_Alf5, Escherichia phage vB_EcoM_AYO145A, Escherichia phage vB_EcoM_DalCa, Escherichia phage vB_EcoM_EC01230-10, Escherichia phage vB_EcoM_ECOO78, Escherichia phage vB_EcoM_Fl, Escherichia phage vB_EcoM_G4498, Escherichia phage vB_EcoM_G4507, Escherichia phage vB_EcoM_G50, Escherichia phage vB_EcoM_G8, Escherichia phage vB_EcoM_G9062, Escherichia phage vB_EcoM_Goslar, Escherichia phage vB_EcoM_IME537, Escherichia phage vB_EcoM_JS09, Escherichia phage VB_ECOM_KAW1E185, Escherichia phage vB_EcoM_KWBSE43-6, Escherichia phage vB_EcoM_Lutter, Escherichia phage vB_EcoM_NBG2, Escherichia phage vB_EcoM_OE5505, Escherichia phage vB_EcoM_Ozark, Escherichia phage vB_EcoM_PhAPEC2, Escherichia phage vB_EcoM_PHB05, Escherichia phage vB_EcoM_Schickermooser, Escherichia phage vB_EcoM_VR20, Escherichia phage vB_EcoM_VR25, Escherichia phage vB_EcoM_VR26, Escherichia phage vB_EcoM_VR7, Escherichia phage vB_EcoM_WFC, Escherichia phage vB_EcoM_WFH, Escherichia phage vB_EcoM-12474III, Escherichia phage vB_EcoM-4HA13, Escherichia phage vB_EcoM-ep3, Escherichia phage vB_EcoM-fFiEco06, Escherichia phage vB_EcoM-G28, Escherichia phage vB_EcoM-Rol21c4YLVW, Escherichia phage vB_EcoM-UFV13, Escherichia phage vB_EcoM-VpaEl, Escherichia phage vb_EcoM-VR5, Escherichia phage vB_EcoP_24B, Escherichia phage vB_EcoP_ACG-C91, Escherichia phage vB_EcoP_B, Escherichia phage vB_EcoP_C, Escherichia phage vB_EcoP_F, Escherichia phage vB_EcoP_G7C, Escherichia phage vB_EcoP_K, Escherichia phage vB_EcoP_PhAPEC5, Escherichia phage vB_EcoP_PhAPEC7, Escherichia phage vB_EcoP_S523, Escherichia phage vB_EcoP_SU10, Escherichia phage vB_EcoS Sal79lw, Escherichia phagevB_EcoS_ACG-M12, Escherichia phage vB_EcoS_AHP42, Escherichia phage vB_EcoS_AHS24, Escherichia phage vB_EcoS_AKFV33, Escherichia phage vB_EcoS_AKS96, Escherichia phage vB_Ecos_CEB_EC3a, Escherichia phage vB_EcoS_ESCO41, Escherichia phage vB_EcoS_FFH_l, Escherichia phage vB_EcoS_G29-2, Escherichia phage vB_EcoS_HdH2, Escherichia phage vB_EcoS_IME347, Escherichia phage vB_EcoS_IME542, Escherichia phage vB_EcoS_NBD2, Escherichia phage vB_EcoS_PHB17, Escherichia phage vB_EcoS_SH2, Escherichia phage vB_EcoS_swi2, Escherichia phage vB_EcoS_W011D, Escherichia phage vB_EcoS-12210I, Escherichia phage vB_EcoS-95, Escherichia phage vB_EcoS-DELF2, Escherichia phage vB_EcoS-Golestan, Escherichia phage vB_EcoS-IME253, Escherichia phage vB_vPM_PD06, Escherichia phage vB_vPM_PD112, Escherichia phage VEc3, Escherichia phage VEc33, Escherichia phage VEcB, Escherichia phage vojen, Escherichia phage WA45, Escherichia phage WG01, Escherichia phage Wphi, Escherichia phage wV7, Escherichia phage wV8, Escherichia phage YD-2008. S, Escherichia phage YUEELO1, Escherichia phage YZ1, Escherichia phage ZG49, Escherichia phageJJdtsur, Escherichia phage_Vecl3, Escherichia typing phage 1 and Escherichia virus KFS-EC.

[0400] The tail phage may be a Pseudomonas tail phage. Examples of Pseudomonas tail phages include Pseudomonas aeruginosa PS75 adTyT-supercontl.7, Pseudomonas phage 119X, Pseudomonas phage 14-1, Pseudomonas phage 17A, Pseudomonas phage 22PfluR64PP, Pseudomonas phage 73, Pseudomonas phage Achelous, Pseudomonas phage Alpheus, Pseudomonas phage Andromeda, Pseudomonas phage antinowhere, Pseudomonas phage B3, Pseudomonas phage Bf7, Pseudomonas phage Bjorn, Pseudomonas phage BrSPl, Pseudomonas phage crassa, Pseudomonas phage D3, Pseudomonas phage D3112, Pseudomonas phage datas, Pseudomonas phage DL60, Pseudomonas phage DL62, Pseudomonas phage DL68, Pseudomonas phage DMS3, Pseudomonas phage Dobby, Pseudomonas phage EL, Pseudomonas phage Epal4, Pseudomonas phage Epa5, Pseudomonas phage EPa61, Pseudomonas phage Epa7, Pseudomonas phage F_HA0480sp / Pal651, Pseudomonas phage F116, Pseudomonas phage F8, Pseudomonas phage gh-1, Pseudomonas phage H66, Pseudomonas phage Henninger, Pseudomonas phage inbricus, Pseudomonas phage JBD67, Pseudomonas phage JD18, Pseudomonas phage JG004, Pseudomonas phage JG024, Pseudomonas phage KNP, Pseudomonas phage KPP10, Pseudomonas phage KPP12, Pseudomonas phage KPP21, Pseudomonas phage KPP25, Pseudomonas phage Lana, Pseudomonas phage LBL3, Pseudomonas phage LIT1, Pseudomonas phage Littlefix, Pseudomonas phage LKA1, Pseudomonas phage LKD16, Pseudomonas phage LKO4, Pseudomonas phage LMA2, Pseudomonas phage LPB1, Pseudomonas phage LUZ19, Pseudomonas phage LUZ24, Pseudomonas phage LUZ7, Pseudomonas phage M6, Pseudomonas phage MP1412, Pseudomonas phage MP22, Pseudomonas phage MP29, Pseudomonas phage MP38, Pseudomonas phage MPK6, Pseudomonas phage MPK7Pseudomonas phage Nerthus, Pseudomonas phage NH-4, Pseudomonas phage nickie, Pseudomonas phage Njord, Pseudomonas phage Noxifer, Pseudomonas phage NP1, Pseudomonas phage NV1, Pseudomonas phage PA01, Pseudomonas phage PA1 / KOR / 2010, Pseudomonas phage PA10, Pseudomonas phage PA11, Pseudomonas phage PA26, Pseudomonas phage PA5, Pseudomonasphage PA7, Pseudomonas phage PA8P1, Pseudomonas phage PaBG, Pseudomonas phage PAE1, Pseudomonas phage PaGUll, Pseudomonas phage PAK_P1, Pseudomonas phage PAK_P2, Pseudomonas phage PAK_P3, Pseudomonas phage PAK_P4, Pseudomonas phage PaMxll, Pseudomonas phage PaMx25, Pseudomonas phage PaMx28, Pseudomonas phage PaMx41, Pseudomonas phage PaMx74, Pseudomonas phage PaPl, Pseudomonas phage PaP3, Pseudomonas phage PaP4, Pseudomonas phage PAXYB1, Pseudomonas phage PB1, Pseudomonas phage Persinger, Pseudomonas phage Pf-10, Pseudomonas phage Pfl, Pseudomonas phage Pfl ERZ-2017, Pseudomonas phage pfl6, Pseudomonas phage Pf3, Pseudomonas phage Pf3, Pseudomonas phage pf8_ST274-AUS411, Pseudomonas phage PFP1, Pseudomonas phage phCDa, Pseudomonas phage phi-2, Pseudomonas phage Phi-Si, Pseudomonas phage phi 12, Pseudomonas phage phi 13, Pseudomonas phage phi 15, Pseudomonas phage phi2954, Pseudomonas phage phi3, Pseudomonas phage phi6, Pseudomonas phage phi8, Pseudomonas phage PhiCHU, Pseudomonas phage phiCTX, Pseudomonas phage phiIBB-PAA2, Pseudomonas phage phiIBB-PF7A, Pseudomonas phage phikF77, Pseudomonas phage phiKMV, Pseudomonas phage phiKTN6, Pseudomonas phage phiKZ, Pseudomonas phage phiMK, Pseudomonas phage phiNFS, Pseudomonas phage phiNN, Pseudomonas phage phiNV3, Pseudomonas phage phiPMW, Pseudomonas phage phiPsal7, Pseudomonas phage phiPSA2, Pseudomonas phage phiPsa374, Pseudomonas phage PhiR.18, Pseudomonas phage phiYY, Pseudomonas phage PMBT14, Pseudomonas phage PMBT3, Pseudomonas phage PMG1, Pseudomonas phage PollyC, Pseudomonas phage PP7, Pseudomonas phage PPPL-1, Pseudomonas phage PPpW-4, Pseudomonas phage PRR1, Pseudomonas phage PspYZUOl, Pseudomonas phage PspYZU05, Pseudomonas phage PspYZU08, Pseudomonas phage PT2, Pseudomonas phage PT5, Pseudomonas phage R12, Pseudomonas phage R26, Pseudomonas phage RLP, Pseudomonas phage shl2, Pseudomonas phage SL1, Pseudomonas phage SL2, Pseudomonas phage SMI, Pseudomonas phage SN, Pseudomonas phage tabernarius, Pseudomonas phage tf, Pseudomonas phage TL, Pseudomonas phage UFV-P2, Pseudomonas phage uligo, Pseudomonas phage UNO-SLW1, Pseudomonas phage vB_Pae_BR319a, Pseudomonas phage vB_Pae_PS44, Pseudomonas phage vB_Pae-Kakheti25, Pseudomonas phage vB_Pae-SS2019XI, Pseudomonas phage vB_PaeM_Cl-14_Ab28, Pseudomonas phage vB_PaeM_C2-10_Ab02, Pseudomonas phage vB_PaeM_C2-10_Abl, Pseudomonas phage vB_PaeM_CEB_DPl, Pseudomonas phage vB_PaeM_E215, Pseudomonas phage vB_PaeM_E217, Pseudomonas phage vB_PaeM_Gl, Pseudomonas phage vB_PaeM_LSl, Pseudomonas phage vB_PaeM_MAGl, Pseudomonas phage vB_PaeM_PAOl_Ab03, Pseudomonas phage vB_PaeM_PS24, Pseudomonas phage vB_PaeM_SCUT-Sl, Pseudomonas phage vB_PaeM_SCUT-Sl, Pseudomonas phage vB_PaeM_USP_l, Pseudomonas phage vB_PaeP_130_113, Pseudomonas phage vB_PaeP_C2-10_Ab09, Pseudomonas phage vB_PaeP_C2-10_Ab22, Pseudomonas phage vB_PaeP_PAOl_l-15pyo, Pseudomonas phage vB_PaeP_PAOl_Ab05, Pseudomonas phage vB_PaeP_PPA-ABTNL, Pseudomonas phage vB_PaeS_PAOl_Abl8, Pseudomonas phage vB_PaeS_PAOl_Abl9, Pseudomonas phage vB_PaeS_PM105, Pseudomonas phage vB_PaeS_SCH_Ab26, Pseudomonas phage vB_PsyM_KILl, Pseudomonas phagevB_PsyM_KIL4, Pseudomonas phage vB_PsyP_3MF5, Pseudomonas phage VCM, Pseudomonas phage VSW-3, Pseudomonas phage WRT, Pseudomonas phage YMC11 / O6 / C171_PPU_BP, Pseudomonas phage YuA, Pseudomonas phage ZC01, Pseudomonas phage ZC03, Pseudomonas phage ZC08, Pseudomonas phage Zigelbrucke, Pseudomonas phage Zuri, Pseudomonas sp. S4_EA_lb YA0848_38 and Pseudomonas virus Pal93.

