Production and secretion of indole-3-acetic acid (IAA) in bacteria

Abstract

The present invention relates to modified bacteria and plasmids (e.g., conjugate plasmids) engineered to express indole-3-acetic acid (IAA), pharmaceutical compositions containing the same and their use in the treatment of various metabolic and cardiovascular diseases.

Description

Background Art

[0001] Metabolic disorders have emerged as a public health threat, with an estimated prevalence of approximately one quarter worldwide. Strategies and therapeutic interventions are needed to prevent metabolic disorders, particularly type 2 diabetes (T2D), obesity, and non-alcoholic fatty liver disease (NAFLD).

[0002] Gut microbiome dysbiosis has been associated with these metabolic conditions, leading to an inflammatory state due to increased intestinal epithelial permeability (Taleb, Frontiers in Immunology, 10: 2113, 2019). The intestinal epithelium is maintained by the presence of tight junctions and adherens junctions in epithelial cells and acts as a protective barrier against external substances. However, in inflammatory diseases, this barrier can become dysfunctional, leading to increased permeability and movement of microbial components such as lipopolysaccharides (LPS) into the blood. This can lead to the observed inflammation, which is implicated in the development of metabolic cardiovascular disease.

[0003] Recent evidence has linked indole-3-acetic acid (IAA), a gut microbiota-derived metabolite from dietary tryptophan, to resistance to liver damage and sebaceous gland disease in mice (Ji et al., Nutrient 11:2062, 2019; Li et al., Frontiers in Pharmacology, 12:769501, 2021), as well as improved epithelial barrier function in mice (Laurans et al., Nature Medicine, 24:1113, 2018) and piglets (Liang et al., Frontiers in Microbiology, 9:1736, 2018). In humans, reduced levels of tryptophan and IAA levels, and increased levels of kynurenine, have been observed in fecal samples from patients with obesity and T2D compared to healthy subjects, and similarly, changes in tryptophan metabolism toward more kynurenine and less IAA in humans with obesity and T2D have been observed (Laurans et al., 2018, supra; Natividad et al., Cell Metabolism, 28:737, 2018).

[0004] IAA and other tryptophan-derived compounds have previously been characterized as aryl hydrocarbon receptor (AHR) agonists, identifying a direct effect of the gut microbiome and its metabolites on gut homeostasis. In vitro data confirm that IAA induces activation of the aryl hydrocarbon receptor (AHR) in intestinal immune cells, increasing the production of anti-inflammatory interleukins such as IL-17 and IL-22, which are important for antimicrobial immunity and mucosal barrier integrity (Laurans et al., 2018, supra). In addition, IAA has been shown to attenuate lipogenesis in hepatocytes induced by cytokines and free fatty acids (Krishnan et al., Cell Reports, 23: 1099, 2018).

[0005] Romasi & Lee, J. Microbiol. Biotechnol., 23(12), 1726-1736, 2013, doi: http: / / dx.doi.org / 10.4014 / jmb.1308.08082 discloses IAA-producing Escherichia coli (E. coli) comprising ipdC, aspC and iad1 genes.

[0006] WO2017 / 123418 (Synlogic, Inc) discloses, inter alia, genetic circuits for inclusion in bacteria to produce indole metabolites and derivatives.

[0007] WO2021 / 242897 (Synlogic, Inc) discloses a gene cassette for producing IAA, which is operably linked to an inducible promoter.

[0008] There remains a need for efficient in situ production of IAA, for example in the gut microbiome, which has beneficial effects in vivo. Summary of the invention

[0009] The inventors have advantageously recognized that the secretion level of indole-3-acetic acid (IAA) can be increased by modifying bacteria to include various heterologous export proteins. As known in the art, the amount of specific molecules diffused to the local environment can be limited by molecular diffusion through bacterial membranes (both Gram-negative and Gram-positive bacteria). Without being bound by theory, in general, bacterial membranes are hydrophobic, so the more hydrophilic a small molecule is, the less likely it is to cross the membrane. However, certain molecules that are harmful to cells (such as toxins, bacteriocins, etc.) are more often exported together with certain molecules that provide specific extracellular functions (such as molecules related to quorum sensing, iron acquisition, etc.). In addition, some molecules are actively transported through the membrane by dedicated export proteins (for example, excessive amino acids can be exported from the cell to maintain steady state, such as alanine export protein alaE and leucine export protein leuE found in Escherichia coli), or export proteins of a certain class of molecules (for example, setA transport proteins found in Escherichia coli export various sugar molecules). However, most IAA transport proteins to date have been found in plant species. Therefore, in many bacteria, the secretion of IAA is limited by diffusion rate. The inventors have engineered bacteria to express heterologous export proteins that are capable of exporting IAA across bacterial membranes. Unexpectedly, heterologous export proteins are able to form and function within bacterial membranes. This results in increased secretion of IAA into the local environment and contributes to a reduced fitness disadvantage of IAA expression within bacteria because (without being bound by theory) IAA is moved out of the cytoplasm and / or periplasm into the local environment.

[0010] In addition, there remains a need for consistent and high-level IAA biosynthesis from bacteria, particularly for therapeutic applications. This is achieved by the present invention in several ways:

[0011] First, previous attempts to include heterologous genes for biosynthesis of IAA on plasmids have required control of inducible and / or weak promoters. This has been necessary because when constitutive promoters are used to control heterologous gene expression of IAA on plasmids, it results in plasmid instability and high levels of genetic mutations within the heterologous gene. These mutations and instabilities ultimately lead to gene inactivation and can even cause bacterial cell death. The inventors unexpectedly realized that providing heterologous genes directly into the chromosome of bacteria results in stable expression of the heterologous gene without the associated plasmid instability and deleterious genetic mutations.

[0012] Furthermore, the inventors were able to use constitutive promoters to control the expression of heterologous genes contained by the chromosome of the bacteria, which allowed for higher and more consistent IAA expression. Surprisingly, and in contrast to plasmid production, as might be expected, this higher production appeared to have little effect on the fitness of the bacteria.

[0013] The inventors have also selected specific heterologous gene combinations for IAA production and extracellular export (e.g. Figure 2 , 5 and 7), which may offer advantages over other genes in one or more of the following aspects: production stability, production level, pathway efficiency, and optimal temperature range for in vivo production.

[0014] Therefore, the present invention provides the following:

[0015] In the first configuration:

[0016] Provided are modified bacteria or plasmids (e.g., conjugative plasmids) for producing and secreting indole-3-acetic acid (IAA), wherein the bacteria comprises one or more heterologous genes for the biosynthesis of IAA, and wherein:

[0017] a. The bacterium further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium; or

[0018] b. The plasmid further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium, which comprises the plasmid and produces and secretes IAA.

[0019] In the second configuration:

[0020] Provided are modified bacteria or plasmids (e.g., conjugative plasmids) for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding:

[0021] i. indole-3-pyruvate decarboxylase (ipdC) from a Pantoea species, such as Pantoea agglomerans;

[0022] ii. Tryptophan-pyruvate aminotransferase 1 (taa1) from a species of the genus Arabidopsis, such as Arabidopsis thaliana; and

[0023] iii. indole-3-acetaldehyde dehydrogenase (iad1) from a species of the genus Ustilago, such as Ustilago maydis;

[0024] The heterologous genes (i) to (iii) are each or all under the control of one or more constitutive promoters.

[0025] In the third configuration:

[0026] Provided are host (eg, donor) cells comprising a plasmid (eg, a conjugative plasmid) as described herein.

[0027] In the fourth configuration:

[0028] Provided are pharmaceutical compositions comprising a modified bacterium as described herein, or comprising a host (donor) cell comprising a plasmid (e.g., a conjugative plasmid) as described herein, and a pharmaceutically acceptable excipient or carrier

[0029] In the fifth configuration:

[0030] Provided is a method of producing IAA in the intestine of a subject, comprising administering to the subject a modified bacterium as described herein, a host (donor) cell as described herein, or a pharmaceutical composition as described herein.

[0031] In the sixth configuration:

[0032] Provided are methods of treating metabolic diseases, such as cardiovascular metabolic diseases, comprising administering to a subject in need thereof a modified bacterium as described herein, a host (donor) cell as described herein, or a pharmaceutical composition as described herein.

[0033] In the seventh configuration:

[0034] Provided is a modified bacterium as described herein, a host (donor) cell as described herein, or a pharmaceutical composition as described herein for use as a medicament.

[0035] In the eighth configuration:

[0036] Provided is a method of producing a modified bacterium as described herein, wherein the heterologous gene is contained by the chromosome of the bacterium, the method comprising using recombineering to introduce the heterologous gene into the chromosome of the bacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : A schematic diagram of the biosynthetic pathway for converting indole to indole-3-acetic acid (IAA) is shown. Note that in the absence of specific genes for converting IAA to IBA, it is believed (without being bound by theory) that it is an equilibrium reaction that spontaneously converts IAA to IBA.

[0038] Figure 2: Layout of the IAA biosynthetic pathway. Plasmid p1915 contains ipdC, aspC, iad1 and tnaB under the control of the cI-based temperature-sensitive promoter Pts. Plasmid p1138 contains the purple pigment amilCP under the control of the same Pts. Both plasmids carry the chloramphenicol resistance gene and use the p15A origin.

[0039] Figure 3 : In vitro IAA production using b5674 (E. coli MG1655 containing p1915). IAA production in bacterial supernatants was measured by Salkowski assay after overnight culture in M9 medium containing 1 mM tryptophan. Strain b5674 contained the p1915 plasmid engineered to produce IAA, while strain b5673 contained a control plasmid lacking the IAA pathway.

[0040] Figure 4 : Stability of pathway after in vivo passaging. Production of IAA was quantified in all colonies recovered from fecal samples from the active group (b5674) up to 54 hours post-inoculation after a single inoculation.

[0041] Figure 5 : Layout of plasmids with temperature-induced IAA biosynthetic pathway and export proteins. Plasmids p1880, p1881 and p1882 contain ipdC, aspC and iad1 under the control of the temperature-sensitive promoter Pts based on cI. All plasmids carry the chloramphenicol resistance gene and use the p15A origin. In addition, plasmids p1881 and 1882 carry the export proteins pin2 and aec expressed by the promoter driving the expression of the resistance marker, respectively.

[0042] Figure 6 : In vitro IAA production with and without exportin. IAA production in bacterial supernatants was measured by Salkowski assay after overnight culture in M9 medium containing 5 mM tryptophan. Strains b5626, b5627, and b5629 contain plasmids p1880, p1881, and p1882, respectively, carrying IAA pathways without exportin, with exportin pin2, and with exportin aec, respectively (Pts-ipdC-aspC-iad1).

[0043] Figure 7; The layout of the constructs of the IAA biosynthetic pathway and output protein containing constitutive expression. All plasmids contain chloramphenicol resistance markers and cloDF-based starting points with copy numbers of 10-20. Plasmid p2526 contains ipdC, aspC and iad1 under the control of the promoter Pc-aga. Plasmid p2527 additionally contains the aec output protein expressed by the promoter driving the expression of the resistance marker. Plasmids p2528 and p2529 are similar to the above, wherein taa1 replaces aspC. The IAA pathway inserted into the chromosome of b6131 contains ipdC, taa1, iad1 and aec under the control of the promoter Pc-tga.

[0044] Figure 8 : Pathway optimization with taa1 and aec leads to improved IAA secretion. IAA production in bacterial supernatants was measured by Salkowski assay after overnight cultivation in M9 medium containing 1 mM tryptophan. The modified plasmids were integrated into the same backbone strains carrying either of the two aminotransferases (aspC / taa1) with and without the aec exporter. Strains b7196 (aspC); b7197 (aspC, aec); b7198 (taa1); b7199 (taal, aec)

[0045] Fig. 9 : Optimize the chromosomal integration of the pathway. After overnight culture in M9 medium containing 1mM tryptophan, IAA production in bacterial supernatant was measured by Salkowski assay. Comparison of bacterial strain b6131 (symbioflor G6 / 7ΔtnaCA, chromosome Ptga-ipdC-taa1-iad1-aec in ΔtrpR) and bacterial strain b7199 (symbioflor G6 / 7ΔtnaCA, ΔtrpR, containing plasmid p1882 with Paga-ipdC-taa1-iad1, aec) production between. Bacterial strain b6570 (symbioflor G6 / 7ΔtnaCA, ΔtrpR) serves as a negative control. There is no significant difference between the IAA production levels between bacterial strains b7199 and b6131.

[0046] Fig.10 : Pathway stability after passage in vivo. Until 7 days after inoculation (Day 8), the production of IAA was quantified in all colonies recovered from fecal samples from the active group (strain b6131). After overnight cultivation in M9 medium containing 1 mM tryptophan, IAA production in bacterial supernatants was measured by Salkowski assay. The production capacity remained at the level of the b6131 strain, which was not administered in vivo and can be used as a control.

[0047] Fig.11 : IAA production relative to growth. IAA production in bacterial supernatants was measured by Salkowski assay after overnight cultivation in M9 medium containing 1 mM tryptophan. IAA production remained stable at both 30°C and 37°C. Strain b6570 was included as a control.

[0048] Figure 12: Results of LC-MS analysis. The substances quantified are tryptophan (TRP), indole-3-acetic acid (IAA), indole (IND) and indole-3-ethanol (HEI). A) and B) The performance of different chromosomal deletions was evaluated in the presence and absence of a plasmid carrying the IAA production pathway. C) The pathway was integrated into the chromosome of a double mutant strain (b6570, ΔtrpRΔtnaCA) and the resulting strain (b6131, ΔtrpRΔtnaCA IAA via csiR) was studied in the presence of different substrate combinations.

[0049] Fig.13 : Quantification of IAA in supernatants of ON cultures grown in M9 medium with 1 mM tryptophan. b6570 (no IAA) served as a negative control, while b6131 (pathway at csiR) served as a benchmark. Strains b7848 (pathway at aslA) and b7829 (pathway at uspG) produced IAA at levels comparable to b6131.

[0050] Fig.14 : Quantification of IAA in supernatants of ON cultures grown anaerobically in M9 medium with 1 mM tryptophan. b6570 (no IAA pathway) served as negative control, while b6131 (pathway at csiR) served as benchmark. Unpaired two-tailed t-test comparing the two groups yielded p-values ​​of 1.2-10.

[0051] Fig.15 : Determination of CFU in faeces showing the levels for individual animals in Groups 5 and 6 and further with the geometric mean indicated (black line).

[0052] Fig.16 : IAA detected in nmol / g feces by LC-MS for Groups 5 and 6 that have shown stable colonization.

[0053] Fig.17 : Tryptophan detected by LC-MS in nmol / g feces for groups 5 and 6 which have shown stable colonization. At 24 hours, all analyses have shown levels below the detection limit in group 5 animals.

[0054] Fig.18: Indole detected by LC-MS in nmol / g feces for Groups 5 and 6 that have shown stable colonization.

[0055] Fig.19 : Schematic representation of the modifications introduced into the conjugative β10 plasmid. The inserted DNA contains the three genes necessary for the production of IAA from tryptophan (ipdC, taa1 and iad1) and the transporter aec that moves IAA outside the cell. In addition, a chloramphenicol resistance marker was added for selection.

[0056] Fig. 20 : IAA produced after overnight aerobic growth in M9 supplemented with 1 mM tryptophan. The IAA pathway was integrated into a conjugative vector (CGV) and conjugated into symbioflor G6 / 7 (SBF) wild-type E. coli and E. hormaechei strains. The level of IAA produced by the strain carrying CGV was as high as that observed by SBF, wild-type E. coli and E. hormaechei, because it was from an "SBF lead candidate" that contained the pathway in its chromosome.

[0057] Fig.21 : CFU counts (CFU / g) in feces during the in vivo study. The day 1 sample was obtained before the first dose, while the day 30 sample (including days 29 and 30) was obtained just before the termination of the mice. The data revealed that at day 3, approximately 2×10 9 CFU / g feces, and showed a slow decline to 4×10 5 CFU / g feces and appeared to remain stable around this level for the remainder of the study.

[0058] Fig. 22 : IAA (nmol / g) quantification in the feces of mice. Day 1 shows the level in mice before dosing begins. The first time point on day 3 shows an average value of 26 nmol / g feces, which drops to about 5.5 nmol / g feces on day 10 and drops to 2.5 nmol / g feces on the day of termination.

[0059] Fig.23 : Bar graphs show the total number of recipients (R) and transconjugants (T) after conjugation of plasmids p2464 (β10, control) and p2806 (β10 with IAA pathway) from b8524 (donor) to b5652 (recipient E. coli).

[0060] Fig.24: Shows CFU levels in feces. Pre-sampling was performed on day 1 and followed by a single oral dose on day 1. Animals were given prebiotics in drinking water until day 11. Prebiotics did not affect colonization of strain b8344 as colonization remained relatively stable after removal. Shows colonization stabilized at approximately 1×10 7 CFU / g feces.

[0061] Fig.25 : Shows fecal levels of IAA measured by LCMS before a single oral dose on day 1, and subsequently after doses on days 4, 11, and 16. The mean of the pre-sample was less than 1 nmol / g feces, and then IAA increased on day 4 and reached a peak of 54 nmol / g feces on day 11. On day 16, fecal samples contained a mean of 42 nmol / g feces. DETAILED DESCRIPTION

[0062] definition

[0063] As used herein, "auxotrophic" describes a mutation in a bacterium, or a bacterium that contains a plasmid as described herein and produces and secretes IAA (i.e., a recipient bacterium when the plasmid is a conjugative plasmid), that results in the bacterium or the bacterium that contains the plasmid (i.e., results in the recipient bacterium) being unable to produce a compound required for its growth. Thus, auxotrophic bacteria and auxotrophic bacteria that contain a plasmid as described herein and produce and secrete IAA (i.e., auxotrophic recipient bacterium when the plasmid is a conjugative plasmid) require additional nutrients for cell proliferation and ultimately survival compared to unmutated bacteria or unmutated bacteria that contain a plasmid as described herein and produce and secrete IAA (i.e., unmutated recipient bacterium when the plasmid is a conjugative plasmid).

[0064] As used herein, "auxin" refers to a group of naturally occurring plant hormones that regulate growth, particularly by stimulating cell elongation in the stem. Auxins play a role in cell division and differentiation, fruit development, root formation from cuttings, inhibition of lateral branching (apical dominance), and defoliation (abscission). The most important naturally occurring auxin is indole-3-acetic acid (IAA), which is formed by the breakdown of the amino acid tryptophan or glycosides. Other naturally occurring auxins include 4-chloroindole-3-acetic acid (4-Cl-IAA), 2-phenyllactic acid (PAA), indole-3-butyric acid (IBA), and indole-3-propionic acid (IPA).

[0065] As used herein, "conjugative plasmid" is such a plasmid, when contained in a bacterial cell ("host" or "donor" cell used interchangeably herein), the plasmid can be transferred to another bacterium ("recipient" cell) by a bacterial conjugation mechanism. Bacterial conjugation is the unidirectional and horizontal transmission of genetic information from one bacterium to another bacterium. Conjugative plasmids are generally divided into two categories: mobile plasmids and autonomous transfer plasmids. Mobile plasmids at least include other genetic information transferred to the recipient cell on the transfer origin (oriT), relaxase and plasmids, and need to be provided by, for example, a second plasmid or the chromosome of the donor cell. The auxiliary function provided by the chromosome of the donor cell realizes plasmid transfer. On the other hand, in addition to the genetic information transferred to the recipient cell on the plasmid, the autonomous transfer plasmid also contains all the molecular mechanisms required for autonomous transfer on the same plasmid (for example, for the initiation of pili formation and gene transfer). Both mobile plasmids and autonomous transfer plasmids include a relaxase gene, which recognizes the transfer origin (oriT), and catalyzes the initial cutting of oriT in the donor to produce a DNA chain from the plasmid to be transferred, and the final connection of the transfer DNA of the reconstructed conjugative plasmid in the recipient cell. Thus, in one embodiment, the conjugative plasmid is a mobile plasmid. In another embodiment, the conjugative plasmid is an autonomous transfer plasmid. In another embodiment, the conjugative plasmid comprises 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.

[0066] Bacterial cells with conjugative autonomous transfer plasmids contain surface structures (pili) encoded by the conjugation mechanism on the plasmid, which involves the connection of donor cells and recipient cells and the transfer of genetic information contained in 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 effective. For example, the F plasmid of Escherichia coli, the pCF10 plasmid of Enterococcus faecalis and the pXO16 plasmid of Bacillus thuringiensis use different mechanisms to establish mating pairs, and the size of mating aggregates is different, and they have different host ranges in Gram-negative bacteria (F) and Gram-positive bacteria (pCF10 and pXO16). Their plasmid sizes are also different; 54, 100 and 200 kb respectively. However, it is worth noting that despite the differences in origin and size, these conjugation systems can maintain effective conjugation transfer in liquid culture medium. The conjugation process allows the protection of plasmid DNA from environmental nucleases, and therefore effective delivery of plasmid DNA into recipient cells can be obtained. The conjugation function is encoded by natural plasmids. Numerous conjugative plasmids (and transposons) are known, which can transfer related genes within a species (narrow host range) or between multiple species (wide host range). Transmissible plasmids widely span the bacterial field, and similar systems for horizontal gene transfer by pilus structure have recently been described for archaea. The modified conjugative plasmid is described in more detail in WO2021 / 037732 (SNIPR BiomeApS), which is incorporated herein in its entirety. The characteristics of such conjugative plasmids and bacterial cells comprising them as described in the claims submitted in WO2021 / 037732 are also incorporated herein by reference.

[0067] "Constitutive promoter" refers to a promoter capable of promoting 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.

[0068] As used herein, "export protein" refers to a membrane-integrated protein that is capable of exporting IAA outside the bacterial cell. Specific export proteins used in the present disclosure are described elsewhere herein. As discussed elsewhere herein, in some bacteria, there is no endogenous export protein for IAA, and therefore the cell must rely on the diffusion of IAA to remove IAA from the cell. Without being bound by theory, IAA export in Gram-negative bacteria may be particularly problematic due to the presence of both the inner and outer membranes. Therefore, the addition of heterologous export proteins may provide further benefits in this type of bacterial cell. The ability of any given protein to act as an export protein can be easily measured by those skilled in the art, and methods are provided in the examples below. The identification of putative export proteins can be achieved by literature searches and by using protein and genetic databases such as GenBank that are well known to those skilled in the art.

[0069] "Heterologous" refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is introduced exogenously into a given cell. A heterologous gene includes a natural gene or a fragment thereof that has been introduced into a cell. For example, a heterologous gene may include a natural coding sequence that is part of a chimeric gene to include a natural coding sequence that is part of a chimeric gene to include a non-natural regulatory region that is reintroduced into the cell. A heterologous gene may also include a natural gene or a fragment thereof that is introduced into a non-natural cell. Thus, a heterologous gene may be foreign or natural to a recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses a non-natural amount of nucleic acid and / or a polypeptide that it encodes; and / or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. For a plasmid, such as a conjugative plasmid, a heterologous gene is a gene that has been modified to be included on such a plasmid (e.g., a conjugative plasmid).

[0070] "Inducible promoter" refers to a promoter of a coding sequence or gene whose transcription is under its control and / or to which it is operably linked in the presence of an inducer of the promoter. The inducer may be one or more environmental conditions and / or one or more inducing molecules.

[0071] A "kill switch" refers to a biocontainment system that is included in a bacterium or plasmid (e.g., a conjugative plasmid) and is designed to destroy the bacterium, or in the case of a plasmid (e.g., a conjugative plasmid), the plasmid alone or the plasmid and the bacteria containing the plasmid together, when no longer contained within its desired environment (e.g., within a microbiome within a subject, such as the intestinal microbiome). Such means are well known in the art and are controllable, for example, by the addition of non-naturally occurring substances (e.g., synthetic amino acids), temperature, and the like.

[0072] As used herein, "microbiome" refers to the whole of microorganisms in a particular environment, such as in / on an organism, in a marine environment (e.g., the sea), and / or in a terrestrial environment (e.g., soil). In some embodiments, a microbiome may refer to the whole of microorganisms located or stably maintained, for example, on the surface and deep layers of the skin, in the saliva and oral mucosa, conjunctiva, and gastrointestinal tract of an organism. A microbiome may be present in any organ described elsewhere herein.

[0073] "Prevention" as used herein with respect to the transcription or expression of a gene or protein refers to the complete elimination of transcription or expression.

[0074] As used herein, "reduction" in relation to the transcription or expression of a gene or protein refers to a reduction in gene transcription or expression, e.g., a reduction of at least 50% in transcription or expression. In one embodiment, the reduction is a reduction of at least 60%, at least 70%, at least 80%. In one embodiment, the reduction is a reduction of at least 85%. In one embodiment, the reduction is a reduction of at least 90%. In one embodiment, the reduction is a reduction of at least 95%. In one embodiment, the reduction is a reduction of at least 97%. In one embodiment, the reduction is a reduction of at least 99%. In one embodiment, the reduction is a reduction of 100%.

[0075] Genes for the biosynthesis of IAA

[0076] The genes (including production of IAA, heterologous export proteins, any kill switch genes) inserted into the modified bacteria and plasmids (e.g., conjugative plasmids) of the present invention can be from a variety of sources, such as plants, mammals, bacteria. In one embodiment, one or more heterologous genes (including production of IAA, heterologous export proteins, any kill switch genes) are from prokaryotic origin. In one embodiment, one or more heterologous genes (including production of IAA, heterologous export proteins, any kill switch genes) are from bacterial origin in which such genes are present (e.g., from any bacterial strain described elsewhere herein, such as any strain listed in Table 3).

[0077] Specific sources of heterologous genes for IAA production and secretion used in the various pathways described herein are described below.

[0078] As will be apparent to those skilled in the art, the following list may not cover all known genes that catalyze the reaction. Further genes can be identified, for example, by literature search, using known databases (e.g., GenBank, Kegg, EMBL or protein databases such as Uniprot, etc.), by keyword searches, putative annotations and / or sequence homology to known enzymes (proteins and / or DNA sequences). A list of databases can be found here: https: / / en.wikipedia.org / wiki / List_of_biological_databases.

[0079] Genes for converting indole to tryptophan include, but are not limited to, trpB. The tryptophan synthase beta subunit (trpB) may be from Escherichia, such as Escherichia coli. TrpB includes paralogs TrpB1 and TrpB2.