[0401] The tail phage may be a Fusobacterium aA phage. The tail phage may be a Faecalibacterium tail phage. The tail phage may be a Lactobacillus tail phage. The tail phage may be a Streptococcus tail phage. The tail phage may be a Providencia tail phage. The tail phage may be a Salmonella tail phage. The tail phage may be a Helicobacter tail phage. The tail phage may be a Shigella tai I phage. The tail phage may be a Staphy / ocuccus \\ phage. The tail phage may be a Clostridioides aW phage. The tail phage may be an Acinetobacter W phage.

[0402] Any of the tail phages described herein may be used to deliver the transmissible element to the recipient bacterium. Where a tail phage is used to deliver the transmissible element to the recipient bacterium, the recipient bacterium is of a species that is capable of being infected by the phage.

[0403] The phage may be a filamentous phage.

[0404] The filamentous phage may be a Vibrio filamentous phage. The filamentous phage may be a Xanthomonas filamentous phage. The filamentous phage may be a Raistonia filamentous phage. The filamentous phage may be an Escherichia filamentous phage. The filamentous phage may be an Enterobacteria filamentous phage. The filamentous phage may be an Erwinia filamentous phage. The filamentous phage may be a Pseudomonas filamentous phage. The filamentous phage may be a Salmonella filamentous phage. The filamentous phage may be a Stenotrophomonas filamentous phage.

[0405] In any example described herein, the transmissible element may be capable of being introduced into the recipient bacterium without heat stock. In any example described herein, the transmissible element may be capable of being introduced into a recipient bacterium without electroporation. In any example described herein, the transmissible element may be capable of being introduced into a recipient bacterium without in vitro transformation techniques, including chemical induction (e.g. using calcium).

[0406] In some examples, the transmissible element (for example, a phagemid or conjugative plasmid) provides at least one plasmid to the recipient bacterium. In one example, the transmissible element or recipient bacterium comprises a first plasmid. In another example, the transmissible element or recipient bacterium comprises a first and a second plasmid. In some examples, the recipient bacterium comprises a first plasmid (or a second plasmid) and the transmissible element provides a second (or a first plasmid) to the recipient bacterium, such that the recipient bacterium comprises both a first and a second plasmid.

[0407] Thus, in one example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) arecomprised by a first plasmid. In another example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) and any further nucleotide sequence of (c) are comprised by a first plasmid. In another example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a), any further nucleotide sequence of (c) and the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f), and / or any kill switch) are comprised by a first plasmid. In another example, the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f), and / or any kill switch) are comprised by a first plasmid. The first plasmid may be comprised by a transmissible element. The first plasmid may be comprised by a recipient bacterium.

[0408] In an alternative example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) and any further nucleotide sequence of (c) are comprised by a first plasmid, and the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f), and / or any kill switch) are comprised by a second plasmid. The first and second plasmid may be comprised by a transmissible element. The first and second plasmid may be comprised by a recipient bacterium. The first plasmid may be comprised by a transmissible element, and the second plasmid may be comprised by a recipient bacterium which is already comprised by the microbiome (e.g. of the GI tract) of the subject, and the transmissible element transmits the first plasmid to the recipient bacterium when the transmissible element is present in the microbiome. Alternatively, the second plasmid may be comprised by a transmissible element, and the first plasmid may be comprised by a recipient bacterium which is already comprised by the microbiome (e.g. of the GI tract) of the subject, and the transmissible element transmits the second plasmid to the recipient bacterium when the transmissible element is present in the microbiome.

[0409] In other examples, the recipient bacterium comprises some of the nucleotide sequences described herein in its chromosome (see elsewhere herein), and the transmissible element provides a plasmid to the recipient bacterium.

[0410] Bacteria

[0411] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a gram-negative bacterium.

[0412] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a gram-positive bacterium.

[0413] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium is a strain selected from any of the strains in Table 4.In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be an E. colisXravcx. In one example, the E. colisXravcx is an E. coli x vn from phylogroup A. In one example, the E. coH strain is an E. coH strain from phylogroup Bl. In one example, the E. coH strain is an E. coH strain from phylogroup E. In one example, the E. coH strain is an E. coH strain which is present in a probiotic product. The probiotic product may be colinfant New Born (e.g. strain AO 34 / 86). The probiotic product may be symbioflor2 (e.g. strain Gl / 2, G4 / 9, G5, G6 / 7, and G8). The probiotic product may be Mutaflor (e.g. E. co / / Nissle).

[0414] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a strain belonging to a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibaciiius, Lactiplantibacillus, Leviiactobaciiius, Ligilactobacillus, Limosilactobacillusa Lactococcus.

[0415] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides enus. In one example, the genera is a Bacteroides genus. In one example, the genera is a Bifidobacterium genus.

[0416] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus, Bacteroides theta iotaomicron, Lactobacillus gasseri, Lacticaseibaciiius paracasei, Lactiplantibacillus plantarum, Leviiactobaciiius brevis, Ligilactobacillus saiivarius, Limosilactobacillus reuteri and Lactococcus iactis.

[0417] In any example herein, the e bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron.

[0418] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a strain belonging to the genus Spiruiina.

[0419] In any example herein, the bacterium (e.g. engineered bacterium), donor bacterium and / or recipient bacterium may be a strain belonging to the genus Cyanobacteria. o

[0420] In one example, the donor (host) bacterium comprising the transmissible elements described herein has been engineered to remove some or all (e.g. all) prophage genes present in the bacterial genome. The donor (host) bacterium may be devoid of some or all (e.g. all) prophage genes.

[0421] In one example, the donor (host) bacterium comprising the transmissible elements described herein has been engineered to remove any identified pathogenicity factors (such as hiyA, hiyB, hiyC and / or hiyDor any combination thereof) present in the bacterial genome. The donor (host) bacterium may be devoid of pathogenicity factors (such as hiyA, hiyB, hiyC and / or hiyD or any combination thereof).In some examples, the bacterium is an engineered bacterium which comprises a plasmid which comprises some or all of the various nucleotide sequences described herein. In one example, the bacterium comprises a first plasmid. In another example, the bacterium comprises a first and a second plasmid.

[0422] Thus, in one example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) are comprised by a first plasmid. In another example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) and any further nucleotide sequence of (c) are comprised by a first plasmid. In another example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a), any further nucleotide sequence of (c) and the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f), and / or any kill switch) are comprised by a first plasmid. The first plasmid may be comprised by a bacterium.

[0423] In an alternative example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) and any further nucleotide sequence of (c) are comprised by a first plasmid, and the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f), and / or any kill switch) are comprised by a second plasmid. The first and second plasmid may be comprised by a bacterium.

[0424] In another example, the chromosome of the bacterium comprises any of the nucleotide sequences described herein (including, in part (a), for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate; in part (b), for the production of a first molecule of interest (MOI); in part (c), for the import of xylitol, in part (d) for exporting the MOI; in part (e), sequences encoding signal peptides; importers of any second MOI or further substrates; nucleic acid modifiers (e.g. in part (f)); and / or kill switch genes).

[0425] In one example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) are comprised by the chromosome of the bacterium or recipient bacterium. In one example, the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) and the further nucleotide sequence of (c) are comprised by the chromosome of the bacterium or recipient bacterium. In this example, the bacterium or recipient bacterium may further comprise a plasmid comprising the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f)).

[0426] In another example, the chromosome of the bacterium or recipient bacterium may comprise the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any orall of the further nucleotide sequences of (d), (e) and / or (f); and any kill switch genes). In this example, the bacterium or recipient bacterium may further comprise a plasmid comprising the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) and the further nucleotide sequence of (c).

[0427] In a particular example, the chromosome of the bacterium or recipient bacterium comprises the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a), the at least one nucleotide sequence for the production of the first MOI of (b), the further nucleotide sequence of (c), and optionally any of the further nucleotide sequences of (d), (e) and / or (f), or any kill switch genes). In an example, the chromosome of the bacterium or recipient bacterium comprises the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a), the at least one nucleotide sequence for the production of the first MOI of (b), the further nucleotide sequence of (c), and all of the further nucleotide sequences of (d), (e) and / or (f), or any kill switch genes).

[0428] In a particular example, the bacterium (e.g. engineered bacterium), or the donor or recipient bacterium (e.g. comprising a transmissible element), each as described herein is capable of colonising a compartment in the GI tract of a subject selected from the ileum, cecum, colon and contents thereof, or of any combination of the foregoing. In an example, the bacterium, or the donor or recipient bacterium, each as described herein is capable colonising a compartment in the GI tract of a subject selected from the ileum, cecum, colon, jejunum and duodenum and contents thereof, or of any combination of the foregoing. In an example, the bacterium, or the donor or recipient bacterium, each as described herein is capable colonising a compartment in the GI tract of a subject selected from the jejunum and duodenum and contents thereof, or of any combination of the foregoing. In an example, the bacterium, or the donor or recipient bacterium, each as described herein is capable of colonising the ileum (or the contents of the ileum) in the GI tract of a subject. In an example, the bacterium, or the donor or recipient bacterium, each as described herein is capable of colonising the cecum (or the contents of the cecum) in the GI tract of a subject. In an example, the bacterium, or donor or recipient bacterium, each as described herein is capable of colonising the colon (or the contents of the colon) in the GI tract of a subject. The bacterium (e.g. engineered or recipient) may be an ileum bacterium. The bacterium (e.g. engineered or recipient) may be a cecum bacterium. The bacterium (e.g. engineered or recipient) may be a colon bacterium. The bacterium (e.g. engineered or recipient) may be a combination of one or more (or all) of an ileum bacterium, a cecum bacterium and a colon bacterium. Methods for profiling the human intestinal environment are described in Shalon et a!., " Profiling the human intestinal environment under physiological conditions", Nature, 617, 581-591, 2023, doi:https: / / doi.org / 10.1038 / s41586-023-05989-7, which methods are incorporated herein by reference.In an example, the bacterium, donor or recipient bacterium is capable of growing in exhausted media supplemented with xylitol. Additionally or alternatively, the bacterium, or donor or recipient bacterium is capable of growing in M9 media. In these examples, the cell density (CFU) of the bacterium, or donor or recipient bacterium may increase at least 2-fold more after 24 hours on said media than a bacterium, or donor or recipient bacterium which does not comprise the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a). The increase may be at least 3-fold. The increase may be at least 4-fold. The supplemented xylitol may be at a concentration of 2%.

[0429] Formulations and compositions comprising the bacteria or transmissible elements

[0430] There is provided a pharmaceutical composition comprising a population of bacteria (e.g. engineered bacteria) as described herein, and a pharmaceutically acceptable excipient or carrier.

[0431] There is provided a pharmaceutical composition comprising a population of donor (host) bacteria as described herein, and a pharmaceutically acceptable excipient or carrier.

[0432] There is provided a pharmaceutical composition comprising a transmissible element as described herein, and a pharmaceutically acceptable excipient or carrier. There is provided a pharmaceutical composition comprising a plurality of transmissible elements as described herein, and a pharmaceutically acceptable excipient or carrier.

[0433] The population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements may be formulated in a pharmaceutical composition comprising a diluent, excipient or carrier. The formulation may be comprised within a medical device (such as an ampoule, a syringe, or an inhaler) or may be formulated in a tincture, a capsule or a slow-release formulation. The formulation may be an oral tablet, comprised within a blister pack.

[0434] In one example, the pharmaceutical composition comprising the population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements as described herein is formulated for oral or rectal administration. In a particular example, the pharmaceutical composition is formulated for oral administration. In one example, the pharmaceutical composition is formulated as a capsule or coated tablet.