[0080] Genes for converting tryptophan to tryptamine include, but are not limited to, tdc.Tryptophan decarboxylase (tdc) may be from Catharanthus, such as Catharanthus roseus, or Clostridium, such as Clostridium sporogenes.

[0081] Genes for converting tryptamine to IAA1d include, but are not limited to, tynA. Monoamine oxidase (tynA) may be derived from Escherichia, such as Escherichia coli.

[0082] Genes for converting IAA1d to IAA include, but are not limited to, iad1 and aao1. Indole-3-acetaldehyde dehydrogenase (iad1) may be from Ustilago, such as Ustilago maydis. Indole-3-acetaldehyde oxidase (aao1) may be from Arabidopsis, such as Arabidopsis. In one embodiment, iad1 comprises the nucleotide sequence of Seq ID No: 42. In one embodiment, the gene has at least 80% identity (e.g., 85%, 90% or 95%, particularly 90% identity) to Seq ID No: 42, and converts IAA1d to IAA.

[0083] Genes for converting tryptophan to IPyA include, but are not limited to, staO, aro9, aspC, taa1, and trpDH. L-tryptophan oxidase (staO) can be from Streptomyces, such as Streptomyces species TP-A0274. L-tryptophan aminotransferase (aro9) can be from S. cerevisiae. Aspartate aminotransferase (aspC) can be from Escherichia coli. L-tryptophan-pyruvate aminotransferase (taa1) can be from Arabidopsis, such as Arabidopsis. Tryptophan dehydrogenase (trpDH) can be from Nostoc, such as a strain of Nostoc punctiforme, such as Nostoc punctiforme NIES-2108. In one embodiment, aspC comprises the nucleotide sequence of Seq ID No: 39. In one embodiment, the gene has at least 80% identity (e.g., 85%, 90% or 95%, particularly 90% identity) to Seq ID No: 39 and converts tryptophan to IPyA. In one embodiment, taa1 comprises the nucleotide sequence of Seq ID No: 40. In one embodiment, the gene has at least 80% identity (e.g., 85%, 90% or 95%, particularly 90% identity) to Seq ID No: 40 and converts tryptophan to IPyA.

[0084] trpDH may be disadvantaged due to its reported instability, see Matsui et al., J. Biotechnol., 196-197:27-32, 2015, doi: 10.1016 / j.jbiotec.2015.01.010, which is incorporated herein in its entirety. Even at low temperatures (4°C), trpDH (EC 1.4.1.19) showed >80% reduction in activity after 24 hours. Depending on the desired application, aspC may be less attractive due to its range of substrates / catalytic reactions. aspC has been identified as a multifunctional enzyme that catalyzes the synthesis of aspartate, phenylalanine, tyrosine, and other compounds via transamination reactions. In contrast, while taa1 can catalyze reactions with tryptophan, phenylalanine, and tyrosine, taa1 has a much higher affinity for tryptophan than its other substrates and is therefore likely to provide higher levels of tryptophan conversion to IPyA than aspC.

[0085] Genes for converting IPyA to IAAld include, but are not limited to, IpdC. Indole-3-pyruvate decarboxylase (IpdC) may be from Enterobacter, such as Enterobacter cloacae, or Pantoea, such as Pantoea agglomerans, particularly Pantoea agglomerans. In one embodiment, IpdC comprises the nucleotide sequence of Seq ID No: 41. In one embodiment, the gene has at least 80% identity (e.g., 85%, 90% or 95%, particularly 90% identity) to Seq ID No: 41, and converts IPyA to IAAld.

[0086] Genes for converting IPyA to IAA include, but are not limited to, YUC genes. The indole-3-pyruvate monooxygenase (YUC) gene can be from Arabidopsis, such as Arabidopsis thaliana. The YUC gene can also be from Escherichia, such as Escherichia coli, such as YUC2 and YUC6.

[0087] Genes for converting tryptophan to IAM include, but are not limited to, iaaM. Tryptophan 2-monooxygenase (iaaM) may be from Pseudomonas, such as Pseudomonas savastanoi.

[0088] Genes for converting IAM to IAA include, but are not limited to, iaaH. Indoleacetamide hydrolase (iaaH) may be from Pseudomonas, such as Pseudomonas sassafras.

[0089] Genes for converting tryptophan to IAOx include, but are not limited to, CYP79B2 and CYP79B3. Tryptophan N-monooxygenase (CYP79B2) can be from Arabidopsis, such as Arabidopsis thaliana. Tryptophan N-monooxygenase (CYP79B3) can be from Arabidopsis, such as Arabidopsis thaliana.

[0090] Genes for converting IAOx to IAN include, but are not limited to, CYP7 1A13. Indoleacetaldehyde oxime dehydratase (CYP71A13) may be from Arabidopsis, such as Arabidopsis thaliana. The enzyme is described in more detail in Nafisi et al., The Plant Cell, 19(6), 2039-2052, 2007, which is incorporated herein in its entirety.

[0091] Genes for converting IAN to IAA include, but are not limited to, nit1, nit2, and nit3. The nitrilase may be from Arabidopsis, such as Arabidopsis thaliana.

[0092] Genes for converting IAN to IAM include, but are not limited to, nthAB. Nitrile hydratase (nthAB) can be from Arabidopsis, such as Arabidopsis thaliana. nthAB can be from a Pseudomonas strain, such as Pseudomonas strain UW4. The enzyme is described in more detail in Duca et al., Applied and Environmental Microbiolog, 80(15), 2014, doi: 10.1128 / AEM.00649-14

[0093] Genes for converting chorismate to anthranilate include, but are not limited to, trpE. Anthranilate synthase (trpE) may be from Escherichia, such as Escherichia coli.

[0094] Genes for converting anthranilate to N-(5-phosphoribosyl)-anthranilate include, but are not limited to, trpD. The phosphoribosyltransferase (trpD) may be from Escherichia, such as Escherichia coli.

[0095] Genes for converting N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate) include, but are not limited to, trpC. InGP synthase (trpC) may be derived from Escherichia, such as Escherichia coli.

[0096] Genes for converting 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate to indole-3-glycerolphosphate include, but are not limited to, trpA. Tryptophan synthase (trpA)) may be derived from Escherichia, such as Escherichia coli.

[0097] Production of Indole-3-acetic acid (IAA)

[0098] As discussed elsewhere herein, IAA is a therapeutically useful molecule, and there remains a need for efficient in situ production of IAA.

[0099] A modified bacterium for secreting IAA is provided, wherein the bacterium comprises one or more genes for the biosynthesis of IAA and further comprises a heterologous gene encoding an exporter protein capable of exporting IAA outside the bacterium.

[0100] Also provided are plasmids for secreting IAA (e.g., conjugative plasmids), wherein the plasmid comprises a heterologous gene encoding an export protein capable of exporting IAA out of the bacterium comprising the plasmid (i.e., the recipient bacterium when the plasmid is a conjugative plasmid), and the bacterium comprising the plasmid comprises one or more genes for producing IAA.

[0101] In some embodiments, the bacteria or bacteria comprising a plasmid (e.g., a conjugative plasmid) comprise one or more endogenous genes for producing IAA. In these embodiments, the addition of a heterologous gene encoding an exporter protein as described herein is expected to improve the secretion of IAA into the external environment of the bacterial cell or bacterial cell comprising a plasmid (e.g., a conjugative plasmid).

[0102] Also provided are modified bacteria for producing and secreting IAA, wherein the bacteria comprises one or more heterologous genes for the biosynthesis of IAA, and further comprises a heterologous gene encoding an exporter protein capable of exporting IAA outside the bacteria.

[0103] Also provided are plasmids for producing and secreting IAA (e.g., conjugative plasmids), wherein the plasmid comprises one or more heterologous genes for the biosynthesis of IAA, and further comprises a heterologous gene encoding an export protein, the export protein being capable of exporting the IAA outside of bacteria that contain the plasmid and produce it, and the bacteria secrete the IAA (i.e., a recipient bacterium when the plasmid is a conjugative plasmid).

[0104] In any of the embodiments described herein for modified bacteria, the bacteria used to produce and secrete IAA further comprises a modification in an endogenous tryptophanase (tnaA). The TnaA protein is responsible for converting tryptophan into indole. When active in the bacteria described herein, TnaA activity reduces the amount of tryptophan that can be used in other pathways for producing IAA (see Figure 1 ). Therefore, the inactivation of the tnaA gene will increase the amount of tryptophan available for the biosynthesis of IAA. In the examples herein (see, e.g., Example 2.1), the inventors used a deletion of tnaCA, which, in addition to the effect of increasing the amount of available tryptophan by reducing its conversion into indole, further increased the amount of tryptophan in the cell by the deletion of the tnaC leader peptide and the intergenic region found immediately upstream of tnaA. Bacterial cells may include a gene (tnaB) encoding an import protein for tryptophan located immediately downstream of tnaA, referred to as TnaB. The deletion of the leader sequence including the TnaC peptide removes the tryptophan-dependent regulation of the TnaB import protein for tryptophan, thereby further increasing the amount of tryptophan available for the biosynthesis of IAA.

[0105] Thus, in any embodiment, the bacteria used to produce and secrete IAA further comprises a modification in endogenous tryptophanase (tnaA) and / or tnaC that reduces the expression (or prevents the expression) of said tnaA and / or tnaC. In any embodiment, the modification in endogenous tnaA and / or tnaC is a deletion of one or more nucleotides in endogenous tnaA and / or tnaC that prevents or reduces (e.g., prevents) the transcription or expression of said tnaA. In another embodiment, the modification is a deletion in endogenous tnaC that is a deletion comprising a deletion of nucleotides that express a TnaC peptide at least when transcribed. In another embodiment, the deletion prevents the transcription or expression of tnaA and tnaC.

[0106] In any embodiment described herein about modified bacteria, the bacterium for producing and secreting IAA includes the modification in endogenous tryptophan transcription repressor (trpR).TrpR gene forms a homodimer combined with tryptophan when transcribed.After tryptophan combines, TrpR combines with the DNA of the promoter of trp operon, and spatially blocks RNA polymerase to combine and start transcription.Therefore, the mutation in endogenous trpR causes the transcription of trp operon to increase.The effect is triple: (i) de-represses the synthesis of chorismic acid (therefore increasing the chorismic acid amount in the cell), (ii) de-represses trpEDCA, which converts chorismic acid into indole (therefore increasing the indole amount in the cell), and (iii) de-represses tryptophan and indole to enter the intracellular mtr proton symporter (therefore increasing the tryptophan and indole amount in the cell).

[0107] Therefore, in any embodiment, the bacterium for producing and secreting IAA further comprises a modification in an endogenous tryptophan transcriptional repressor (trpR) gene that reduces the expression of the trpR (or prevents its expression). In any embodiment, the modification in endogenous trpR is the deletion of one or more nucleotides in the endogenous trpR gene, which prevents or reduces (e.g., prevents) the transcription or expression of trpR. In another embodiment, the modification of endogenous trpR causes TrpR to no longer be able to form homodimers (e.g., the modification is the deletion or mutation of one or more amino acids in the dimerization interface, which prevents the dimerization of TrpR). In another embodiment, the modification of endogenous trpR causes TrpR to no longer be able to bind to tryptophan (e.g., the modification is the deletion or mutation of one or more amino acids in the tryptophan binding site, which prevents the conformational changes in the TrpR dimer after tryptophan binding).

[0108] The crystal structure of TrpR has been solved (see, e.g., Schevitz et al., Nature, 317(6040), 782-786, 198, doi: 10.1038 / 317782a0, incorporated herein by reference in its entirety), and several studies on its dimerization and mutation effects have been published (see, e.g., Pal et al., Structure, 25, 867-877, 2017, http: / / dx.doi.org / 10.1016 / j.str.2017.04.015 and Sprenger et al., Acta Crystallogr. F. Struct. Biol. Commun., 77(Pt 7), 215-225, 2021, doi: 10.1107 / S2053230X21006142, both incorporated herein by reference in their entirety). Using this information and similar publications, one can identify potential sites for modification (eg, deletion or substitution).

[0109] In any of the embodiments herein, further genes may be included in the bacteria or plasmid (eg, conjugative plasmid) to increase the production of tryptophan.

[0110] Thus, in any embodiment, the bacterium or plasmid (eg, conjugative plasmid) of the invention comprises one or more heterologous genes for tryptophan biosynthesis from chorismate ((3R,4R)-3-[(1-carboxyvinyl)oxy]-4-hydroxycyclohexa-1,5-diene-1-carboxylic acid).

[0111] In one embodiment, the one or more heterologous genes for the biosynthesis of tryptophan comprise (or consist of): a gene for converting chorismate to anthranilate (optionally, a gene for anthranilate synthase (trpE)), a gene for converting anthranilate to N-(5-phosphoribosyl)-anthranilate (optionally, a gene for phosphoribosyltransferase (trpD)), a gene for converting N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate (optionally, a gene for (trpF)), a gene for converting 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate to indole-3-glycerolphosphate (optionally, a gene for InGP synthase (trpC)), and a gene for converting indole-3-glycerolphosphate to indole (optionally, a gene for tryptophan synthase (trpA)).