[0435] The formulation comprising the population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements described herein may be freeze dried prior to encapsulation. In one example, the pharmaceutical composition comprising the population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements as described herein is a lyophilised formulation. In one example, the pharmaceutical composition comprising the population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements as described herein is an encapsulated formulation to be released in the lower gut of a subject.Acceptable carriers, excipients, or stabilizers are non-toxic to subjects at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatine, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; and metal complexes (e.g. Zn-protein complexes). In limited circumstances, due to stability of the vectors, the formulation may include preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). A skilled formulator is aware of agents which are compatible with the different modes of delivery of the bacteria, conjugative plasmids or host (e.g. donor) bacteria described herein.

[0436] The population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements can also be formulated in liposomes. Liposomes containing the population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements are prepared by methods known in the art, such as described in Epstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030; and U. S. Pat. Nos. 4,485,045 and 4,544,545, each of which is incorporated herein by reference in its entirety. Liposomes with enhanced circulation time are disclosed in U. S. Pat. No. 5,013,556, incorporated herein by reference.

[0437] The population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements described herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.

[0438] Sustained-release preparations can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U. S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0439] The formulation may be comprised within a medical device, such as an ampoule, a syringe, or an inhaler.

[0440] Suitable dosage amounts for the population of bacteria (e.g. engineered bacteria) or the population of donor bacteria may range from about 105to 1012bacteria, e.g. approximately 105bacteria, approximately 106bacteria, approximately 107bacteria, approximately 108bacteria, approximately 109bacteria, approximately 1010bacteria, approximately 1011bacteria, or approximately 1012bacteria. The composition may be administered daily, weekly, or monthly. It may be administered multiple times per day (e.g. twice or three times per day).

[0441] In some examples, the population of bacteria (e.g. engineered bacteria), population of donor bacteria, or plurality of transmissible elements are enterically coated for release into the gut or a particular region of the gut, for example, the small or large intestines. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some examples, the coating is degraded in specific pH environments in order to specify the site of release. In some examples, at least two coatings are used. In some examples, the outside coating and the inside coating are degraded at different pH levels.

[0442] There is provided a kit comprising a population of bacteria (e.g. engineered bacteria) as described herein, and at least one dose of xylitol (as described herein) for administration to a subject. There is provided a kit comprising a population of donor bacteria as described herein, and at least one dose of xylitol (as described herein) for administration to a subject. There is provided a kit comprising a plurality of transmissible elements as described herein, and at least one dose of xylitol (as described herein) for administration to a subject. There is provided a kit comprising a pharmaceutical composition as described herein, and at least one dose of xylitol (as described herein) for administration to a subject. The kit may further comprise instructions for use (as described herein).

[0443] Uses of the bacteria, recipient bacteria, transmissible elements and methods disclosed herein The methods described herein can be carried out in vitro. The methods described herein can be carried out in vivo.

[0444] There is provided the use of xylitol (e.g. D-xylitol) for colonisation of a bacterium (e.g. engineered bacterium), or a donor or recipient bacterium (e.g. comprising a transmissible element) described herein in a microbiome of the GI tract of a subject.

[0445] There is provided a method of colonising a microbiome of a subject, said method comprising the steps of: I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject a population of bacteria, a population of donorbacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein. The xylitol may be orally administered to the subject.

[0446] There is also provided a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein for use in colonising a microbiome of the GI tract of a subject, the use comprising I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition. The xylitol may be orally administered to the subject.

[0447] There is provided the use of xylitol (e.g. D-xylitol) for promoting viability of a population of bacteria (e.g. engineered bacterium), or a donor or recipient bacterium (e.g. comprising a transmissible element) described herein in a microbiome of the GI tract of a subject.

[0448] There is provided a method of promoting viability of a population of bacteria in a microbiome of a subject, said method comprising the steps of: I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein.

[0449] There is also provided a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein for promoting viability of a population of bacteria a microbiome of the GI tract of a subject, the use comprising I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition.

[0450] There is provided the use of xylitol (e.g. D-xylitol) for providing a selective advantage to a population of bacteria (e.g. engineered bacterium), or a donor or recipient bacterium (e.g. comprising a transmissible element) described herein in a microbiome of the GI tract of a subject.

[0451] There is provided a method of providing a selective advantage to a population of bacteria in a microbiome of a subject, said method comprising the steps of: I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein.

[0452] There is also provided a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein for providing a selective advantage to a population of bacteria a microbiome of the GI tract of a subject, the use comprising I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition.The xylitol may be as described elsewhere herein. In particular, the xylitol is administered orally.

[0453] The compartments may be any compartment of the GI tract described herein. The xylitol (e.g. amounts, dosing regimen etc) may be as described elsewhere herein. In particular, the subject ingests xylitol. Alternatively, the subject has ingested xylitol. Alternatively, the subject is concurrently on a course of xylitol.

[0454] Steps I. and II. of the method or use can be carried out in either order. Thus, in one example, step I. is carried out before step II. Alternatively, step II. is carried out before step I. The skilled person will understand that multiple doses of xylitol may be administered to the subject, and multiple doses of the population of bacteria, the population of donor bacteria, the plurality of transmissible elements, or the pharmaceutical composition may be administered to the subject.

[0455] The population of bacteria or donor bacteria, the plurality of transmissible elements or the pharmaceutical composition in step II. may be administered orally (e.g. as described elsewhere herein). Alternatively, the population of bacteria or donor bacteria, the plurality of transmissible elements or the pharmaceutical composition in step II. may be administered by faecal transplant.

[0456] In an example, the method or use comprises administering an antibiotic to said subject before administration of the bacteria, transmissible elements or pharmaceutical composition in step II. This may clear a niche in the microbiome e.g. GI tract, into which the bacteria (e.g. engineered bacteria), or donor or recipient bacteria may grow. The antibiotic may be a broad-spectrum antibiotic. Antibiotic compounds are well-known to those in the art.

[0457] The bacteria, or donor or recipient bacteria (or transmissible elements) may be capable of detection in faeces at a higher CFU / g than subjects which have not received xylitol according to step I. The CFU may be at least 20% or 30% higher in faeces of subjects who have received xylitol. The CFU may be at least 40% or 50% higher in faeces of subjects who have received xylitol. The CFU may be at least 60% or 70% higher in faeces of subjects who have received xylitol. The CFU may be at least two-fold higher in faeces of subjects who have received xylitol. The CFU may be at least (approximately) half an order of magnitude higher in faeces of subjects who have received xylitol. The CFU may be at least (approximately) one order of magnitude higher in faeces of subjects who have received xylitol. The CFU may be at (approximately) 1.5 orders of magnitude higher in faeces of subjects who have received xylitol. The CFU may be at least (approximately) 2 orders of magnitude higher in faeces of subjects who have received xylitol. The CFU may be at least (approximately) 2.5 orders of magnitude higher in faeces of subjects who have received xylitol. The CFU may be at least (approximately) 3 orders of magnitude higher in faeces of subjects who have received xylitol. In an example, the subject is a mouse, the xylitol is administered in drinking water and the CFU / g is higher during ingestion (or administration) of the xylitol, as compared to mice not receiving xylitol. Additionally or alternatively, the subject is a mouse, the xylitol is administered in drinking water and the CFU / g is higher for a period of time after termination of ingestion (or administration) of the xylitol,as compared to mice not receiving xylitol. The period of time may be at least 3 days after termination of ingestion (or administration) of the xylitol. The period of time may be at least 4 days after termination of ingestion (or administration) of the xylitol. The period of time may be at least 5 days after termination of ingestion (or administration) of the xylitol. The period of time may be at least a week after termination of ingestion (or administration) of the xylitol.

[0458] In an example, the subject may be suffering from a disease or disorder. Particular diseases and disorders include those which can be treated or prevented by expression or production of the first MOI. Additionally or alternatively, diseases and disorders include those which can be treated or prevented by the removal of the second MOI. Those diseases and disorder are described elsewhere herein. Thus, in these examples, the bacteria, transmissible elements or pharmaceutical composition of step II. comprise the at least one nucleotide sequence of (b) for the production of a first MOI.

[0459] There is provided a method of treating or preventing a disease or condition in a subject in need thereof, said method comprising the steps of: I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein. The method may comprise any of the features described herein.

[0460] There is provided a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition as described herein for use in treating or preventing a disease or disorder in a subject, said use comprising the steps of: I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein. The use may comprise any of the features described herein.

[0461] There is provided the use of a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition as described herein in the manufacture of a medicament for the treatment or prevention of a disease or disorder in a subject, wherein said medicament is for use in combination with xylitol. In an example, the medicament is approved for use in combination with xylitol. The use of xylitol may be sequential to the use of the medicament. The use of xylitol may be simultaneous with the use of the medicament. In an example, the medicament is for use in a method comprising the steps of: I. providing xylitol (e.g. D-xylitol) for ingestion by the subject, whereby the subject ingests the xylitol; and II. administering to the subject a population of bacteria, a population of donor bacteria, a plurality of transmissible elements, or a pharmaceutical composition, each as described elsewhere herein. The use may comprise any of the features described herein.

[0462] Any of the methods or uses herein relating for treatment may also relate to the prevention of the specified disease or condition.There is provided a method of modifying recipient bacteria in the gut of a subject, comprising administering to said subject a population of donor (host) bacteria, or a plurality of transmissible elements (e.g. conjugative plasmids, phage or phagemids), or a pharmaceutical composition as described herein. The subject may comprise one or more strains of bacteria which are capable of the transmissible element being transmitted to said one or more strains of bacteria.

[0463] There is provided method of producing a first MOI in the gut of a subject, comprising administering to said subject a population of bacteria (e.g. engineered bacteria), a population of donor (host) bacteria, or a plurality of transmissible elements (e.g. conjugative plasmids, phage or phagemids), or a pharmaceutical composition as described herein. The subject may comprise one or more strains of bacteria which are capable of the transmissible element being transmitted to said one or more strains of bacteria.

[0464] The subject can be a human or animal subject. The subject can be a mammal such as a nonprimate (e.g. cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g. monkey and human). The subject may be a rodent, mouse or rat. The subject may be a vertebrate, reptile, bird or fish. In particular, the subject is a human.

[0465] There is provided a bacterium (e.g. engineered bacterium), a recipient or donor bacterium (e.g. comprising a transmissible element), or a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation, each as described herein for use as a medicament. There is also provided a bacterium (e.g. engineered bacterium), a recipient or donor bacterium (e.g. comprising a transmissible element), or a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation, each as described herein for use in therapy.

[0466] There is provided a method of treating a metabolic disease comprising administering to a subject in need thereof a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein, wherein the metabolic disease is caused by a deficiency of the first MOI and / or wherein the first MOI is a molecule capable of treating said metabolic disease. The subject may comprise one or more strains of bacteria which are capable of the transmissible element being transmitted to said one or more strains of bacteria.

[0467] There is provided a method of treating a cardiovascular metabolic disease comprising administering to a subject in need thereof a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein, wherein the cardiovascular metabolic disease is caused by a deficiency of the first MOI and / or wherein the first MOI is a molecule capable of treating said cardiovascular metabolic disease. The subject may comprise one or more strains of bacteria which are capable of the transmissible element being transmitted to said one or more strains of bacteria.

[0468] There is provided a method of treating a metabolic disease selected from leaky gut, type 1 diabetes, type 2 diabetes (including complications of type 1 and type 2 diabetes, e.g. insulin sensitivityin type 2 diabetes), metabolic syndrome, Bardet-Biedel syndrome, Prader-Willi syndrome, nonalcoholic fatty liver disease, tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency, Single-minded 1 (SIM1) deficiency, leptin deficiency, leptin receptor deficiency, pro-opiomelanocortin (POMC) defects, proprotein convertase subtilisin / kexin type 1 (PCSK1) deficiency, Src homology 2B1 (SH2B1) deficiency, pro-hormone convertase 1 / 3 deficiency, melanocortin-4-receptor (MC4R) deficiency, Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome, pseudohypoparathyroidism type 1A, Fragile X syndrome, Borjeson-Forsmann-Lehmann syndrome, Alstrom syndrome, Cohen syndrome, and ulnar-mammary syndrome, said method comprising administering to a subject in need thereof a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein. The metabolic disease may be caused by a deficiency of the first MOI and / or the first MOI may be a molecule capable of treating said metabolic disease. The subject may comprise one or more strains of bacteria which are capable of the transmissible element being transmitted to said one or more strains of bacteria.

[0469] There is provided a method of treating a metabolic disease selected from metabolic syndrome, type 2 diabetes (including complications of type 2 diabetes, e.g. insulin sensitivity in type 2 diabetes), and non-alcoholic fatty liver disease, said method comprising administering to a subject in need thereof a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein. The metabolic disease may be caused by a deficiency of the first MOI and / or the first MOI may be a molecule capable of treating said metabolic disease. The subject may comprise one or more strains of bacteria which are capable of the transmissible element being transmitted to said one or more strains of bacteria.

[0470] There is provided a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein for use in the treatment of a metabolic disease.

[0471] There is provided a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein for use in the treatment of a cardiovascular metabolic disease.

[0472] There is provided a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein for use in the treatment of a metabolic disease selected from leaky gut, type 1 diabetes, type 2 diabetes (including complications of type 1 and type 2 diabetes, e.g. insulin sensitivity in type 2 diabetes), metabolic syndrome, Bardet-Biedel syndrome, Prader-Willi syndrome, non-alcoholic fatty liver disease, tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency, Single-minded 1 (SIM1) deficiency, leptin deficiency, leptin receptor deficiency, pro-opiomelanocortin (POMC) defects, proprotein convertase subtilisin / kexin type 1 (PCSK1) deficiency, Src homology 2B1 (SH2B1) deficiency, pro-hormone convertase 1 / 3 deficiency, melanocortin-4-receptor (MC4R)deficiency, Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome, pseudohypoparathyroidism type 1A, Fragile X syndrome, Borjeson-Forsmann-Lehmann syndrome, Alstrom syndrome, Cohen syndrome, and ulnar-mammary syndrome.

[0473] There is provided a donor (host) bacterium, a transmissible element (e.g. a conjugative plasmid, phage or phagemid), or a pharmaceutical formulation as described herein for use in the treatment of a metabolic disease selected from metabolic syndrome, type 2 diabetes (including complications of type 2 diabetes, e.g. insulin sensitivity in type 2 diabetes), and non-alcoholic fatty liver disease.

[0474] Metabolic Syndrome affects approximately 20—30% of the middle-aged population, and represents an increased risk to cardiovascular disorders, the leading cause of death in the United States. Obesity, dyslipidaemia, hypertension, and type 2 diabetes are described as metabolic syndrome. In some examples, the bacteria, conjugative plasmids, host (e.g. donor) cells or pharmaceutical compositions described herein are useful in the treatment, prevention and / or management of metabolic syndrome and / or obesity.

[0475] Metabolic syndrome is a clustering of at least three of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels.

[0476] Metabolic diseases are associated with a variety of physiological changes, including but not limited to elevated glucose levels, elevated triglyceride levels, elevated cholesterol levels, insulin resistance, high blood pressure, hypogonadism, subfertility, infertility, abdominal obesity, pro-thrombotic conditions, and pro-inflammatory conditions.

[0477] Cardiovascular disease includes coronary artery diseases (CAD) such as angina and myocardial infarction, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis, caused inter alia by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, and the like.

[0478] Obesity is a common, deadly, and costly disease in developed countries which impacts all age groups, race, and gender. Obesity can be classified as an inflammatory disease because it is associated with immune activation and a chronic, low-grade systemic inflammation. Endotoxemia, a process resulting from translocation of endotoxic compounds (lipopolysaccharides [LPS]) of gram-negative intestinal bacteria. In the last decade, it has become evident that insulin resistance and T2DM are characterized by low-grade inflammation. In this respect, LPS trigger a low-grade inflammatory response, and the process of endotoxemia can therefore result in the development of insulin resistance and other metabolic disorders. Other anti-inflammatory ALMs as described herein may also be useful in the treatment of type 2 diabetes.In certain examples, the donor (host) bacteria, transmissible elements (e.g. conjugative plasmids, phage or phagemids), or pharmaceutical compositions as described herein decrease tryptophan levels in the subject, e.g. in the serum and / or in the gut, e.g. for the prevention, treatment, and / or management of obesity.

[0479] Metabolic syndrome is an important risk factor for cardiovascular disease incidence and mortality, as well as all-cause mortality. Thus, the detection, prevention, and treatment of the underlying risk factors of the metabolic syndrome are a critical approach to lower the cardiovascular disease incidence in the general population.

[0480] The donor (host) bacteria, transmissible elements (e.g. conjugative plasmids, phage or phagemids), or pharmaceutical compositions as described herein can be administered to the subject in one or more doses. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data, and taking into account age, weight and sex of the subject. It is to be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0481] The disease or condition may be metabolic syndrome or cardiometabolic disease (e.g. selected from obesity, diabetes, insulin resistance and non-alcoholic fatty liver disease). The disease may be inflammatory bowel disease (e.g. selected from Crohn's disease and Ulcerative Colitis). The condition may be irritable bowel syndrome or leaky gut syndrome.Table 4: Example Bacteria

[0482] Optionally, the modified bacteria or the bacteria to which the conjugative plasmid is hosted are / or transferred (i.e. recipient bacteria) are selected from th Table.