[0112] In any embodiment, the bacterium or plasmid (eg, conjugative plasmid) of the invention comprises one or more heterologous genes for tryptophan biosynthesis from anthranilate.

[0113] In one embodiment, the one or more heterologous genes for the biosynthesis of tryptophan comprise (or consist of) a gene for converting anthranilate to N-(5-phosphoribosyl)-anthranilate (optionally, a gene for phosphoribosyltransferase (trpD)), a gene for converting N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate (optionally, a gene for (trpF)), a gene for converting 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate to indole-3-glycerolphosphate (optionally, a gene for InGP synthase (trpC)), and a gene for converting indole-3-glycerolphosphate to indole (optionally, a gene for tryptophan synthase (trpA)).

[0114] In any embodiment, the bacterium or plasmid (eg, conjugative plasmid) of the invention comprises one or more heterologous genes for tryptophan biosynthesis from N-(5-phosphoribosyl)-anthranilate.

[0115] In one embodiment, the one or more heterologous genes for the biosynthesis of tryptophan comprise (or consist of): a gene for converting N-(5-phosphoribosyl)-anthranilate to 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate (optionally, it is the gene for (trpF)), a gene for converting 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate to indole-3-glycerolphosphate (optionally, it is the gene for InGP synthase (trpC)), and a gene for converting indole-3-glycerolphosphate to indole (optionally, it is the gene for tryptophan synthase (trpA)).

[0116] In any embodiment, the bacterium or plasmid (eg, conjugative plasmid) of the invention comprises one or more heterologous genes for tryptophan biosynthesis from 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate.

[0117] In one embodiment, the one or more heterologous genes for the biosynthesis of tryptophan comprise (or consist of) a gene for converting 1-(o-carboxyphenylamino-1-deoxyribulose-5-phosphate to indole-3-glycerolphosphate (optionally, it is a gene for InGP synthase (trpC)), and a gene for converting indole-3-glycerolphosphate to indole (optionally, it is a gene for tryptophan synthase (trpA)).

[0118] In any embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for tryptophan biosynthesis from indole-3-glycerophosphate.

[0119] In one embodiment, the one or more heterologous genes for tryptophan biosynthesis comprise (or consist of): a gene for converting indole-3-glycerolphosphate to indole (optionally, a gene for tryptophan synthase (trpA)).

[0120] In an alternative, in any of the embodiments described herein, the modified bacterium or plasmid (eg, a conjugative plasmid) does not comprise any heterologous genes for the biosynthesis of tryptophan.

[0121] Therefore, in any embodiment described herein, modified bacteria or plasmid (e.g., conjugative plasmid) does not include genes encoding one or more genes selected from trpA, trpB, trpC, trpD and trpE. In one embodiment, modified bacteria or plasmid (e.g., conjugative plasmid) does not include genes encoding trpA, trpC and trpD. In one embodiment, modified bacteria or plasmid (e.g., conjugative plasmid) does not include genes encoding trpA, trpC and trpE. In one embodiment, modified bacteria or plasmid (e.g., conjugative plasmid) does not include genes encoding trpC, trpD and trpE.

[0122] In any of the embodiments described herein, the modified bacterium or plasmid (eg, a conjugative plasmid) does not comprise the genes consisting of trpA, trpB, trpC, trpD, and trpE.

[0123] In any of the embodiments described herein, the modified bacterium or plasmid (eg, conjugative plasmid) does not comprise the genes consisting of trpA, trpC, trpD, and trpE.

[0124] In an alternative, in any of the embodiments described herein, the modified bacteria or plasmid (eg, conjugative plasmid) does not comprise heterologous genes other than the one or more heterologous genes for biosynthesis of IAA.

[0125] In the alternative, in any of the embodiments described herein, the modified bacteria or plasmid (eg, conjugative plasmid) does not comprise heterologous genes other than the one or more heterologous genes for biosynthesis of IAA and any kill switch genes.

[0126] In the alternative, in any of the embodiments described herein for modified bacteria, the bacteria do not comprise heterologous genes other than the one or more heterologous genes for biosynthesis of IAA and any heterologous or modified auxotrophic genes.

[0127] In the alternative, in any of the embodiments described herein for modified bacteria, the bacteria do not comprise heterologous genes other than the one or more heterologous genes for biosynthesis of IAA, any kill switch genes, and any heterologous or modified auxotrophic genes.

[0128] In an alternative, in any of the embodiments described herein, the modified bacteria or plasmid (eg, conjugative plasmid) does not comprise heterologous genes other than the one or more heterologous genes for biosynthesis of IAA and a heterologous gene encoding an exporter protein capable of exporting IAA.

[0129] In an alternative, in any of the embodiments described herein, the modified bacteria or plasmid (e.g., conjugative plasmid) does not contain any heterologous genes other than one or more heterologous genes for biosynthesis of IAA and a heterologous gene encoding an export protein capable of exporting IAA and any kill switch gene.

[0130] In an alternative, in any of the embodiments described herein for the modified bacteria, the bacteria do not comprise heterologous genes other than the one or more heterologous genes for biosynthesis of IAA and a heterologous gene encoding an exporter capable of exporting IAA and any heterologous or modified auxotrophic genes.

[0131] In the alternative, in any of the embodiments described herein for modified bacteria, the bacteria do not comprise heterologous genes other than one or more heterologous genes for biosynthesis of IAA and a heterologous gene encoding an exporter capable of exporting IAA, any kill switch genes, and any heterologous or modified auxotrophic genes.

[0132] In an alternative embodiment, in any of the embodiments described herein wherein the bacterium or a bacterium comprising a plasmid (e.g., a conjugative plasmid) comprises one or more endogenous genes for producing IAA, the modified bacterium or plasmid (e.g., a conjugative plasmid) does not comprise any heterologous genes other than the heterologous gene encoding an export protein capable of exporting IAA.

[0133] In an alternative, in any of the embodiments described herein wherein the bacterium or a bacterium comprising a plasmid (e.g., a conjugative plasmid) comprises one or more endogenous genes for producing IAA, the modified bacterium or plasmid (e.g., a conjugative plasmid) does not comprise heterologous genes other than the heterologous gene encoding the export protein capable of exporting IAA and any kill switch gene.

[0134] In the alternative, in any of the embodiments described herein for a modified bacterium comprising one or more endogenous genes for producing IAA, the bacterium does not comprise heterologous genes other than the heterologous gene encoding an exporter protein capable of exporting IAA and any heterologous or modified auxotrophic genes.

[0135] In the alternative, in any of the embodiments described herein for modified bacteria comprising one or more endogenous genes for producing IAA, the bacteria does not comprise heterologous genes other than the heterologous gene encoding an exporter capable of exporting IAA, any kill switch gene, and any heterologous or modified auxotrophic gene.

[0136] In any embodiment herein, one or more genes for the biosynthesis of IAA are each or all under the control of one or more promoters as described elsewhere herein. In one embodiment, the one or more promoters are one or more constitutive promoters (e.g., any constitutive promoter described elsewhere herein).

[0137] In any of the embodiments herein, one or more genes for the biosynthesis of IAA are contained within an operon under the control of a single promoter as described elsewhere herein. In one embodiment, the promoter is a constitutive promoter (eg, any constitutive promoter described elsewhere herein).

[0138] In any embodiment herein, the gene encoding an export protein capable of exporting IAA to the outside of the bacterium, or to the outside of the bacterium containing the plasmid and producing and secreting IAA (i.e., when the plasmid is a conjugative plasmid, it is exported to the outside of the recipient bacterium) is under the control of a promoter as described elsewhere herein. In one embodiment, the promoter is a constitutive promoter (e.g., any constitutive promoter described elsewhere herein).

[0139] In any embodiment herein, one or more genes for the biosynthesis of IAA and genes encoding export proteins capable of exporting IAA to the outside of bacteria or to the outside of bacteria containing the plasmid and producing and secreting IAA (i.e., when the plasmid is a conjugative plasmid, exported to the outside of the recipient bacteria), are each or all under the control of one or more promoters as described elsewhere herein. In one embodiment, the promoter is a constitutive promoter (e.g., any constitutive promoter described elsewhere herein).

[0140] In any embodiment herein, one or more genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA to the outside of the bacterium or to the outside of the bacterium containing the plasmid and producing and secreting IAA (i.e., when the plasmid is a conjugative plasmid, it is exported to the outside of the recipient bacteria) are all under the control of a single promoter as described elsewhere herein. In one embodiment, the promoter is a constitutive promoter (e.g., any constitutive promoter described elsewhere herein).

[0141] In any embodiment herein, one or more genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA to the outside of the bacterium or to the outside of the bacterium containing the plasmid and producing and secreting IAA (i.e., when the plasmid is a conjugative plasmid, exported to the outside of the recipient bacterium) are contained in an operon under the control of a single promoter as described elsewhere herein. In one embodiment, the promoter is a constitutive promoter (e.g., any constitutive promoter described elsewhere herein).

[0142] In any of the embodiments herein concerning modified bacteria, one or more genes for the biosynthesis of IAA are comprised by the chromosome of the bacterium.

[0143] In any of the embodiments herein concerning modified bacteria, the one or more genes for the biosynthesis of IAA are all contained by the chromosome of the bacterium.

[0144] In any of the embodiments herein concerning the modified bacteria, the gene encoding the exporter protein capable of exporting IAA is comprised by the chromosome of the bacterium.

[0145] In any of the embodiments herein concerning modified bacteria, one or more genes for the biosynthesis of IAA and a gene encoding an exporter protein capable of exporting IAA outside the bacterium are contained by the chromosome of the bacterium.

[0146] In any of the embodiments herein concerning modified bacteria, one or more genes for the biosynthesis of IAA and a gene encoding an exporter protein capable of exporting IAA outside the bacterium are comprised by an operon constituting the chromosome of the bacterium.

[0147] In any of the embodiments herein concerning modified bacteria, one or more genes for the biosynthesis of IAA and genes encoding export proteins capable of exporting IAA outside the bacteria are comprised of an operon under the control of a single promoter as described elsewhere herein, which operon constitutes the chromosome of the bacteria. In one embodiment, the promoter is a constitutive promoter (e.g., any constitutive promoter described elsewhere herein).

[0148] The position that is used for heterologous gene recombinant transformation into the chromosome of bacteria is known to those skilled in the art.For example, the gene can be placed in a desert region in the chromosome, or can replace the gene (or part of a gene or one or more nucleotides) that is deleted to provide another effect, such as providing auxotrophy.

[0149] Recent evidence has linked indole-3-acetic acid (IAA, a gut microbiota-derived metabolite and growth hormone from dietary tryptophan) to resistance to liver damage and sebaceous gland disease in mice (Ji et al., supra; Li et al., supra), and improved epithelial barrier function in mice (Laurans et al., supra) and piglets (Liang et al., supra). In humans, reduced levels of tryptophan and IAA levels, and increased levels of kynurenine in fecal samples from obese and T2D patients compared to healthy subjects have been observed, and similarly, changes in tryptophan metabolism towards more kynurenine and less IAA in humans with obesity and T2D (Laurans et al., 2018, supra; Natividad et al., supra).

[0150] In vitro data confirmed that IAA induced the activation of aryl hydrocarbon receptors (AHR) in intestinal immune cells, increasing the production of anti-inflammatory interleukins such as IL-17 and IL-22, which are important for antimicrobial immunity and mucosal barrier integrity (Laurans et al., 2018, supra). In addition, IAA has been shown to reduce lipogenesis in hepatocytes induced by cytokines and free fatty acids (Krishnan et al., supra). Ji et al., supra, showed that AHR activation by IAA alleviated high-fat diet (HFD)-induced hepatotoxicity in mice. Other indications mediated by IAA are described herein.

[0151] There are various pathways for IAA production, which are schematically shown in Figure 1 Each step of the pathway is controlled by one or more heterologous genes. Figure 1 Any gene of the pathway shown in (or a portion of the pathway if it is desired to initiate IAA production from an intermediate molecule) can be inserted into a bacterium or plasmid (eg, a conjugative plasmid) described herein to produce IAA.

[0152] A modified bacterium for producing and secreting indole-3-acetic acid (IAA) is provided, wherein the bacterium comprises one or more heterologous genes for the biosynthesis of IAA and further comprises a heterologous gene encoding an exporter protein capable of exporting IAA outside the bacterium.

[0153] Provided are plasmids (e.g., conjugative plasmids) for producing and secreting indole-3-acetic acid (IAA), wherein the plasmid comprises one or more heterologous genes for the biosynthesis of IAA, and further comprises a heterologous gene encoding an export protein capable of exporting IAA out of a bacterium that comprises the plasmid and produces and secretes IAA (i.e., when the plasmid is a conjugative plasmid, out of the recipient bacterium).

[0154] Provided are host (eg, donor) cells (as described elsewhere herein) comprising a plasmid (eg, a conjugative plasmid) for producing and secreting indole-3-acetic acid (IAA) as described herein.

[0155] In one embodiment, the bacterium or plasmid (eg, a conjugative plasmid) comprises one or more heterologous genes for the biosynthesis of IAA.

[0156] In one embodiment, the bacterium or plasmid (eg, a conjugative plasmid) comprises one or more heterologous genes for the biosynthesis of IAA from indole.

[0157] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting indole to tryptophan, a gene for converting tryptophan to tryptamine, a gene for converting tryptamine to indole-3-acetaldehyde (IAAld), and a gene for converting IAAld to IAA.