[0483] Abiotrophia Acetobacter lovaniensis Acetobacterium woodii Acholeplasma modicum Acidaminococcus intestini Abiotrophia defectiva Acetobacter malorum Acetofilamentum Acholeplasma morum Acidicaldus Acaricomes Acetobacter nitrogenifigens Acetofilamentum rigidum Acholeplasma multilocale Acidicaldus organivorans Acaricomes phytoseiuli Acetobacter oeni Acetohalobium Acholeplasma oculi Acidimicrobium Acetitomaculum Acetobacter orienta / is Acetohaiobium arabaticum Acholeplasma palmae Acidimicrobium ferrooxidans Acetitomaculum ruminis Acetobacter orleanensis Acetomicrobium Acholeplasma parvum Acidiphilium Acetivibrio Acetobacter pasteurianus Acetomicrobium faecale Acholeplasma pleciae Acidiphilium acidophilum Acetivibrio cellulolyticus Acetobacter pomorum Acetomicrobium flavidum Acholeplasma vituli Acidiphilium angustum Acetivibrio ethanolgignens Acetobacter senegalensis Acetonema Achromobacter Acidiphilium cryptum Acetivibrio multivorans Acetobacter xylinus Acetonema longum Achromobacter denitrificans Acidiphilium multivorum Acetoanaerobium Acetobacterium Acetothermus Achromobacter insolitus Acidiphilium organovorum Acetoanaerobium noterae Acetobacterium bakii Acetothermus paucivorans Achromobacter piechaudii Acidiphilium rubrum Acetobacter Acetobacterium carbinolicum Acholeplasma Achromobacter ruhlandii Acid isoma Acetobacter aceti Acetobacterium Acholeplasma axanthum Achromobacter spanius Acidisoma sibiricum Acetobacter cerevisiae dehaiogenans Acholeplasma brassicae Acidaminobacter Acidisoma tundrae Acetobacter cibinongensis Acetobacterium fimetarium Acholeplasma cavigenitalium Acidaminobacter Acidisphaera Acetobacter estunensis Acetobacterium malicum Acholeplasma equifetale hydrogenoformans Acidisphaera rubrifaciens Acetobacter fabarum Acetobacterium paiudosum Acholeplasma granularum Acidaminococcus Acidithiobacillus Acetobacter ghanensis Acetobacterium tundrae Acholeplasma hippikon Acidaminococcus Acidithiobacillus albertensis Acetobacter indonesiensis Acetobacterium wieringae Acholeplasma laidlawii fermentans Acidithiobacillus caldusAcidithiobacillus ferrooxidans Acinetobacter bayiyi Actinoalloteichus Actinobacuium massiiiense Actinomadura chokoriensis Acidithiobaciiius thiooxidans Acinetobacter bouvetii Actinoaiioteichus Actinobacuium schaaiii Actinomadura citrea Acidobacterium Acinetobacter caicoaceticus cyanogriseus Actinobacuium suis Actinomadura coeruiea Acidobacterium capsuiatum Acinetobacter gerneri Actinoalloteichus Actinomyces urinate Actinomadura echinospora Acidocella Acinetobacter haemoiyticus hymeniacidonis Actinocatenispora Actinomadura fibrosa Acidoceiia aminoiytica Acinetobacter johnsonii Actinoalloteichus spitiensis Actinocatenispora rupis Actinomadura formosensis Acidocella faciiis Acinetobacter junii Actinobaccillus Actinocatenispora Actinomadura hibisca Acidomonas Acinetobacter iwoffi Actinobacillus capsuiatus thaiiandica Actinomadura kijaniata Acidomonas methanoiica Acinetobacter parvus Actinobacillus deiphinicoia Actinocatenispora sera Actinomadura atina Acidothermus Acinetobacter radioresistens Actinobacillus hominis Actinocorallia Actinomadura Hvida Acidothermus cellulolyticus Acinetobacter schindieri Actinobacillus indoiicus Actinocoraiiia aurantiaca Actinomadura Acidovorax Acinetobacter soii Actinobacillus iignieresii Actinocorallia aurea iuteofiuorescens Acidovorax anthurii Acinetobacter tandoii Actinobacillus minor Actinocorallia cavernae Actinomadura macra Acidovorax caeni Acinetobacter tjernbergiae Actinobacillus muris Actinocorallia giomerata Actinomadura madurae Acidovorax cattieyae Acinetobacter towneri Actinobacillus Actinocorallia herbida Actinomadura oiigospora Acidovorax citrulli Acinetobacter ursingii pieuropneumoniae Actinocorallia iibanotica Actinomadura peiietieri Acidovorax defiuvii Acinetobacter venetianus Actinobacillus porcinus Actinocorallia iongicatena Actinomadura rubrobrunea Acidovorax deiafieidii Acrocarpospora Actinobacillus rossii Actinomadura Actinomadura rugatobispora Acidovorax faciiis Acrocarpospora corrugata Actinobacillus scotiae Actinomadura alba Actinomadura umbrina Acidovorax konjaci Acrocarpospora Actinobacillus seminis Actinomadura atramentaria Actinomadura Acidovorax temperans macrocephaia Actinobacillus succinogenes Actinomadura verrucosospora Acidovorax vaierianeiiae Acrocarpospora pieiomorpha Actinobaccillus suis bangiadeshensis Actinomadura vinacea Acinetobacter Actibacter Actinobacillus ureae Actinomadura cateiiatispora Actinomadura viridiiutea Acinetobacter baumannii Actibacter sediminis Actinobacuium Actinomadura chibensis Actinomadura viridisActinomadura yumaensis Actinoplanes consettensis Actinotalea Agrobacterium Aicanivorax borkumensis Actinomyces Actinoplanes deccanensis Actinotaiea fermentans geiatinovorum Aicanivorax jadensis Actinomyces bovis Actinoplanes derwentensis Aerococcus Agrococcus Algicola Actinomyces dentico / ens Actinoplanes digitatis Aerococcus sanguinicoia Agrococcus citreus Aigicoia bacterioiytica Actinomyces europaeus Actinoplanes durhamensis Aerococcus urinae Agrococcus jenensis Alicyclobacillus Actinomyces georgiae Actinoplanes ferrugineus Aerococcus urinaeequi Agromonas Alicyclobacillus Actinomyces gerencseriae Actinoplanes giobisporus Aerococcus urinaehominis Agromonas oiigotrophica disuifidooxidans Actinomyces hordeovuineris Actinoplanes humidus Aerococcus viridans Agromyces Alicyclobacillus sendaiensis Actinomyces howeiiii Actinoplanes itaiicus Aeromicrobium Agromyces fucosus Alicyclobacillus vuicanaiis Actinomyces hyovaginaiis Actinoplanes Hguriensis Aeromicrobium erythreum Agromyces hippuratus Alishewanella Actinomyces israelii Actinoplanes iobatus Aeromonas Agromyces iuteoius Alishewanella fetalis Actinomyces johnsonii Actinoplanes missouriensis Aeromonas aiiosaccharophiia Agromyces medioianus Alkalibacillus Actinomyces meyeri Actinoplanes paiieronii Aeromonas bestiarum Agromyces ramosus Alkalibacillus haloalkaliphilu Actinomyces naesiundii Actinoplanes philippinensis Aeromonas caviae Agromyces rhizospherae Alkalilimnicola Actinomyces neuii Actinoplanes rectiiineatus Aeromonas encheieia Akkermansia Alkalilimnicola ehriichii Actinomyces odontoiyticus Actinoplanes reguiaris Aeromonas enteropeiogenes Akkermansia muciniphila Alkaliphilus Actinomyces oris Actinoplanes Aeromonas eucrenophila Albidiferax Aikaiiphiius oremiandii Actinomyces radingae teichomyceticus Aeromonas ichthiosmia Albidiferax ferrireducens Alkaliphilus transvaaiensis Actinomyces siackii Actinoplanes utahensis Aeromonas jandaei Albidovulum Allochromatium Actinomyces turicensis Actinopolyspora Aeromonas media Albidovulum inexpectatum AHochromatium vinosum Actinomyces viscosus Actinopoiyspora haiophiia Aeromonas popoffii Alcaligenes Alloiococcus Actinoplanes Actinopolyspora mortivaiHs Aeromonas sobria Alcaligenes denitrificans AHoiococcus otitis Actinoplanes auranticoior Actinosynnema Aeromonas veronii Alcaligenes faecaiis Allokutzneria Actinoplanes brasiiiensis Actinosynnema mirum Agrobacterium Alcan ivorax AHokutzneria aibataAltererythrobacter Amphritea japonica Anaerococcus AneurinibaciHus Arcanobacterium Aitererythrobacter Amycolatopsis Anaerococcus hydrogenaiis aneuriniiyticus haemoiyticum ishigakiensis Amyco / atopsis alba Anaerococcus iactoiyticus AneurinibaciHus miguianus Arcanobacterium pyogenes Altermonas Amycolatopsis albidoflavus Anaerococcus prevotii AneurinibaciHus Archangium Aitermonas haiopianktis Amycolatopsis azurea Anaerococcus tetradius thermoaerophiius Archangium gephyra Altermonas macieodii Amycolatopsis coioradensis Anaerococcus vaginalis Angiococcus Arcobacter Alysiella Amycolatopsis iurida Anaerofustis Angiococcus disciformis Arcobacter butzieri Alysiella crassa Amycolatopsis mediterranei Anaerofustis stercorihominis Angulomicrobium Arcobacter cryaerophiius Alysiella fiiiformis Amycolatopsis rifamycinica Anaeromusa Anguiomicrobium tetraedraie Arcobacter haiophiius Aminobacter Amycolatopsis rubida Anaeromusa acidaminophiia Anoxybacillus Arcobacter nitrofigiiis Aminobacter aganoensis Amycolatopsis suiphurea Anaeromyxobacter AnoxybaciHus Arcobacter skirrowii Aminobacter aminovorans Amycolatopsis toiypomycina Anaeromyxobacter pushchinoensis Arhodomonas Aminobacter niigataensis Anabaena dehaiogenans Aquabacterium Arhodomonas aquaeoiei Aminobacterium Anabaena cyiindrica Anaerorhabdus Aquabacterium commune Arsenophonus Aminobacterium mobile Anabaena fios-aquae Anaerorhabdus furcosa Aquabacterium parvum Arsenophonus nasoniae Aminomonas Anabaena variabilis Anaerosinus Aquaspirillum Arthrobacter Aminomonas paucivorans Anaeroarcus Anaerosinus glyceri ' Aquaspirillum poiymorphum Arthrobacter agiiis Ammoniphilus Anaeroarcus burkinensis Anaerovirgula Aquaspiriiium Arthrobacter aibus Ammoniphiius oxaiaticus Anaerobaculum Anaerovirguia muitivorans putridiconchyiium Arthrobacter aurescens Ammoniphilus oxaiivorans Anaerobacuium mobile Ancalomicrobium Aquaspiriiium serpens Arthrobacter Amphibacillus Anaerobiospirillum Ancaiomicrobium adetum Aquimarina chiorophenoHcus Amphibaciiius xyianus Anaerobiospirillum Ancylobacter Aquimarina iatercuia Arthrobacter citreus Amphritea succiniciproducens Ancyiobacter aquaticus Area nobacterium Arthrobacter crystallopoiete Amphritea baienae Anaerobiospirillum thomasii AneurinibaciHus Arthrobacter cumminsiiArthrobacter giobiformis Arthrobacter woluwensis Avibacterium voiantium Azotobacter saiinestris Bainearium Hthotrophicum Arthrobacter Asaia Azoarcus Azotobacter vineiandii Balneatrix histidinoiovorans Asaia bogorensis Azoarcus indigens Bacteriovorax Baineatrix aipica Arthrobacter iiicis Asanoa Azoarcus toiuiyticus Bacteriovorax stoipii Balneola Arthrobacter iuteus Asanoa ferruginea Azoarcus toiuvorans Bacteroides Baineoia vulgaris Arthrobacter methyiotrophus Asticcacaulis Azohydromonas Bacteroides caccae Barnesiella Arthrobacter mysorens Asticcacau / is biprosthecium Azohydromonas austraiica Bacteroides coaguians Barnesieiia viscericoia Arthrobacter nicotianae Asticcacaulis excentricus Azohydromonas iata Bacteroides eggerthii Bartonella Arthrobacter nicotinovorans Atopobacter Azomonas Bacteroides frag His Bartonella aisatica Arthrobacter oxydans A topobacter phocae Azomonas agiiis Bacteroides gaiacturonicus Bartonella bacHHformis Arthrobacter pascens Atopobium Azomonas insignis Bacteroides heicogenes Bartonella ciarndgeiae Arthrobacter Atopobium fossor Azomonas macrocytogenes Bacteroides ovatus Bartonella doshiae phenanthrenivorans Atopobium minutum Azorhizobium Bacteroides pectinophiius Bartonella eiizabethae Arthrobacter Atopobium parvuium Azorhizobium cauiinodans Bacteroides pyogenes Bartonella grahamii poiychromogenes Atopobium rimae Azorhizophilus Bacteroides saiyersiae Bartonella henselae A trhrobacter protophormiae Atopobium vaginae Azorhizophiius paspaii Bacteroides stercoris Bartonella rochaiimae Arthrobacter Aureobacterium Azospirillum Bacteroides suis Bartonella vinson ii psychroiactophiius Aureobacterium barken Azospiriiium brasiiense Bacteroides tectus Bavariicoccus Arthrobacter ramosus Aurobacterium Azospiriiium haiopraeferens Bacteroides Bavariicoccus seiieri Arthrobacter suifonivorans Aurobacterium Hquefaciens Azospiriiium irakense. thetaiotaomicron Bdellovibrio Arthrobacter suifureus Avibacterium Azotobacter Bacteroides uniformis Bdeiiovibrio bacteriovorus Arthrobacter uratoxydans Avi bacterium avium Azotobacter beijerinckii Bacteroides ureoiyticus Bdellovibrio exovorus Arthrobacter ureafaciens Avibacterium ga Hina rum Azotobacter chroococcum Bacteroides vuigatus Beggiatoa Arthrobacter viscosus Avibacterium paragallinarum Azotobacter nigricans Balnea rium Beggiatoa albaBeijerinckia Bifidobacterium breve Bilophila Bog oriel ia caseiiytica Brachybacterium faecium Beijerinckia derxii Bifidobacterium catenuiatum Bilophila wadsworthia Bordetella Brachybacterium Beijerinckia fluminensis Bifidobacterium choerinum Biostraticola Bordeteiia avium paracongiomeratum Beijerinckia indica Bifidobacterium coryneforme Biostraticoia tofi Bordetella bronchiseptica Brachybacterium Beijerinckia mobiiis Bifidobacterium cunicuii Bizionia Bordetella hinzii rhamnosum

[0484] Belliella Bifidobacterium dentium Bizionia argentinensis Bordetella hoimesii Brachybacterium Belliella ba idea Bifidobacterium gaiiicum Blastobacter Bordetella parapertussis tyrofermentans Bellilinea Bifidobacterium gallinarum Biastobacter capsuiatus Bordetella pertussis Brachyspira BeHHinea caidifistuiae Bifidobacterium indicum Blastobacter den itrif. leans Bordetella petrii Brachyspira aivinipuiH Belnapia Bifidobacterium long urn Blastococcus Bordetella trematum Brachyspira hyodysenteriae Beinapia moabensis Bifidobacterium Biastococcus aggregatus Borrelia Brachyspira innocens Berge riel la magnumBifidobacterium Blastococcus saxobsidens Borreiia afzeiii Brachyspira murdochii Bergerieiia den itrif leans merycicum Blastoch loris Borrelia americana Brachyspira piiosicoii Beutenbergia Bifidobacterium minimum Bia stoch ion's viridis Borreiia burgdorferi Bradyrhizobium Beutenbergia cavernae Bifidobacterium Blastomonas Borreiia caroiinensis Bradyrhizobium canariense Bibersteinia pseudocatenuiatum Biastomonas natatoria Borreiia coriaceae Bradyrhizobium eikanii Bibersteinia trehaiosi Bifidobacterium Blastopirellula Borreiia garinii Bradyrhizobium japonicum Bifidobacterium pseudoiongum Biastopireiiuia marina Borreiia japonica Bradyrhizobium Haoningens Bifidobacterium adoiescentis Bifidobacterium pullorum Blautia Bosea Brenneria Bifidobacterium anguiatum Bifidobacterium ruminantium Biautia coccoides Bosea minatitianensis Brenneria a ini Bifidobacterium animaiis Bifidobacterium saecuiare Blautia hansenii Bosea thiooxidans Brenneria nigrifiuens Bifidobacterium asteroides Bifidobacterium subtile Biautia producta Brachybacterium Brenneria quercina Bifidobacterium bifidum Bifidobacterium Biautia wexierae Brachybacterium Brenneria quercina Bifidobacterium bourn thermophiium Bogoriella aiimentarium Brenneria saiicisBrevi bacillus Brevibacterium oxydans Burkhoideria am bi fa ria Buttiauxella B. akibai BrevibaciHus agri Brevibacterium paucivorans Burkholderia andropogonis Buttiauxeiia agrestis B. a ica lophilus BrevibaciHus borsteiensis Brevibacterium stationis Burkholderia anthina Buttiauxella brennerae B. aigicoia BrevibaciHus brevis Brevinema Burkholderia caiedonica Buttiauxeiia ferragutiae B. aiginoiyticus BrevibaciHus centrosporus Brevinema andersonii Burkholderia caryophyiii Buttiauxella gaviniae B. aikaiidiazotrophicus BrevibaciHus choshinensis Brevundimonas Burkholderia cenocepacia Buttiauxeiia izardii B. alkalinitrilicus BrevibaciHus invocatus Brevundimonas alba Burkholderia cepacia Buttiauxeiia noackiae B. aikaiisediminis BrevibaciHus iaterosporus Brevundimonas aurantiaca Burkholderia cocovenenans Buttiauxeiia warmboidiae B. alkalitelluris BrevibaciHus parabrevis Brevundimonas diminuta Burkholderia doiosa Butyrivibrio B. aititudinis BrevibaciHus reuszeri Brevundimonas intermedia Burkholderia fungorum Butyrivibrio fibrisoivens B. aiveayuensis Brevibacterium Brevundimonas Burkholderia giathei Butyrivibrio hungatei B. alvei Brevibacterium abidum subvibrioides Burkholderia giumae Butyrivibrio proteociasticus B. amyioiiquefaciens Brevibacterium album Brevundimonas vancanneytii Burkholderia gramin is Bacillus B. a. subsp.

[0485] Brevibacterium aurantiacum Brevundimonas variabiiis Burkholderia kururiensis B. acidiceier Amyioiiquefaciens Brevibacterium ceiere Brevundimonas vesicuiaris Burkholderia muitivorans B. acidicoia B. a. subsp. Plantarum Brevibacterium epidermidis Brochothrix Burkholderia phenazinium B. acidiproducens B. dipsosauri Brevibacterium Brochothrix campestris Burkholderia piantarii B. acidocaidarius B. drentensis frigoritoierans Brochothrix thermosphacta Burkholderia pyrrocinia B. acidoterrestris B. edaphicus Brevibacterium haiotoierans Brucella Burkholderia siivatiantica B. aeoiius B. ehimensis Brevibacterium iodinum Brucella cam's Burkholderia stabiiis B. aerius B. eiseniae Brevibacterium linens Brucella neotomae Burkholderia thaiiandensis B. aerophiius B. enciensis Brevibacterium iyticum Bryobacter Burkholderia tropica B. agaradhaerens B. endophyticus Brevibacterium mcbreiineri Bryobacter aggregatus Burkholderia unamae B. agri B. endoradicis Brevibacterium otitidis Burkholderia Burkholderia vietnamiensis B. aidingensis B. farraginisB. fastid iosus B. andreesenii B. boron iphii us B. cycioheptanicus B. hum! B. fengqiuensis B. aneuriniiyticus B. borsteiensis B. cytotoxicus B. hwajinpoensis B. firmus B. anthracis B. brevis Miguia B. daiiensis B. idriensis B. flexus B. aquimaris B. butanoiivorans B. decisifrondis B. indicus B. fora minis B. arenosi B. canaveraii us B. decoiorationis B. infantis B. fordii B. arseniciseienatis B. carboniphiius B. deserti B. infernus B. formosus B. arsenicus B. cecembensis B. giucanoiyticus B. insoiitus B. fortis B. aurantiacus B. ceiiuiosiiyticus B. gordonae B. invictae B. fumarioii B. arvi B. centrosporus B. gottheiiii B. iranensis B. funiculus B. aryabhatai B. cereus B. gra minis B. isabeiiae B. fusiformis B. asahii B. chagannorensis B. haimapaius B. isronensis B. gaiactophiius B. atrophaeus B. chitinoiyticus B. haloalkaliphilus B. jeotgaii B. gaiactosidiiyticus B. axarquiensis B. chondroitinus B. halochares B. kaustophiius B. gaiiiciensis B. azotofixans B. choshinensis B. haiodenitrificans B. kobensis B. gelatin! B. azotoformans B. chungangensis B. haiodurans B. kochii B. gibsonii B. badius B. cibi B. ha lophilus B. kokeshiiformis B. ginseng! B. barbaricus B. circuians B. haiosaccharovorans B. koreensis B. ginsengihumi B. bataviensis B. dark!! B. hemicellulosilyticus B. koriensis B. g inseng isoii B. beijingensis B. ciausii B. hemicentroti B. kribbensis

[0486]

[0487] subsp. Marinus) B. berkeieyi B. cohnii B. horneckiae B. larvae B. aminovorans B. beveridgei B. composti B. horti B. iaterosporus B. amyioiyticus B. bogoriensis B. curdianoiyticus B. huizhouensis B. saiexigensB. saiiphiius B. subtilis (eg, B. s. subsp. B. tusciae B. pseudomycoides B. iocisaiis

[0488] B. schiegeiH Inaquosorum; or B. s. subsp. B. vaiidus. B. psychrodurans B. iuciferensis B. sediminis Spizizeni; or B. s. subsp. B. vaiiismortis B. psychrophiius B. iuteoius

[0489] B. seienatarsenatis Subtilis) B. vedderi B. psychrosaccharoiyticus B. iuteus

[0490] B. seienitireducens B. taeanensis B. veiezensis B. psychrotoierans B. macauensis B. seohaeanensis B. tequiiensis B. vietnamensis B. puivifaciens B. macerans B. shacheensis B. thermantarcticus B. vireti B. pumiius B. macquariensis B. shackietonii B. thermoaerophiius B. vuicani B. purgationiresistens B. macyae

[0491] B. siamensis B. thermoamyiovorans B. wakoensis B. pycnus B. maiacitensis B. siivestris B. thermocatenuiatus B. weihenstephanensis B. qingdaonensis B. mannaniiyticus B. simplex B. thermocloacae B. xiamenensis B. qingshengii B. marisfiavi B. si rails B. thermocopriae B. xiaoxiensis B. reuszeri B. marismortui B. smithii B. thermodenitrificans B. zhanjiangensis B. rhizosphaerae B. marmarensis B. soii B. thermogiucosidasius B. peoriae B. rigui B. massiiiensis B. soiimangrovi B. thermoiactis B. persepoiensis B. ruris B. megaterium B. solisalsi B. thermoieovorans B. persicus B. sa fensis B. mesonae B. songkiensis B. thermophiius B. pervagus B. saiarius B. methanoiicus B. sonorensis B. thermoruber B. piakortidis B. iautus B. methyiotrophicus B. sphaericus B. thermosphaericus B. pocheonensis B. iehensis B. miguianus B. sporothermodurans B. thiaminoiyticus B. polygo ' B. ientimorbus B. moja vensis B. stearothermophiius B. th iopara ns B. poiymyxa B. ientus B. muciiaginosus B. stratosphericus B. thuringiensis B. popiiiiae B. Hcheniformis B. muraiis

[0492] B. subterraneus B. tianshenii B. pseudaica lophilus B. iigniniphiius B. murimartini B. trypoxyiicoia B. pseudofirmus B. iitoraiis B. mycoidesB. naganoensis B. B. paraflexus Campylobacter Capnocytophaga ochracea Catenococcus thiocydi nanhaiensis B. B. pasteurii Campylobacter coli Capnocytophaga sputigena Catenu loplanes nanhaiisediminis B. B. patagoniensis Campylobacter concisus Cardiobacterium Catenuloplanes atrovinosus nea / sonii Caenimonas Campylobacter curvus Cardiobacterium hominis Catenuloplanes castaneus B. neidei Caenimonas koreensis Campylobacter fetus Carnimonas Catenuloplanes crispus B. neizhouensis Caldalkalibacillus Campylobacter gracilis Carni monas nigrif leans Catenuloplanes indicus B. niabensis Caldalkalibacillus uzonensis Campylobacter he / veticus Carnobacterium Catenuloplanes japonicus B. niacin! Caldanaerobacter Campylobacter hominis Carnobacterium Catenuloplanes nepa / ensis B. nova / is Ca / danaerobacter Campylobacter alterfunditum Catenuloplanes niger B. oceanisediminis subterraneus hyointestinalis Carnobacterium divergens Chryseobacterium B. odyssey! Caldanaerobius Campylobacter jejuni Carnobacterium funditum Chryseobacterium

[0493] B. okhensis Ca / danaerobius fijiensis Campylobacter lari Carnobacterium gallinarum ba / ustinum

[0494] B. okuhidensis Caldanaerobius Campylobacter mucosa / is Carnobacterium Citrobacter B. o / eronius poiysaccharoiyticus Campylobacter rectus maltaromaticum C ama / onaticus

[0495] B. oryzaecorticis Caldanaerobius zeae Campylobacter showae Carnobacterium mobile C braakii

[0496] B. oshimensis Caldanaerovirga Campylobacter sputorum Carnobacterium viridans C diversus

[0497] B. pabuli Caidanaerovirga Campylobacter upsaliensis Caryophanon C farmer!

[0498] B. pakistanensis acetigignens Capnocytophaga Caryophanon latum C freundii

[0499] B. pallid us Caldicellulosiruptor Capnocytophaga canimorsus Caryophanon tenue C gillenii

[0500] B. pallid us Caidiceiiuiosiruptor bescii Capnocytophaga cynodegmi Catellatospora C koseri

[0501] B. panacisoli Caldicellulosiruptor Capnocytophaga gingiva / is Catellatospora citrea C murHniae

[0502] B. panaciterrae kristjanssonii Capnocytophaga granulosa Catellatospora C pasteurii

[0503] B. pantothenticus Caldicellulosiruptor Capnocytophaga methionotrophica C rodentium

[0504] B. parabrevis owensensis haemotytica Catenococcus C sed / akiiC werkmanii Clostridium Clostridium botulinum, Clostridium ciostrid io forme, Clostridium ganghwense, C youngae aigidixyianoiyticum, Clostridium bowmanii, Clostridium coccoides, Clostridium gasigenes, Coccoch loris Clostridium aigifaecis, Clostridium bryantii, Clostridium cochlea rium, Clostridium ghonii, Coccoch ion's eiabens Clostridium algoriphilum, Clostridium butyricum, Clostridium colletant, Clostridium glycol icum, Corynebacterium Clostridium alkalicellulosi, Clostridium cadaveris, Clostridium coiicanis, Clostridium Corynebacterium fiavescens Clostridium aminophiium, Clostridium caenicoia, Clostridium coiinum, giycyrrhiziniiyticum, Corynebacterium variabiie Clostridium aminovaiericum, Clostridium caminithermaie, Clostridium coiiagenovorans, Clostridium grantii,

[0505] Curtobacterium Clostridium amygdaiinum, Clostridium carboxidivorans, Clostridium cyiindrosporum, Clostridium haemoiyticum, Curtobacterium aibidum Clostridium amyioiyticum, Clostridium earn is, Clostridium difficile, Clostridium ha lophilum, Curtobacterium citreus Clostridium arbusti, Clostridium cavendishii, Clostridium dioiis, Clostridium hasti forme, Clostridium Clostridium arcticum, Clostridium ceiatum, Clostridium disporicum, Clostridium hathewayi, Clostridium absonum, Clostridium argentinense, Clostridium ceierecrescens, Clostridium drakei, Clostridium herbivorans, Clostridium aceticum, Clostridium asparagiforme, Clostridium ceiiobioparum, Clostridium durum, Clostridium hiranonis, Clostridium acetireducens, Clostridium Clostridium Clostridium estertheticum, Clostridium histoiyticu...

Claims

Claims:

1. A bacterium, or transmissible element for transmission to a recipient bacterium, wherein the bacterium or transmissible element comprises:(a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate, wherein one, more or all of the one or more nucleotide sequences is heterologous to the bacterium or recipient bacterium.

2. The bacterium or transmissible element according to claim 1, which further comprises:(b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium.

3. A bacterium, or transmissible element for transmission to a recipient bacterium, wherein the bacterium or transmissible element comprises:(a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose-5-phosphate; and(b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium.

4. The bacterium or transmissible element according to claim 3, or the bacterium according to claim 5 or 6, wherein one, more or all of the one or more nucleotide sequences of (a) is heterologous to the bacterium or recipient bacterium.

5. A bacterium comprising:(a) one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate, operably linked to a promoter which is not operably linked to the nucleotide sequence(s) encoding the peptide product(s) in nature,wherein the promoter is not a native promoter of the pUC13 plasmid.

6. The bacterium according to claim 5, which further comprises:(b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium.

7. The bacterium according to any one of claims 1 to 6, or the transmissible element according to any one of claims 1 to 4, or a recipient or donor bacterium comprising a transmissible element as defined in any one of claims 1 to 4 for use as a medicament.

8. The bacterium according to any one of claims 1 to 6, a donor or recipient bacterium comprising a transmissible element as defined in any one of claims 1 to 4, or the bacterium or transmissible element for the use according to claim 7, wherein the bacterium, or donor or recipient bacterium is capable of colonising a compartment in the GI tract of a subject selected from the ileum, cecum, colon and contents thereof, or of any combination of the foregoing, and / or selected from the jejunum and duodenum and contents thereof or any combination of the foregoing.

9. Use of xylitol for colonisation ofa bacterium according to any one of claims 1 to 6, or 8, ora donor or recipient bacterium comprising a transmissible element as defined in any one of claims 1 to 4 or 8,in a microbiome of the GI tract of a subject.

10. The use according to claim 9, wherein the subject has been administered (e.g. orally), in particular ingests or has ingested, xylitol.