[0158] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting indole to tryptophan, a gene for converting tryptophan to indole-3-pyruvate (IPyA), a gene for converting IPyA to IAA1d, and a gene for converting IAA1d to IAA.

[0159] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting indole to tryptophan, a gene for converting tryptophan to indole-3-pyruvate (IPyA), and a gene for converting IPyA to IAA.

[0160] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of) a gene for converting indole to tryptophan, a gene for converting tryptophan to indole-3-acetamide (IAM), and a gene for converting IAM to IAA.

[0161] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of) a gene for converting indole to tryptophan, a gene for converting tryptophan to indole-3-acetaldoximine (IAOx), a gene for converting IAOx to indole-3-acetonitrile (IAN), and a gene for converting IAN to IAA.

[0162] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting indole to tryptophan, a gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx), a gene for converting IAOx to indole-3-acetonitrile (IAN), a gene for converting IAN to IAM, and a gene for converting IAM to IAA.

[0163] In one embodiment, the one or more heterologous genes for biosynthesis of IAA are such that production of IAA is comparable (eg, statistically similar, eg, within statistical error) in vitro at 30° C. and 37° C. In vitro methods are described in Example 2.5 below.

[0164] In one embodiment, the bacterium or plasmid (eg, a conjugative plasmid) comprises one or more heterologous genes for IAA biosynthesis from tryptophan.

[0165] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of) a gene for converting tryptophan to tryptamine, a gene for converting tryptamine to indole-3-acetaldehyde (IAAld), and a gene for converting IAAld to IAA.

[0166] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting tryptophan to indole-3-pyruvate (IPyA), a gene for converting IPyA to IAA1d, and a gene for converting IAA1d to IAA.

[0167] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting tryptophan to indole-3-pyruvate (IPyA) and a gene for converting IPyA to IAA.

[0168] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of) a gene for converting tryptophan to indole-3-acetamide (IAM) and a gene for converting IAM to IAA.

[0169] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx), a gene for converting IAOx to indole-3-acetonitrile (IAN), and a gene for converting IAN to IAA.

[0170] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx), a gene for converting IAOx to indole-3-acetonitrile (IAN), a gene for converting IAN to IAM, and a gene for converting IAM to IAA.

[0171] In another embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for the biosynthesis of IAA from tryptamine.

[0172] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting tryptamine to IAA1d and a gene for converting IAA1d to IAA.

[0173] In another embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for IAA biosynthesis from IAA1d.

[0174] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting IAA1d to IAA.

[0175] In another embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for IAA biosynthesis from IPyA.

[0176] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting IPyA to IAA.

[0177] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting IPyA to IAA1d and a gene for converting IAA1d to IAA.

[0178] In another embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for IAA biosynthesis from an IAM.

[0179] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting IAM to IAA.

[0180] In another embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for IAA biosynthesis from IAOx.

[0181] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting IAOx to IAN and a gene for converting IAN to IAA.

[0182] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of) a gene for converting IAOx to IAN, a gene for converting IAN to IAM, and a gene for converting IAM to IAA.

[0183] In another embodiment, the bacteria or plasmids (eg, conjugative plasmids) of the invention comprise one or more heterologous genes for IAA biosynthesis from IAN.

[0184] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of) a gene for converting IAN to IAM and a gene for converting IAM to IAA.

[0185] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise (or consist of): a gene for converting IAN to IAA.

[0186] In another embodiment, the bacterium or plasmid (eg, conjugative plasmid) may contain genes from more than one of the pathways described above.

[0187] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise:

[0188] i. indole-3-pyruvate decarboxylase (ipdC);

[0189] ii tryptophan-pyruvate aminotransferase 1 (taa1); and

[0190] iii. Indole-3-acetaldehyde dehydrogenase (iad1).

[0191] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise:

[0192] i. indole-3-pyruvate decarboxylase (ipdC) from Pantoea species;

[0193] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis thaliana; and

[0194] iii. Indole-3-acetaldehyde dehydrogenase (iad1) from Ustilago species.

[0195] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA comprise:

[0196] i. Indole-3-pyruvate decarboxylase (ipdC) from Pantoea agglomerans;

[0197] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis thaliana; and

[0198] iii. Indole-3-acetaldehyde dehydrogenase (iad1) from Ustilago maydis.

[0199] In some embodiments, the modified bacteria or plasmid (e.g., conjugative plasmid) may not contain a heterologous gene encoding an export protein capable of exporting IAA. However, in any of the following embodiments, the bacteria may further contain a heterologous gene encoding an export protein capable of exporting IAA outside the bacteria. Similarly, in any of the following embodiments, the plasmid (e.g., conjugative plasmid) may further contain a heterologous gene encoding an export protein capable of exporting IAA outside the bacteria containing the plasmid and producing and secreting IAA (i.e., when the plasmid is a conjugative plasmid, it is exported to the recipient bacteria).

[0200] Further, in any of the following embodiments, the heterologous gene for biosynthesis of IAA and / or the heterologous gene encoding an exporter capable of exporting IAA can be located in a single operon. The single operon can be under the control of a constitutive promoter.

[0201] Provided are modified bacteria or plasmids (e.g., conjugative plasmids) for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding (optionally in downstream order):

[0202] i. indole-3-pyruvate decarboxylase (ipdC) from Pantoea species such as Pantoea agglomerans;

[0203] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis species, such as Arabidopsis thaliana; and

[0204] iii. indole-3-acetaldehyde dehydrogenase (iad1) from a Ustilago species, such as Ustilago maydis;

[0205] Each or all of the heterologous genes i. to iii. are under the control of one or more constitutive promoters.

[0206] Provided are modified bacteria or plasmids (e.g., conjugative plasmids) for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding (optionally in downstream order):

[0207] i. indole-3-pyruvate decarboxylase (ipdC) from Pantoea species such as Pantoea agglomerans;

[0208] ii tryptophan-pyruvate aminotransferase 1 (taal) from Arabidopsis species, such as Arabidopsis thaliana; and

[0209] iii. indole-3-acetaldehyde dehydrogenase (iad1) from a Ustilago species, such as Ustilago maydis;

[0210] Among them, the heterologous genes i. to iii. are all under the control of a single constitutive promoter.

[0211] Provided are modified bacteria for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding (optionally in downstream order):

[0212] i. Indole-3-pyruvate decarboxylase (ipdC) from Pantoea agglomerans;

[0213] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis thaliana; and

[0214] iii. indole-3-acetaldehyde dehydrogenase (iad1) from Ustilago maydis;

[0215] Among them, the heterologous genes i. to iii. are all under the control of a single constitutive promoter.

[0216] Provided are plasmids (e.g., conjugative plasmids) for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding (optionally in downstream order):

[0217] i. Indole-3-pyruvate decarboxylase (ipdC) from Pantoea agglomerans;

[0218] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis thaliana; and

[0219] iii. indole-3-acetaldehyde dehydrogenase (iad1) from Ustilago maydis;

[0220] Among them, the heterologous genes i. to iii. are all under the control of a single constitutive promoter.

[0221] Provided are modified bacteria for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding:

[0222] i. Indole-3-pyruvate decarboxylase (ipdC) from Pantoea agglomerans;

[0223] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis thaliana; and

[0224] iii. indole-3-acetaldehyde dehydrogenase (iad1) from Ustilago maydis;

[0225] wherein the heterologous genes i. to iii. are all under the control of a single constitutive promoter, and

[0226] The bacterium further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium.

[0227] Provided are plasmids (e.g., conjugative plasmids) for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding:

[0228] i. Indole-3-pyruvate decarboxylase (ipdC) from Pantoea agglomerans;

[0229] ii tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis thaliana; and

[0230] iii. indole-3-acetaldehyde dehydrogenase (iad1) from Ustilago maydis;

[0231] wherein the heterologous genes i. to iii. are all under the control of a single constitutive promoter, and

[0232] The plasmid further comprises a heterologous gene encoding an export protein capable of exporting IAA to the outside of bacteria that contain the plasmid and produce and secrete IAA (ie, when the plasmid is a conjugative plasmid, to the outside of the recipient bacteria).

[0233] In one embodiment, the heterologous gene encoding an exporter capable of exporting IAA is contained within an operon comprising the following genes in downstream order:

[0234] i. optionally indole-3-pyruvate decarboxylase (ipdC) from a Pantoea species, such as Pantoea agglomerans;

[0235] ii, optionally tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis species, such as Arabidopsis thaliana; and

[0236] iii. optionally an indole-3-acetaldehyde dehydrogenase (iad1) from a Ustilago species, such as Ustilago maydis; and

[0237] iv. A heterologous gene encoding an IAA exporter.

[0238] Optionally wherein the operon is comprised by the chromosome of the modified bacterium.

[0239] In one embodiment, the heterologous gene encoding an exporter capable of exporting IAA is contained within an operon comprising the following genes in downstream order:

[0240] i. optionally indole-3-pyruvate decarboxylase (ipdC) from a Pantoea species, such as Pantoea agglomerans;

[0241] ii, optionally tryptophan-pyruvate aminotransferase 1 (taa1) from Arabidopsis species, such as Arabidopsis thaliana; and

[0242] iii. optionally an indole-3-acetaldehyde dehydrogenase (iad1) from a Ustilago species, such as Ustilago maydis; and

[0243] iv. a heterologous gene encoding an IAA export protein, and

[0244] Wherein the operon is contained by the chromosome of the modified bacterium.

[0245] Export protein

[0246] In plants, IAA is an amphipathic weak acid that diffuses across the cell membrane only when protonated (IAAH) and is membrane-impermeable at neutral cytosolic pH. In bacteria that have been engineered to produce IAA, previous attempts have relied on simple diffusion of IAA across the bacterial membrane, which has resulted in suboptimal levels of IAA being secreted into the local environment.

[0247] The inventors have advantageously recognized that the secretion level of IAA can be increased by modifying bacteria to include various heterologous export proteins. As known in the art, the amount of specific molecules diffused to the local environment can be limited by molecular diffusion through bacterial membranes (both Gram-negative and Gram-positive bacteria). Without being bound by theory, in general, bacterial membranes are hydrophobic, so the more hydrophilic a small molecule is, the less likely it is to cross the membrane. However, certain molecules that are harmful to cells (such as toxins, bacteriocins, etc.) are more often exported together with certain molecules that provide specific extracellular functions (such as molecules related to quorum sensing, iron acquisition, etc.). In addition, some molecules are actively transported through the membrane by dedicated export proteins (for example, excess amino acids can be exported from the cell to maintain steady state, such as alanine export protein alaE and leucine export protein leuE found in Escherichia coli), or export proteins of a certain class of molecules (for example, setA transport proteins found in Escherichia coli export various sugar molecules). However, most IAA transport proteins to date have been found in plant species. Therefore, in many bacteria, the secretion of IAA is limited by diffusion rate. The inventors have engineered bacteria to express heterologous export proteins that are capable of exporting IAA across bacterial membranes. Unexpectedly, heterologous export proteins are able to form and function within bacterial membranes. This results in increased secretion of IAA into the local environment and contributes to a reduced fitness disadvantage of IAA expression within bacteria because (without being bound by theory) IAA is moved out of the cytoplasm and / or periplasm into the local environment.

[0248] In one embodiment, the heterologous gene encoding the exporter capable of exporting IAA is from a plant species. In one embodiment, the plant species is an Arabidopsis species such as Arabidopsis thaliana. In one embodiment, the exporter is from a bacterial species. In one embodiment, the bacterial species is a Pantoea species such as Pantoea agglomerans. In one embodiment, the exporter is not capable of exporting proteins outside the bacterial cell.

[0249] In one embodiment, the exportin is not an ABC family protein transporter.

[0250] Auxin efflux proteins are a large class of molecules that are predominantly expressed in plants. These molecules promote the output of naturally occurring auxins (mainly IAA) from plant cells, rather than other transporter systems used by cells for transporting macromolecules such as peptides and proteins. In plants, auxin efflux proteins are widely divided into two categories: PIN (Pin-formed) family protein transporters and ABC (ATP-binding cassette transporter) family protein transporters. The latter class comprises many subfamilies, as described in further detail below. Despite the differences between the structures of plant cells and bacterial cells, the inventors surprisingly showed in the examples below that the PIN family protein transporters from Arabidopsis mediate the auxin efflux from bacterial cells without the need for additional plant-specific factors.

[0251] In one embodiment, the heterologous gene encoding the export protein capable of exporting IAA encodes an auxin efflux protein. In one embodiment, the auxin efflux protein is from a plant species. In one embodiment, the plant species is an Arabidopsis species such as Arabidopsis thaliana. In one embodiment, the auxin efflux protein is from a bacterial species. In one embodiment, the bacterial species is a Pantoea species such as Pantoea agglomerans. In one embodiment, the export protein is a PIN family protein transporter that is capable of exporting IAA outside the bacterial cell. In one embodiment, the export protein is an ABC family protein transporter that is capable of exporting IAA outside the bacterial cell.

[0252] Methods for determining whether any given export protein is capable of exporting IAA outside the cell are known to those skilled in the art. For example, as shown in the Examples below, bacteria producing IAA can be produced with and without the export protein of interest, and the amount of IAA in the supernatant is measured. When the export protein is capable of exporting IAA outside the bacteria, the amount of IAA visible in the supernatant increases. A test that can be used is the Salkowski colorimetric assay, as described in Example 3.3 below.

[0253] A specific protein in the bacterium Pantoea agglomerans C1 was recently discovered (see Luziatelli et al., Microorganisms, 8(2): 153, 2020, which is incorporated herein by reference in its entirety) and identified as a protein transporter of the auxin efflux carrier (AEC) family (see Luziatelli et al., Table 4 on page 9, Contig 1: 1779607-1780566, supra), with the gene name aec.

[0254] Based on predicted functionality from sequence information (e.g., based on the sequences described in Luziatelli et al., supra), GenBank predicts that there are more than 1500 "auxin efflux carrier" family protein transporters in bacteria, many of which remain to be studied and functionally confirmed. The functionality of predicted auxin efflux carrier (AEC) family protein transporters can be determined using methods described elsewhere herein. Without being bound by theory, auxin efflux carrier (AEC) family protein transporters of bacterial origin may allow for more efficient IAA export from bacteria than auxin efflux carrier (AEC) family protein transporters of other origins.

[0255] Therefore, in one embodiment, the export protein is an auxin efflux carrier (AEC) family protein transporter. In one embodiment, the export protein is an auxin efflux carrier (AEC) family protein transporter from a bacterial species. In one embodiment, the export protein is an auxin efflux carrier (AEC) family protein transporter from a Pantoea species, such as Pantoea agglomerans. In one embodiment, the export protein is an aec from a Pantoea species, such as Pantoea agglomerans. In one embodiment, the export protein comprises the nucleotide sequence of SEQ ID No: 2.

[0256] PIN family protein transporters :

[0257] The bias of auxin transporters has been attributed to a highly regulated polar-localized efflux complex characterized by the PIN-FORMED (PIN) family of protein transporters. PINs have been shown to align with auxin transporters and are essential for normal polarized organ development and auxin movement in plants.

[0258] PIN auxin efflux carriers are distantly related to some fungal transporters with 9-11 transmembrane helices, but are thought to have differentiated into discrete groups early in the evolution of vascular plants. The PIN nomenclature is derived from the PIN-FORMED inflorescence phenotype associated with the loss of PIN1, which is the main mediator of polar auxin flow function in angiosperm development. The Arabidopsis genome contains eight PIN genes, five of which encode full-length PINs (PIN1, 2, 3, 4, and 7), and three of which encode short PINs (PIN5, 6, and 8). Short PIN proteins lack the long central hydrophilic loop found in full-length PINs and are located in the inner membrane structure in which they are thought to function in the steady-state auxin compartmentalization, although the power to maintain the inner membrane auxin gradient has not yet been defined. The auxin efflux directly mediated by PIN1, 2, 4, 5, and 7 has been confirmed in multiple heterologous systems (Geisler et al., The Plant Journal, 44, 179-194, 2005; et al., Science, 312, 914-918, 2006; Blakeslee et al., Plant Cell., 19(1): 131-147, 2007, each of which is incorporated herein by reference).

[0259] Therefore, in one embodiment, the export protein is a PIN family protein transporter. In one embodiment, the export protein is a PIN family protein transporter that can export IAA to the outside of the bacterial cell. In one embodiment, the export protein is a PIN family protein transporter from a plant species. In one embodiment, the export protein is a PIN1 protein transporter, such as a plant species, such as a species of Arabidopsis, such as a PIN1 protein transporter of Arabidopsis. In one embodiment, the export protein is a PIN2 protein transporter, such as a plant species, such as a species of Arabidopsis, such as a PIN2 protein transporter of Arabidopsis. In one embodiment, the export protein is a PIN3 protein transporter, such as a plant species, such as a species of Arabidopsis, such as a PIN3 protein transporter of Arabidopsis. In one embodiment, the export protein is a PIN4 protein transporter, such as a plant species, such as a species of Arabidopsis, such as a PIN4 protein transporter of Arabidopsis. In one embodiment, the export protein is a PIN5 protein transporter, such as a plant species, such as a species of Arabidopsis, such as a PIN5 protein transporter of Arabidopsis. In one embodiment, the export protein is a PIN6 protein transporter, such as a PIN6 protein transporter from a plant species, such as a species of the genus Arabidopsis, such as Arabidopsis thaliana. In one embodiment, the export protein is a PIN7 protein transporter, such as a PIN7 protein transporter from a plant species, such as a species of the genus Arabidopsis, such as Arabidopsis thaliana. In one embodiment, the export protein is a PIN2 or PIN7 protein transporter, such as a PIN2 or PIN7 protein transporter from a plant species, such as a species of the genus Arabidopsis, such as Arabidopsis thaliana. In one embodiment, the heterologous gene encoding the export protein comprises the nucleotide sequence of Seq ID No: 3. In one embodiment, the export protein is any PIN described in Table 1 below.

[0260] ABC protein :

[0261] ATP binding cassette (ABC) transporter uses ATP to actively pump substrates across the membrane into or out of the cell for its electrochemical gradient. Therefore, ABC export proteins (in contrast to import proteins) can be used to transport IAA outside bacterial cells. ABC proteins have an ATP binding cassette, also known as a nucleotide binding domain (NBD). NBD contains several highly conserved motifs, including Walker A and Walker B sequences, ABC signature motifs, H loops and Q loops. ABC transporters also contain transmembrane domains (TMDs), which each contain several hydrophobic alpha helices. The ABC transporter core unit consists of four domains, two NBDs and two TMDs. Two NBDs bind and hydrolyze ATP together (thereby providing a driving force for transport), while TMDs participate in substrate recognition and translocation across lipid membranes.

[0262] The ABC family of protein transporters comprises a number of subfamilies which are generally classified into seven subfamilies ABCA, ABCB, ABCC, ABCD, ABCE, ABCF and ABCG, each of which comprises several sub-names, such as MDR (multidrug resistance) or PGP (P-glycoprotein).

[0263] For review of ABC family protein transporters, see Rees et al., Nature Reviews Molecular Cell Biology, 10, 218-227, 2009, doi: 10.1038 / nrm2646 and Vasiliou et al., Hum Genomics, 3(3), 281-290, 2009, doi: 10.1186 / 1479-7364-3-3-281, each of which is incorporated herein by reference in its entirety.

[0264] Subclasses of particular interest in the present disclosure are the ABCB subfamily of ABC family protein transporters, particularly those from plant origin. Plant ABCB exporters have been shown to export IAA (see Geisler et al., FEBS Letters, 580(4), 1094-102, 2006, doi: 10.1016 / j.febslet.2005.11.054, incorporated herein by reference in its entirety). The auxin transport activity of Arabidopsis, maize and sorghum ABCB1 and Arabidopsis ABCB4 and ABCB19 has been demonstrated in plants and heterologous systems. In plants, ABCB1 and ABCB19 function primarily in the maintenance of long-distance auxin transport fluxes and the movement of auxin out of apical tissues. In Arabidopsis, ABCB19 acts as a rate-limiting negative regulator of the auxin-dependent tropism response. ABCB4 appears to be an inducible auxin efflux transporter, as it mediates auxin import at very low IAA concentrations but rapidly resumes much stronger export activity as IAA concentrations increase. Thus, ABCB functions in long-distance auxin transport, loading auxin into these streams, apical dominance, root elongation, and phototropism, but its role in gravitropism and root meristem is unclear.

[0265] In one embodiment, the export protein is an ABC family protein transporter. In one embodiment, the export protein is an ABC family protein transporter capable of exporting IAA outside the bacterial cell. In one embodiment, the export protein is an ABC family protein transporter that does not export the protein outside the bacterial cell. In one embodiment, the export protein is an ABC family protein transporter that can export IAA outside the bacterial cell, but does not export the protein outside the bacterial cell. In one embodiment, the ACB family protein transporter is from a plant species. In one embodiment, the ACB family protein transporter is from an Arabidopsis species such as Arabidopsis thaliana.

[0266] Therefore, in one embodiment, the export protein is an ABCA subfamily protein transporter. In one embodiment, the export protein is an ABCA subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCA subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCA subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana. In one embodiment, the export protein is an ABCA-PGP subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCA-PGP subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCA-PGP subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana.

[0267] Therefore, in one embodiment, the export protein is an ABCB subfamily protein transporter. In one embodiment, the export protein is an ABCB subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCB subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCB subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana. In one embodiment, the export protein is an ABCB-PGP subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCB-PGP subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCB-PGP subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana.

[0268] Therefore, in one embodiment, the export protein is an ABCD subfamily protein transporter. In one embodiment, the export protein is an ABCD subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCD subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCD subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana. In one embodiment, the export protein is an ABCD-PGP subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCD-PGP subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCD-PGP subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana.

[0269] Therefore, in one embodiment, the export protein is an ABCG subfamily protein transporter. In one embodiment, the export protein is an ABCG subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCG subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCG subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana. In one embodiment, the export protein is an ABCG-PGP subfamily protein transporter capable of exporting IAA to the outside of the bacterial cell. In one embodiment, the export protein is an ABCG-PGP subfamily protein transporter from a plant species. In one embodiment, the export protein is an ABCG-PGP subfamily protein transporter from an Arabidopsis species, such as Arabidopsis thaliana.

[0270] In one embodiment, the exportin is any of the ABC family protein transporters described in Table 1 below.

[0271] Table 1: Bacterial and plant auxin efflux proteins

[0272]

[0273]

[0274] Promoter

[0275] Previous attempts to include heterologous genes for the biosynthesis of IAA on plasmids have required control of inducible and / or weak promoters. This has been necessary because when constitutive promoters are used to control heterologous gene expression of IAA on plasmids, it results in plasmid instability and high levels of genetic mutations within the heterologous gene. These mutations and instabilities ultimately lead to gene inactivation and can even lead to bacterial cell death. The inventors unexpectedly realized that providing heterologous genes directly into the chromosome of bacteria results in stable expression of heterologous genes without the associated plasmid instability and deleterious genetic mutations.

[0276] Thus, in one embodiment, the one or more heterologous genes for biosynthesis of IAA are each or all under the control of one or more constitutive promoters (eg, any of the constitutive promoters described herein).

[0277] In one embodiment, the one or more heterologous genes for the biosynthesis of IAA are contained within an operon under the control of a single constitutive promoter (eg, any of the constitutive promoters described herein).

[0278] In one embodiment, the gene encoding an export protein capable of exporting IAA out of the bacterium, or out of the bacterium that contains the plasmid and produces and secretes IAA (i.e., out of the recipient bacterium when the plasmid is a conjugative plasmid), is under the control of a constitutive promoter (e.g., any constitutive promoter described elsewhere herein).

[0279] In one embodiment, one or more heterologous genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA out of the bacterium, or out of a bacterium that contains the plasmid and produces and secretes IAA (i.e., out of a recipient bacterium when the plasmid is a conjugative plasmid), are each or all under the control of one or more constitutive promoters (e.g., any of the constitutive promoters described elsewhere herein).

[0280] In one embodiment, one or more heterologous genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA out of the bacterium, or out of a bacterium that contains the plasmid and produces and secretes IAA (i.e., out of a recipient bacterium when the plasmid is a conjugative plasmid), are all under the control of a single constitutive promoter (e.g., any of the constitutive promoters described elsewhere herein).

[0281] In one embodiment, one or more heterologous genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA out of the bacterium, or out of a bacterium that contains the plasmid and produces and secretes IAA (i.e., out of a recipient bacterium when the plasmid is a conjugative plasmid), are contained within an operon under the control of a single constitutive promoter (e.g., any of the constitutive promoters described elsewhere herein).

[0282] In one embodiment, the promoter is a constitutive promoter that results in stable production of IAA for at least 5 days (e.g., at least 6 days, at least 7 days, or at least 8 days) in vitro or in vivo. In one embodiment, stability is in vivo. Methods for determining in vivo stability are described in Example 2.4 below.

[0283] In one embodiment, the promoter is a promoter based on the sequence of the tac promoter. The tac promoter is based on a combination of promoters from the trp and lac operons, see de Boer et al., PNAS, 80(1), 21-25, 1983. doi: 10.1073 / pnas.80.1.21, which is incorporated herein in its entirety. Several tac-based promoters have been reported in the art, see, for example, Zhang et al., Microb. Cell Fact, 16: 84, 2017, doi: 10.1186 / s12934-017-0700-2, which is incorporated herein in its entirety. In one embodiment, the promoter is the Pc-tga promoter. In one embodiment, the promoter has the nucleotide sequence of Seq ID No: 1.

[0284] In another embodiment, the promoter is a constitutive promoter that does not result in mutations in one or more genes for biosynthesis of IAA and genes encoding export proteins capable of exporting IAA after at least 5 days (e.g., at least 6 days, at least 7 days, or at least 8 days) in vitro or in vivo. In one embodiment, stability is in vivo. Methods for determining in vivo stability are described in Example 2.4 below.