11. The use according to claim 9 or 10, wherein the colonisation is of a compartment in the GI tract selected from the ileum, cecum, colon and contents thereof, or of any combination of the foregoing, and / or selected from the jejunum and duodenum and contents thereof or any combination of the foregoing.

12. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 11, wherein the one or more nucleotide sequences of (a) encodes a xylitol dehydrogenase (XOHDH).

13. The bacterium, recipient bacterium, transmissible element or use according to claim 12, wherein the XOHDH is from a species selected from an Aspergillus, a G / uconobacter, a Saccharomyces, a Pichia, a Candida, a Providencia and a Morganella species, for example from Aspergillus flavus, G / uconobacter oxydans, Saccharomyces Cerevisiae, Pichia stipites, Candida tropicaiis, Providencia stuartii, and Morganella morganii, in particular Morganella morganii14. The bacterium, recipient bacterium, transmissible element or use according to claim 12 or claim 13, wherein the XOHDH comprises an amino acid sequence of SEQ ID No:1; or a sequence having at least 80% (in particular at least 90%) identity to SEQ ID No:1 and converts xylitol to xylulose, or wherein the nucleotide sequence encoding the XOHDH comprises a nucleotide sequence of SEQ ID No:2; or a sequence having at least 80% (in particular at least 90%) identity to SEQ ID No:2 and the XOHDH converts xylitol to xylulose.

15. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 12 to 14, wherein the XOHDH is capable of converting D-xylitol to D-xylulose,optionally wherein the XOHDH, when isolated, has a KM of less than 100 mM (for example less than 50 mM or less than 20 mM) in an in vitro assay.

16. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 15, wherein the one or more nucleotide sequences of (a) encodes a sugar isomerase (SI).

17. The bacterium, recipient bacterium, transmissible element or use according to claim 16, wherein the SI is from a species selected from a Morganeiia or Escherichia species, for example from Morganella morganiior E. coli, or wherein the SI is from a species or strain listed in Table 1.

18. The bacterium, recipient bacterium, transmissible element or use according to claim 16 or claim 17, wherein the SI comprises an amino acid sequence of SEQ ID No:3; or a sequence having at least 80% (in particular at least 90%) identity to SEQ ID No:3 and converts xylose to xylulose, or wherein the nucleotide sequence encoding the SI comprises a nucleotide sequence of SEQ ID No:4; or a sequence having at least 80% (in particular at least 90%) identity to SEQ ID No:4 and the SI converts xylose to xylulose.

19. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 16 to 18, wherein the SI is capable of converting D-xylose to D-xylulose,optionally wherein the SI, when isolated, has a KM of less than 10 mM in an in vitro assay.

20. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 19, wherein the one or more nucleotide sequences of (a) encodes a xylulokinase (XK).

21. The bacterium, recipient bacterium, transmissible element or use according to claim 20, wherein the XK is from a Morganeiia species, for example from Morganella morganii, or wherein the XK is from an Escherichia species, for example from E. coli.

22. The bacterium, recipient bacterium, transmissible element or use according to claim 20 or claim 21, wherein the XK comprises an amino acid sequence of SEQ ID No:5; or a sequence having at least 80% (in particular at least 90%) identity to SEQ ID No: 5 and converts xylulose to xylulose- 5-phosphate,or wherein the nucleotide sequence encoding the XK comprises a nucleotide sequence of SEQ ID No: 6; or a sequence having at least 80% (in particular at least 90%) identity to SEQ ID No: 6 and the XK converts xylulose to xylulose-5-phosphate.

23. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 20 to 22, wherein the XK is capable of converting D-xylulose to D-xylulose-5-phosphate, optionally wherein the XK, when isolated, has a KM of less than 400 pM (e.g. less than 300 pM) in an in vitro assay.

24. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 23, wherein the one or more nucleotide sequences of (a) encodes a xylitol oxidase (AldO).

25. The bacterium, recipient bacterium, transmissible element or use according to claim 24, wherein the AldO is from a species selected from a Streptomyces species, for example from Streptomyces coelicolor.

26. The bacterium, recipient bacterium, transmissible element or use according to claim 23 or claim 24, wherein the AldO is capable of converting D-xylitol to D-xylose,optionally wherein the AldO, when isolated, has a KM of less than 1 mM (e.g. less than 0.5 mM, or less than 0.4 mM) in an in vitro assay.

27. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 26, wherein the one or more nucleotide sequences of (a) encodes a xylose reductase.

28. The bacterium, recipient bacterium, transmissible element or use according to claim 27, wherein the xylose reductase is from a species selected from a Yamadazyma species, for example from Yamadazyma tenuis, or wherein the xylose reductase is from a Neurospora species, for example from Neurospora crassa.

29. The bacterium, recipient bacterium, transmissible element or use according to claim 27 or claim 28, wherein the xylose reductase is capable of converting D-xylitol to D-xylose,optionally wherein the xylose reductase, when isolated, has a KM of less than 300 mM in an in vitro assay.

30. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 29, wherein the bacterium, recipient bacterium or transmissible element further comprises:(c) a further nucleotide sequence encoding a transporter which is capable of importing xylitol into the bacterium or recipient bacterium.

31. The bacterium, recipient bacterium, transmissible element or use according to claim 30, wherein the transporter is from a species selected from a Morganeiia species, for example from Morganella morganii.

32. The bacterium, recipient bacterium, transmissible element or use according to claim 30 or claim 31, wherein the transporter is selected from a xylose transporter (e.g. xylFGI, optionally from a Escherichia species, e.g. E. coli), a phosphotransferase transporter (e.g. frul, optionally from a Streptococcus species, e.g. Streptococcus mutans), hxt11 (optionally from a Saccharomyces species, e.g. Saccharomyces cerevisiae), hxt15 (optionally from a Saccharomyces species, e.g. Saccharomyces cerevisiae) and plt4 (optionally from a Lotus species, e.g. Lotus Japonicus).

33. The bacterium, recipient bacterium, transmissible element or use according to claim 30 or claim 31, wherein the transporter is an ABC transporter.

34. The bacterium, recipient bacterium, transmissible element or use according to claim 30, claim 31 or claim 33, wherein the transporter comprises amino acid sequences of SEQ ID No: 7, SEQ ID No:8 and SEQ ID No:9; or sequences having at least 80% (in particular at least 90%) identity to each of SEQ ID No:7, SEQ ID No:8 and SEQ ID No:9 and is capable of importing xylitol into the bacterium or recipient bacterium,or wherein the nucleotide sequence encoding the transporter comprises nucleotide sequences of SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12; or sequences having at least 80% (in particular at least 90%) identity to each of SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12 and the transporter is capable of importing xylitol into the bacterium or recipient bacterium.

35. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 34, wherein the bacterium, recipient bacterium, or transmissible element comprises:(a) one or more nucleotide sequences encoding peptide product(s) for the conversion of xylitol to xylulose-5-phosphate, wherein the peptide products comprise i. a xylitol dehydrogenase (XOHDH) (optionally as defined in any one of claims 13 to 15), ii. a sugar isomerase (SI) (optionally as defined in any one of claims 17 to 19), and iii. a xylulokinase (XK) (optionally as defined in any one of claims 21 to 23); and(b) at least one nucleotide sequence for the production of a first molecule of interest (MOI), wherein at least one of the at least one nucleotide sequence is heterologous to the bacterium or recipient bacterium (optionally wherein the MOI is as defined in any one of claims 36 to 47); and(c) a further nucleotide sequence encoding an ABC transporter which is capable of importing xylitol into the bacterium or recipient bacterium (optionally from a species selected from a Morganella species, for example from Morganella morganii, and further optionally as defined in claim 34).

36. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 8 or 10 to 35, wherein the at least one nucleotide sequence of (b) encodes the first MOI.

37. The bacterium, recipient bacterium, transmissible element or use according to claim 36, wherein the first MOI is a therapeutic molecule, such as a peptide molecule (e.g. an antibody fragment, a hormone such as GLP-1, an interleukin, a cytokine, a chemokine, an eukaryotic growth factor, or an enzyme) or a small molecule (e.g. a metabolite such as L-DOPA, indole-3-acetic acid, butyrate etc).

38. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 2 to 4 or 6 to 37, wherein the bacterium, recipient bacterium or transmissible element further comprises:(d) a further nucleotide sequence encoding an exporter which is capable of exporting the first MOI out of the bacterium or recipient bacterium.

39. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 2 to 4 or 6 to 38, wherein the bacterium, recipient bacterium or transmissible element further comprises:(e) a further nucleotide sequence encoding a signal peptide for the secretion of the first MOI to the periplasm of the bacterium or recipient bacterium; to the cell surface of the bacterium or recipient bacterium; or to the extracellular space outside of the bacterium or recipient bacterium.

40. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 2 to 4 or 6 to 39, wherein the bacterium, recipient bacterium or transmissible element comprises a nucleotide sequence which encodes, in 5' to 3' direction, a promoter, a sequence encoding a signal peptide, at least one nucleotide sequence for the expression of the first MOI and optionally a further nucleotide sequence encoding one or more (e.g. one) exporter(s) of the first MOI from the bacterium or recipient bacterium.

41. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 2 to 4 or 6 to 35, wherein the at least one nucleotide sequence of (b) encodes one or more protein(s) for the conversion of a second MOI to the first MOI.

42. The bacterium, recipient bacterium, transmissible element or use according to claim 41, wherein the at least one nucleotide sequence of (b) encoding one or more protein(s) is comprised by one or more operons (e.g. by one operon).

43. The bacterium, recipient bacterium, transmissible element or use according to claim 42, wherein the one or more operons are each under the control of a constitutive promoter.

44. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 41 to 43, wherein the first MOI is selected from a therapeutic molecule (e.g. a peptide molecule) and a beneficial or therapeutic metabolite, in particular a beneficial or therapeutic metabolite, or the second MOI is a detrimental metabolite.

45. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 41 to 44, wherein the bacterium, recipient bacterium or transmissible element further comprises:(d) a further nucleotide sequence encoding an exporter which is capable of exporting the first MOI out of the bacterium or recipient bacterium;or wherein the at least one nucleotide sequence of (b) further encodes:one or more (e.g. one) importer(s) of the second MOI, and / orone or more (e.g. one) importer(s) of any further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

46. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 45, wherein the first MOI comprises; or wherein the bacterium, recipient bacterium or transmissible element further comprises (f) a further nucleotide sequence encoding; a nucleic acid modifier selected from the group consisting of:a) a base editor;b) a prime editor;c) an RNAi system;d) a CRISPRi system;e) a nuclease (e.g. a CRISPR / Cas system, a TALEN or a zinc finger); andf) a nickase.

47. The bacterium, recipient bacterium, transmissible element or use according to claim 46, wherein the nucleic acid modifier is a CRISPR / Cas nuclease or nickase (e.g. nuclease).

48. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein the precursor substrate is xylitol.

49. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein the precursor substrate is a D precursor substrate (e.g. D-xylitol), and wherein all products (e.g. xylose, xylulose and / or xylulose-5-phosphate) derived from said precursor substrate (e.g. D-xylitol) are D products (e.g. D-xylose, D-xylulose and / or D-xylulose-5-phosphate).

50. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein:i) the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) are each or all (e.g. all) under the control of a promoter (e.g. an inducible promoter, for example a native inducible promoter); and / orii) the at least one nucleotide sequence for the production of the first MOI of (b) are each or all (e.g. all) under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); andiii) optionally, the further nucleotide sequence encoding the transporter of (c) is under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); andiv) further optionally the further nucleotide sequence encoding the exporter of (d) is under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); andv) further optionally the further nucleotide sequence encoding the signal peptide of (e) is under the control of a promoter (e.g. a constitutive promoter, for example a native constitutive promoter); andvi) further optionally, the further nucleotide sequence encoding a nucleic acid modifier of (f) is under the control of a promoter (e.g. an inducible promoter).

51. The bacterium, recipient bacterium, transmissible element or use according to claim 50, wherein the promoter of any of parts i) to vi) (in particular each of parts i) and iii), or all of parts i) to vi)) is an inducible promoter, for example a native inducible promoter of the transporter of (c) and / or a native inducible promoter of any of the peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a).