[0285] Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive E. coli cs promoter (e.g., osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive E. coli σ2 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive E. coli σ70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), an E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), a GlnRS promoter (BBa_K088007), a lacZ promoter (BBa_K119000; BBa_K119001), a 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), M13K07 gene BIII promoter (BBa_M13109), M13K07 gene BIII promoter (BBa_M13110), M13K07 gene BIII promoter (BBa_M13111), M13K07 gene BIII promoter (BBa_M13112), M13K07 gene BIII promoter (BBa_M13113), M13K07 gene BIII promoter (BBa_M13114), M13K07 gene BIII promoter (BBa_M13115), M13K07 gene BIII promoter (BBa_M13116), M13K07 gene BIII promoter (BBa_M13117), M13K07 gene BIII promoter (BBa_M13118), M13K07 gene BIII promoter (BBa_M13119), M13K07 gene BIII promoter (BBa_M1312 subtilis σA promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), constitutive Bacillus subtilis σB promoter (e.g., promoter ctc (BBa_K143010) or promoter gsiB (BBa_K143011)), Salmonella promoter (e.g., from Salmonella Pspv2 from Bacteria (BBa_K112706), Pspv from Salmonella (BBa_K112707)), 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 bacteriophage SP6 promoter (e.g. SP6 promoter (BBa_J64998)). ;

[0286] One method for measuring the strength of activity is by measuring the Anderson score for any given promoter. The activity of the reporter gene is measured by the relative fluorescence of the promoter when used in the control plasmid EX-Ptet-S-rbsRFP-P "RFP reporter gene" (see http: / / parts.igem.org / Part:BBa_J61002) in strain TG1 grown to saturation in LB medium. BBa_J23119 is the "consensus" promoter sequence and the strongest member of the family. The presence of NheI and AvrII restriction sites within these promoter portions makes it a scaffold for further modification. For more information, see http: / / parts.igem.org / Part:BBa_J23114.

[0287] Thus, in one embodiment, the constitutive promoter is a strong constitutive promoter (e.g., a promoter having an Anderson score (AS) of AS ≥ 0.4, such as ≥ 0.5). In another embodiment, the promoter has an Anderson score between 0.1 and 0.4 or between 0.1 and 0.5.

[0288] Table 2: Anderson promoter collection

[0289]

[0290] a: Also shown in the Anderson catalog, see:

[0291] http: / / parts.igem.org / Promoters / Catalog / Anderson

[0292] b: Intensity is the Anderson score (AS), e.g. intensity 1 is AS1. The reporter activity of the promoter is given as the relative fluorescence of the plasmid in strain TG1 grown to saturation in LB medium. A suitable plasmid is EX-Ptet-S-rbsRFP-P "RFP reporter gene" as described at http: / / parts.igem.org / Part:BBa_J61002; insertion of the promoter element between the XbaI and SpeI sites results in the RFP reporter gene.

[0293] In some embodiments, it may be desirable to include a promoter that can be induced to produce IAA only under certain conditions. For example, one or more genes for the biosynthesis of IAA can be under the control of a promoter that is active only under certain environmental conditions. For example, an inducible promoter is active under environmental conditions specific to the intestinal tract of a subject. In one embodiment, an inducible promoter is active under environmental conditions specific to the upper gastrointestinal tract of a subject (e.g., bile acids). In one embodiment, an inducible promoter is active under environmental conditions specific to the lower gastrointestinal tract of a subject (e.g., anaerobic conditions). In one embodiment, an inducible promoter is active under hypoxic or anaerobic conditions specific to the intestinal tract of a subject (e.g., upper gastrointestinal tract and / or lower gastrointestinal tract).

[0294] In another embodiment, the inducible promoter is a temperature-sensitive promoter, such as a promoter that is active at physiological temperature (e.g., about 35 to 39° C., such as about 36 to 38° C., such as about 37° C.). For a discussion of this type of 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 Stiiling et al., Mol. Cell, 68, 686-697.e683, 2017, which is incorporated herein in its entirety.

[0295] In another embodiment, the promoter is active only in the presence of certain molecules present in the local physiological environment (e.g., molecules present only in the intestine). Thus, these inducible promoters can turn the production and secretion of IAA on and off when in a desired location (e.g., the intestine). Such inducible promoters are described herein.

[0296] Other promoters of interest are promoters that have been designed to be active only when the bacterial cell is in a certain state, for example when it is "stress active". Such stress active promoters (SPA) are described in GB2303409.3, which is incorporated herein in its entirety and for its disclosure of SPA, in particular with respect to any of the promoters of SEQ ID No: 1 to 10 disclosed therein (disclosed herein as Seq ID No: 60-69) or any of claims 77 to 86 therein.

[0297] Therefore, in one embodiment, the promoter is a promoter selected from RelB, BolA, Hya, YiaG and RpoH promoters. The promoter may be a promoter selected from the following: RelB promoter sequence, σ70; BolA promoter sequence, σS, σ70; Hya promoter sequence, σS, σ70; YiaG promoter sequence, σS; RpoH promoter sequence P1, σ70; RpoH promoter sequence P2, σS; RpoH promoter sequence P3, σ24; RpoH promoter sequence P4, σ70; RpoH promoter sequence P5, σ70; and RpoH promoter sequence P6, σ54. The promoter may be a promoter having a nucleotide sequence selected from any one of Seq ID Nos: 60 to 69, or a nucleotide sequence having 90% (or 95%) homology thereto.

[0298] In one embodiment, one or more genes for biosynthesis of IAA are each or all under the control of one or more inducible promoters (eg, any of the inducible promoters described herein).

[0299] In one embodiment, one or more genes for the biosynthesis of IAA are contained within an operon under the control of a single inducible promoter (eg, any of the inducible promoters described herein).

[0300] In one embodiment, the gene encoding an export protein capable of exporting IAA to the outside of the bacterium, or to the outside of the bacterium that contains the plasmid and produces and secretes IAA (i.e., to the outside of the recipient bacterium when the plasmid is a conjugative plasmid), is under the control of an inducible promoter (e.g., any of the inducible promoters described elsewhere herein).

[0301] In one embodiment, one or more genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA out of the bacteria, or out of bacteria that contain the plasmid and produce and secrete IAA (i.e., when the plasmid is a conjugative plasmid, out of the recipient bacteria), are each or all under the control of one or more constitutive promoters (e.g., any of the constitutive promoters described elsewhere herein).

[0302] In one embodiment, one or more genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting IAA out of the bacterium, or out of a bacterium that contains the plasmid and produces and secretes the IAA (i.e., out of a recipient bacterium when the plasmid is a conjugative plasmid), are all under the control of a single inducible promoter (e.g., any of the inducible promoters described elsewhere herein).

[0303] In one embodiment, one or more genes for the biosynthesis of IAA and a gene encoding an export protein capable of exporting the IAA out of the bacterium, or out of a bacterium that contains the plasmid and produces and secretes the IAA (i.e., out of a recipient bacterium when the plasmid is a conjugative plasmid), are contained in an operon under the control of a single inducible promoter (e.g., any of the inducible promoters described elsewhere herein).

[0304] bacteria

[0305] In any of the embodiments herein, the modified bacterium, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (ie, the recipient bacterium when the plasmid is a conjugative plasmid) can be a Gram-negative bacterium.

[0306] In any of the embodiments herein, the modified bacterium, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (ie, the recipient bacterium when the plasmid is a conjugative plasmid) can be a Gram-positive bacterium.

[0307] In any of the embodiments herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (ie, the recipient bacterium when the plasmid is a conjugative plasmid) is a strain selected from any of the strains in Table 3.

[0308] In any embodiment herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (i.e., the recipient bacteria when the plasmid is a conjugative plasmid) can be an Escherichia coli strain. In one embodiment, the Escherichia coli strain is an Escherichia coli strain from phylogroup A. In one embodiment, the Escherichia coli strain is an Escherichia coli strain from phylogroup B1. In one embodiment, the Escherichia coli strain is an Escherichia coli strain from phylogroup E. In one embodiment, the Escherichia coli strain is an Escherichia coli strain present in a probiotic product. The probiotic product can be colinfant New Born (e.g., strain A034 / 86). The probiotic product can be symbiofior2 (e.g., strains G1 / 2, G4 / 9, G5, G6 / 7, and G8). The probiotic product can be Mutaflor (e.g., Escherichia coli Nissle).

[0309] In any of the embodiments herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (i.e., the recipient bacterium when the plasmid is a conjugative plasmid) can be a strain belonging to a genus selected from the group consisting of Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, and Lactococcus.

[0310] In any embodiment herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (i.e., the recipient bacteria when the plasmid is a conjugative plasmid) can be a strain belonging to a genus selected from Bifidobacterium or Bacteroides. In one embodiment, the genus is Bacteroides. In one embodiment, the genus is Bifidobacterium.

[0311] In any of the embodiments herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (i.e., the recipient bacterium when the plasmid is a conjugative plasmid) can be a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lactobacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus salivarius, Limosilactobacillus reuteri, reuteri and Lactococcus lactis.

[0312] In any of the embodiments herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (i.e., the recipient bacterium when the plasmid is a conjugative plasmid) can be a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris, and Bacteroides thetaiotaomicron.

[0313] In any of the embodiments herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (ie, the recipient bacterium when the plasmid is a conjugative plasmid) may be a strain belonging to the genus Spirulina.

[0314] In any of the embodiments herein, the modified bacteria, the bacterial host (donor) cell comprising the conjugative plasmid, or the bacterial cell comprising the plasmid (ie, the recipient bacterium when the plasmid is a conjugative plasmid) may be a strain belonging to the genus Cyanobacteria.

[0315] In one embodiment of a modified bacterium for producing and secreting IAA, the bacterium may comprise further metabolic pathways that enable the bacterium to metabolize exogenously added compounds (e.g., rare carbohydrates) and selectively grow thereon, or may only be present in a local environment (e.g., the intestinal tract, such as the lower gastrointestinal tract or the upper gastrointestinal tract). These metabolic pathways provide an adaptive advantage for the modified bacterium and help it effectively compete against other natural bacteria in the environment. This results in an increase in the relative abundance of the modified bacterium. Therefore, in one embodiment, the modified bacterium may comprise further metabolic pathways that enable the bacterium to metabolize exogenously added compounds (e.g., rare carbohydrates) and selectively grow thereon. In one embodiment, the modified bacterium may comprise further metabolic pathways that enable the bacterium to metabolize and selectively grow in the presence of compounds that are only present in a local environment (e.g., the intestinal tract, such as the lower gastrointestinal tract or the upper gastrointestinal tract).

[0316] In one embodiment of modified bacteria or donor (host) cells comprising a conjugative plasmid for producing and secreting IAA, the bacteria are capable of maintaining a stable colonization level in a mouse model for at least 5 days, optionally as measured by the number of colony forming units (CFU) in feces. In another embodiment, the bacteria are capable of maintaining a stable colonization level in a mouse model for at least 6 days, optionally as measured by the number of colony forming units (CFU) in feces. In another embodiment, the bacteria are capable of maintaining a stable colonization level in a mouse model for at least 7 days, optionally as measured by the number of colony forming units (CFU) in feces. In another embodiment, the bacteria are capable of maintaining a stable colonization level in a mouse model for at least 8 days, optionally as measured by the number of colony forming units (CFU) in feces.

[0317] The level of colonization can be measured by the CFU level in the feces. Thus, in one embodiment, the colonization level is at least (approximately) 1×10 4 In another embodiment, the colonization level is at least (approximately) 1×10 5 In another embodiment, the colonization level is at least (approximately) 1×10 6 In another embodiment, the colonization level is at least (approximately) 1×10 7 CFU / g feces. In another embodiment, the colonization level is at least (about 11×10 8 CFU / g feces.

[0318] In one embodiment of a modified bacterium or donor (host) cell comprising a conjugative plasmid for producing and secreting IAA, the bacteria are capable of producing IAA in a mouse model for at least (approximately) 24 hours, optionally by measuring IAA levels in feces. In another embodiment, the bacteria are capable of producing IAA in a mouse model for at least (approximately) 48 hours, optionally by measuring IAA levels in feces. In another embodiment, the bacteria are capable of producing IAA in a mouse model for at least (approximately) 72 hours, optionally by measuring IAA levels in feces. In another embodiment, the bacteria are capable of producing IAA in a mouse model for at least (approximately) 96 hours, optionally by measuring IAA levels in feces.

[0319] In one embodiment, the IAA level is at least (about) 10 nmol / g feces. In another embodiment, the IAA level is at least (about) 15 nmol / g feces. In another embodiment, the IAA level is at least (about) 20 nmol / g feces.

[0320] In any of these embodiments regarding colonization or IAA secretion, levels are measured in an in vivo mouse model. The mouse model can be performed as described herein for Group 6 in Example 3.2.2.

[0321] In one embodiment of a modified bacterium or donor (host) cell comprising a conjugative plasmid for producing and secreting IAA, the bacterium or bacterial cell has been engineered to remove some or all (e.g., all) prophage genes present in the genome of the bacterium or bacterial cell. The modified bacterium or bacterial cell strain may lack some or all (e.g., all) prophage genes.

[0322] In one embodiment of a modified bacterium or donor (host) cell comprising a conjugative plasmid for producing and secreting IAA, the bacterium or bacterial cell has been engineered to remove any identified virulence factor (e.g., hlyA, hlyB, hlyC and / or hlyD, or any combination thereof) present in the genome of the bacterium or bacterial cell. The bacterium or bacterial cell strain may lack a virulence factor (e.g., hlyA, hlyB, hlyC and / or hlyD, or any combination thereof).