52. The bacterium, recipient bacterium, transmissible element or use according to claim 51, wherein all of parts i) to vi) are under the control of a single inducible promoter.

53. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 50 to 52, wherein the inducible promoter comprises a nucleotide sequence of SEQ ID No: 13, and the promoter controls expression of repressor encoded by a nucleotide sequence of SEQ ID No: 14, or controls expression of a nucleotide sequence encoding a repressor having an amino acid sequence of SEQ ID No: 15, and wherein the nucleotide sequence encoding the repressor is downstream (3′) of the promoter sequence of SEQ ID No: 13.

54. The bacterium, recipient bacterium, transmissible element or use according to claim 50, wherein the promoter of any of parts i) to vi) (e.g. all of parts ii), iv), v) and vi)) is a constitutive promoter.

55. The bacterium, recipient bacterium, transmissible element or use according to claim 54, wherein all of parts i) to vi) are under the control of a single constitutive promoter.

56. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a) (and optionally the further nucleotide sequence of (c)) are comprised by a first plasmid.

57. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (c), (d), (e) and / or (f)) are comprised by a plasmid, for example a second plasmid when dependent on claim 56.

58. The bacterium, recipient bacterium or use according to any of claims 1 to 55 or claim 57, wherein the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a), and optionally any further nucleotide sequence of (c), are comprised by the chromosome of the bacterium.

59. The bacterium, recipient bacterium or use according to any of claims 1 to 55 or 58, wherein the at least one nucleotide sequence for the production of the first MOI of (b) (and optionally any or all of the further nucleotide sequences of (d), (e) and / or (f)) are comprised by the chromosome of the bacterium.

60. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein the bacterium or recipient bacterium is:A. capable of growing in exhausted media supplemented with xylitol, and / orB. capable of growing in M9 media.

61. The bacterium, recipient bacterium, transmissible element or use according to claim 60, wherein the cell density (CFU) of the bacterium or recipient bacterium increases at least 2-fold more after 24 hours on said media than a bacterium or recipient bacterium which does not comprise the one or more nucleotide sequences encoding peptide product(s) for the conversion of a precursor substrate (e.g. xylitol) to xylulose or xylulose-5-phosphate of (a), e.g. when the xylitol is at a concentration of 2%.

62. The bacterium, recipient bacterium, transmissible element or use according to any preceding claim, wherein the bacterium, recipient bacterium or transmissible element further comprises a kill switch.

63. The bacterium, recipient bacterium, transmissible element or use according to according to any preceding claim, wherein the bacterium or recipient bacterium is a gram negative bacterium.

64. The bacterium, recipient bacterium, transmissible element or use according to any one of claims 1 to 62, wherein the bacterium or recipient bacterium is a strain selected from any of the strains in Table 4.

65. The bacterium, recipient bacterium, transmissible element or use according to according to any one of claims 1 to 62, wherein the bacterium or recipient bacterium is an E. co / / strain, for example an E. coH ovn phylogroup A, Bl and / or E, or an E. coH strain which is present in a probioticproduct, such as colinfant New Born (e.g. strain AO 34 / 86) or symbioflor2 (e.g. strain Gl / 2, G4 / 9, G5, G6 / 7, and G8), or Mutaflor (e.g. E. co / / Nissle).

66. The bacterium, recipient bacterium, transmissible element or use according to according to claim 64, wherein the bacterium or recipient bacterium is a strain belonging to a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibaciiius, Lactipiantibaciiius, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus, (e.g. a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibaciiius paracasei, Lactipiantibaciiius plantarum, Levilactobacillus brevis, Ligilactobacillus saiivarius, Limosilactobacillus reuteri and Lactococcus iactis), in particular a strain belonging to a Bifidobacterium genus or a Bacteroides genus (e.g. a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron).

57. The transmissible element or use according to any preceding claim, wherein the transmissible element is a conjugative plasmid.

68. The bacterium or use according to any one of claims 1 to 66, wherein the bacterium is a recipient bacterium which comprises a transmissible element (e.g. as defined in any one of claims 1 to 4, 12 to 57 or 60 to 66) which is a conjugative plasmid.

69. The bacterium or use according to any one of claims 1 to 66, wherein the bacterium is a donor bacterium which comprises a transmissible element (e.g. as defined in any one of claims 1 to 4, 12 to 57 or 60 to 66) which is a conjugative plasmid.

70. The transmissible element or use according to any one of claims 1 to 66, wherein the transmissible element is selected from a phage or a phagemid (e.g. a phagemid which is packaged in a phage particle).

71. The bacterium or use according to any one of claims 1 to 66, wherein the bacterium is a recipient bacterium which comprises a transmissible element (e.g. as defined in any one of claimsl to 4, 12 to 57 or 60 to 66) which is a plasmid which has been introduced by a phage or packaged phagemid.

72. The bacterium or use according to any one of claims 1 to 66, wherein the bacterium is a recipient bacterium which comprises a transmissible element (e.g. as defined in any one of claims 1 to 4, 12 to 57 or 60 to 66) which is a prophage which has been introduced by a phage.

73. The bacterium or use according to any one of claims 1 to 66, wherein the bacterium is a host bacterium comprising a transmissible element (e.g. as defined in any one of claims 1 to 4, 12 to57 or 60 to 66) which is a phage or phagemid which is used for the production of said phage or of a packaged phagemid.

74. A pharmaceutical composition comprising (i) a population of bacteria as defined in any one of claims 1 to 8 or 12 to 66, (ii) a population of donor bacteria as defined in claim 69, or (iii) a plurality of transmissible elements as defined in any one of claims 1 to 4, 12 to 57, 60 to 66 or 70,and a pharmaceutically acceptable excipient or carrier.

75. A pharmaceutical composition according to claim 74, which is formulated for oral or rectal administration, preferably oral administration, for example formulated as a capsule or coated tablet.

76. A pharmaceutical composition according to claim 74 or claim 75, which is a lyophilised formulation or is an encapsulated formulation to be released in the lower gut of a subject.

77. A kit comprising:(i) a population of bacteria as defined in any one of claims 1 to 8 or 12 to 66, (ii) a population of donor bacteria as defined in claim 69, (iii) a plurality of transmissible elements as defined in any one of claims 1 to 4, 12 to 57, 60 to 66, or 70, or (iv) a pharmaceutical composition as defined in any one of claims 74 to 76; andat least one dose of xylitol for administration to a subject; andoptionally instructions for use.

78. A method of colonising a microbiome of a subject, said method comprising the steps of:I. providing xylitol (e.g. D-xylitol) for ingestion by (e.g. for oral administration to) the subject, whereby the subject ingests the xylitol;II. administering to the subject (i) a population of bacteria as defined in any one of claims 1 to 8 or 12 to 66, (ii) a population of donor bacteria as defined in claim 69, (iii) a plurality of transmissible elements as defined in any one of claims 1 to 4, 12 to 57, 60 to 66 or 70, or (iv) a pharmaceutical composition as defined in any one of claims 74 to 76.

79. A (i) population of bacteria as defined in any one of claims 1 to 8 or 12 to 66, (ii) population of donor bacteria as defined in claim 69, (iii) plurality of transmissible elements as defined in any one of claims 1 to 4, 12 to 57, 60 to 66 or 70, or (iv) the pharmaceutical composition as defined in any one of claims 74 to 76, for use in colonising a microbiome of the GI tract of a subject, the use comprising:I. providing xylitol (e.g. D-xylitol) for ingestion by (e.g. for oral administration to) the subject, whereby the subject ingests the xylitol;II. administering to the subject (i) the population of bacteria, (ii) the population of donor bacteria, (iii) the plurality of transmissible elements, or (iv) the pharmaceutical composition.

80. The method according to claim 78 or the use according to claim 79, wherein the subject is suffering from a disease or disorder, optionally which can be treated or prevented by the first MOI or by the removal of the second MOI.

81. The method according to claim 78 or claim 80, or the use according to claim 79 or claim 80, wherein the method or use comprises administering an antibiotic (e.g. a broad spectrum antibiotic) to said subject before administration of the bacteria, transmissible elements or pharmaceutical composition in step II.

82. The method according to any one of claims 78, 80 or 81, or the use according to any one of claims 79 to 81, wherein step I. is carried out before step II.

83. The method according to any one of claims 78, 80 or 81, or the use according to any one of claims 79 to 81, wherein step II. is carried out before step I.

84. The method according to any one of claims 78, or 80 to 83, or the use according to any one of claims 79 to 83, wherein the xylitol is provided in the form of a composition for oral administration.

85. The method according to any one of claims 78, or 80 to 84, or the use according to any one of claims 79 to 84, wherein the xylitol is in the form of (i) a lyophilised formulation, (ii) an encapsulated formulation to be released in the lower gut of the subject, (iii) a powder for reconstitution in water or other ingestible liquid, (iv) a solution for reconstitution in water or other ingestible liquid, or (v) a tablet formulation for oral administration.

86. The method according to any one of claims 78, or 80 to 85, or the use according to any one of claims 79 to 85, wherein the xylitol is provided in a dosage form comprising 5, 10, 15 or 20g of xylitol, optionally wherein the xylitol is provided in a sachet.

87. The method according to any one of claims 78, or 80 to 86, or the use according to any one of claims 79 to 86, wherein the xylitol is ingested (or administered) at least twice a day.

88. The method according to any one of claims 78, or 80 to 87, or the use according to any one of claims 79 to 87, wherein the bacteria, transmissible elements or pharmaceutical composition of step II. is / are administered orally.

89. The method according to any one of claims 78, or 80 to 87, or the use according to any one of claims 79 to 87, wherein the bacteria, transmissible elements or pharmaceutical composition of step II. is / are administered by faecal transplant.

90. The method according to any one of claims 78, or 80 to 89, or the use according to any one of claims 79 to 89, wherein the bacteria or recipient bacteria colonise a compartment in the GI tract selected from the ileum, cecum, colon and contents thereof, or of any combination of the foregoing, and / or selected from the jejunum and duodenum and contents thereof or any combination of the foregoing.

91. The method according to any one of claims 78, or 80 to 90, or the use according to any one of claims 79 to 90, wherein the bacteria or recipient bacteria are capable of detection in faeces at a higher CFU / g than in subjects which have not received xylitol according to step I.

92. The method according to claim 91, or the use according to claim 91, wherein the subject is a mouse, the xylitol is administered in drinking water and wherein the CFU / g is higher during ingestion (or administration) of the xylitol and / or is higher for a period of time (e.g. at least 3 days, 4 days or 5 days) after termination of ingestion (or administration) of the xylitol, as compared to mice not receiving xylitol.

93. A method of treating or preventing a disease or disorder in a subject, said method comprising carrying out the method as defined in any one of claims 78 or 80 to 92.

94. A (i) population of bacteria as defined in any one of claims 1 to 8 or 12 to 66, (ii) population of donor bacteria as defined in claim 69, (iii) plurality of transmissible elements as defined in any one of claims 1 to 4, 12 to 57, 60 to 66 or 70, or (iv) the pharmaceutical composition as defined in any one of claims 74 to 76, for use in treating or preventing a disease or disorder in a subject, said use comprising carrying out the method as defined in any one of claims 78, or 80 to 92.

95. Use of a (i) population of bacteria as defined in any one of claims 1 to 8 or 12 to 66, (ii) population of donor bacteria as defined in claim 69, (iii) plurality of transmissible elements as defined in any one of claims 1 to 4, 12 to 57, 60 to 66 or 70, or (iv) pharmaceutical composition as defined in any one of claims 74 to 76, in the manufacture of a medicament for the treatment or prevention of a disease or disorder in a subject, wherein said medicament is administered in combination (sequentially or simultaneously) with xylitol, optionally according to a method as defined in any one of claims 78, or 80 to 92.

96. The method according to claim 93, or the use according to claim 94 or claim 95, wherein the bacteria, transmissible elements or pharmaceutical composition of step II. comprise the at least one nucleotide sequence of (b) for the production of a first MOI.

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