[0323] Conjugative plasmid

[0324] A conjugative plasmid is a plasmid that, when contained within a bacterial cell (the "donor" cell), is capable of transfer to another bacterium (the "recipient" cell) via the bacterial conjugation mechanism. Bacterial conjugation is the unidirectional and horizontal transfer of genetic information from one bacterium to another. Conjugative plasmids are generally divided into two categories: mobilizable plasmids and autonomously transferred plasmids.

[0325] In any embodiment, the conjugative plasmid is capable of being transferred to a recipient bacterial cell. In any embodiment, the conjugative plasmid is transferred to a bacterial cell (i.e., a recipient cell). The recipient cell can be any bacterial cell described elsewhere herein (e.g., a Gram-negative bacterial cell).

[0326] Provided are host (donor) cells comprising a conjugative plasmid as described herein.The host (donor) cell may be any bacterial cell (eg, a Gram-negative bacterial cell) described elsewhere herein.

[0327] Movable plasmids at least comprise a transfer origin (oriT), a relaxase, and other genetic information on the plasmid that is transferred to a recipient cell. They require auxiliary functions provided by, for example, a second plasmid or the chromosome of a donor cell to achieve plasmid transfer. In one embodiment, a conjugative plasmid is a mobile plasmid. In one embodiment, a conjugative plasmid is a mobile plasmid that comprises a transfer origin (oriT) and a relaxase.

[0328] In addition to the genetic information on the plasmid that is transferred to the recipient cell, the autonomous transfer plasmid also contains all the molecular machinery required for autonomous transfer on the same plasmid (e.g., for pilus formation and initiation of gene transfer). In one embodiment, the conjugative plasmid is an autonomous transfer plasmid. In one embodiment, the conjugative plasmid is an autonomous transfer plasmid that contains all the molecular machinery required for autonomous transfer. In one embodiment, the conjugative plasmid contains oriT and encodes all the proteins required to mobilize the plasmid for conjugative transfer between cells.

[0329] The modified conjugative plasmid is described in more detail in WO2021 / 037732 (SNIPR Biome ApS), which is incorporated herein in its entirety. The characteristics of such conjugative plasmids and bacterial cells comprising them as described in the claims filed in WO2021 / 037732 are also incorporated herein by reference.

[0330] Thus, in one embodiment, the conjugative plasmid lacks the hypC2 nucleotide sequence or a homologue thereof, such as the hypC2 nucleotide sequence of SeqID No: 70. The conjugative plasmid may comprise the OriT of the IncX plasmid. The conjugative plasmid may be an IncX plasmid. The conjugative plasmid may be a β10 plasmid.

[0331] The conjugative plasmid (or any other plasmid described herein) can be a plasmid based on any plasmid found in the bacterium disclosed herein. For example, the plasmid can be an Enterobacteriaceae plasmid. In one embodiment, the plasmid is an Escherichia coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter plasmid. In one embodiment, the plasmid can be from a genus selected from: Bifidobacterium, Bacteroides, Lactobacillus, Lactobacillus casei, Plant Bacillus, Promoting Lactobacillus, Lactobacillus, Mucolytic Lactobacillus and Lactococcus. In one embodiment, the plasmid can be from a strain belonging to a genus selected from Bifidobacterium or Bacteroides. In one embodiment, the genus is Bacteroides. In one embodiment, the genus is Bifidobacterium. In one embodiment, the plasmid may be from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lactobacillus paracasei, Plantarum lactis, Lactobacillus breve, Lactobacillus salivarius, Lactobacillus reuteri and Lactococcus lactis. In one embodiment, the plasmid may be from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris and Bacteroides thetaiotaomicron. In one embodiment, the plasmid may be from a genus or species disclosed in Table 3.

[0332] In one example, the conjugative plasmid can be replicated in the bacterial cell from any bacterial genus or species described herein. For example, the conjugative plasmid can be replicated in Escherichia coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter host (donor) cells. In one embodiment, the conjugative plasmid can be replicated in the bacterial cell from the genus selected from: Bifidobacterium, Bacteroides, Lactobacillus, Lactobacillus casei, Plant Bacillus, Probiotic Lactobacillus, Lactobacillus, Mucus Lactobacillus and Lactococcus. In one embodiment, the conjugative plasmid can be replicated in the bacterial cell from the bacterial strain belonging to the genus selected from Bifidobacterium or Bacteroides. In one embodiment, the genus is Bacteroides. In one embodiment, the genus is Bifidobacterium. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lactobacillus paracasei, Plantarum lactis, Lactobacillus breve, Lactobacillus salivarius, Lactobacillus mucilaginosus, and Lactococcus lactis. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris, and Bacteroides thetaiotaomicron. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a genus or species disclosed in Table 3.

[0333] In one embodiment, the conjugative plasmid (or any other plasmid described herein) can be hosted in an Enterobacteriaceae cell. In one embodiment, the plasmid can be hosted in an Escherichia coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter cell. In one embodiment, the plasmid can be hosted in a bacterial cell from a genus selected from: Bifidobacterium, Bacteroides, Lactobacillus, Lactobacillus casei, Plant Bacillus, Promoting Lactobacillus, Lactobacillus, Mucolytic Lactobacillus and Lactococcus. In one embodiment, the plasmid can be hosted in a bacterial cell from a strain belonging to a genus selected from Bifidobacterium or Bacteroides. In one embodiment, the genus is Bacteroides. In one embodiment, the genus is Bifidobacterium. In one embodiment, the plasmid is capable of replicating in a bacterial cell from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lactobacillus paracasei, Plantarum lactis, Lactobacillus breve, Lactobacillus salivarius, Lactobacillus reuteri and Lactococcus lactis. In one embodiment, the plasmid is capable of hosting in a bacterial cell from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris and Bacteroides thetaiotaomicron. In one embodiment, the plasmid is capable of hosting in a bacterial cell from a genus or species disclosed in Table 3.

[0334] In one embodiment, the conjugative plasmid can be conjugated and transferred to an Enterobacteriaceae cell. In one embodiment, the plasmid can be conjugated and transferred to an Escherichia coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter cell. In one embodiment, the conjugative plasmid can be conjugated and transferred to a bacterial cell from the genus selected from: Bifidobacterium, Bacteroides, Lactobacillus, Lactobacillus casei, Plant Bacillus, Promoting Lactobacillus, Lactobacillus, Mucolytic Lactobacillus and Lactococcus. In one embodiment, the conjugative plasmid can be conjugated and transferred to a bacterial cell from a strain belonging to a genus selected from Bifidobacterium or Bacteroides. In one embodiment, the genus is Bacteroides. In one embodiment, the genus is Bifidobacterium. In one embodiment, the conjugative plasmid is capable of conjugative transfer to a bacterial cell from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lactobacillus paracasei, Plantarum lactis, Lactobacillus breve, Lactobacillus salivarius, Lactobacillus reuteri and Lactococcus lactis. In one embodiment, the conjugative plasmid is capable of conjugative transfer to a bacterial cell from a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorpha, Bacteroides vulgaris and Bacteroides thetaiotaomicron. In one embodiment, the conjugative plasmid is capable of conjugative transfer to a bacterial cell from a genus or species disclosed in Table 3.

[0335] In one embodiment, the conjugative plasmid is a conjugative plasmid isolated from E. coli. In another embodiment, the conjugative plasmid is a β10 conjugative plasmid from E. coli (eg, as shown in Example 4 herein).

[0336] In one embodiment, the donor (host) cell comprising a conjugative plasmid as described herein is an E. coli (e.g., symbioflor E. coli, e.g., G6 / 7) host cell (e.g., as described in Example 4 herein). In another embodiment, the donor (host) cell comprising a conjugative plasmid as described herein is an Enterobacter hallii host cell (e.g., as described in Example 4 herein).

[0337] In one embodiment, the recipient cell is an Escherichia coli recipient strain comprising a conjugative plasmid as described herein. Plasmid is introduced from donor (host) cells as described herein via conjugation (e.g., as shown in Example 4 herein). The Escherichia coli recipient strain can be included by, for example, a natural microorganism group in the intestinal tract of a subject. In another embodiment, the recipient cell is a Klebsiella recipient strain comprising a conjugative plasmid as described herein. Plasmid is introduced from donor (host) cells as described herein via conjugation (e.g., as shown in Example 4 herein). The Klebsiella recipient strain can be included by, for example, a natural microorganism group in the intestinal tract of a subject.

[0338] Alternatively, delivery of one or more heterologous genes for the biosynthesis of IAA and / or heterologous genes encoding export proteins capable of exporting IAA is delivered by phage particles. Therefore, phage particles are provided, which contain one or more heterologous genes for the biosynthesis of IAA (as described elsewhere herein) and / or heterologous genes encoding export proteins capable of exporting IAA (as described elsewhere herein) to the outside of bacteria infected by the phage. In one embodiment, the one or more heterologous genes for the biosynthesis of IAA and / or heterologous genes encoding export proteins capable of exporting IAA can be contained by a phagemid within the phage particle. In another embodiment, the one or more heterologous genes for the biosynthesis of IAA and / or heterologous genes encoding export proteins capable of exporting the IAA can be contained in the chromosome of the phage particle (e.g., in its chromosome). The phage chromosome or phagemid may further contain a kill switch as described elsewhere herein.

[0339] Kill Switch

[0340] In some embodiments, it may be desirable to include a kill switch in a bacterium or plasmid (e.g., in a conjugative plasmid). A kill switch is a biological containment system that is included in a bacterium or plasmid (e.g., a conjugative plasmid) and is designed to destroy bacteria when no longer contained in its desired environment (e.g., in a microbiome in a subject, such as an intestinal microbiome), or in the case of a plasmid (e.g., a conjugative plasmid), only the plasmid itself or the plasmid and the bacteria comprising the plasmid together. Such means are well known in the art and can be regulated, for example, by adding non-naturally occurring substances (e.g., synthetic amino acids), temperature, etc. Specific examples of promoters and kill switches (particularly for removing plasmids from bacteria) are provided in WO2023 / 012109A2 (SNIPR Biome, ApS).

[0341] Bacteria containing a kill switch have been modified for in vitro research purposes, such as to limit the spread of biofuel production microorganisms outside a laboratory environment. Bacteria modified for in vivo administration to treat diseases can also be programmed to die at a specific time after the expression and delivery of IAA, or die after the subject has experienced the therapeutic effect. For example, in some embodiments, after a period of time after the oxygen level-dependent expression of IAA, the kill switch is activated to kill bacteria, remove plasmids (e.g., conjugative plasmids) from bacteria containing plasmids, or kill bacteria containing plasmids (i.e., recipient bacteria when the plasmid is a conjugative plasmid). In some embodiments, the kill switch is activated in a delayed manner after the oxygen level-dependent expression of IAA. Alternatively, bacteria or bacteria containing plasmids (i.e., recipient bacteria when the plasmid is a conjugative plasmid) can be modified to die after the bacteria have spread outside the disease site. Specifically, it may be useful to prevent long-term colonization of a subject by bacteria or bacteria containing a plasmid (i.e., the recipient bacteria when the plasmid is a conjugative plasmid), spread of bacteria or bacteria containing a plasmid (i.e., the recipient bacteria when the plasmid is a conjugative plasmid) outside of an area of ​​interest within the subject (e.g., outside the intestinal tract), or spread of bacteria or bacteria containing a plasmid (i.e., the recipient bacteria when the plasmid is a conjugative plasmid) into the environment outside the subject (e.g., spread into the environment via the feces of the subject).

[0342] The kill switch can be designed so that the toxin is produced in response to environmental conditions or external signals (e.g., kills bacteria in response to external cues), or alternatively, is designed so that the toxin is produced once the environmental conditions are no longer present or the external signal ceases. Examples of such promoters are also described elsewhere herein. The toxin is toxic to bacteria that produce and secrete IAA.

[0343] The switch that controls toxin production can be based on, for example, transcriptional activation, translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli, such as anaerobic, reactive oxygen species, temperature, bile acids, pH, lactate, caffeine, or other biosensors. These switches can be activated by a single environmental factor, or several activators configured with AND, OR, NAND, and NOR logic may be required to induce cell death. For example, the AND riboregulator switch is activated by tetracycline, isopropyl PDI-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill cells. IPTG induces the expression of endolysin and perforin mRNA, which is then de-repressed by the addition of arabinose and tetracycline. All three inducers must be present to promote cell death.

[0344] Thus, in some embodiments, the bacterium or bacterium comprising the plasmid (i.e., the recipient bacterium when the plasmid is a conjugative plasmid) is further programmed to die or specifically degrade the plasmid (e.g., a conjugative plasmid) upon sensing an exogenous environmental signal, such as in a hypoxic environment.

[0345] The kill switch may be permissive, such that the bacterium or recipient bacteria will continue to thrive in its environment unless defined conditions change. Cell survival or proliferation may be hampered by the expression of toxins and lytic 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), cleavage and degradation of bacterial chromosomes by Cas proteins (e.g., as discussed in Caliando et al., Nat. Commun., 6, 6989, 2015; and Rottinghaus et al., Nature Comms., 13(672), 2022, each of which is incorporated herein by reference in its entirety), or degradation of essential proteins (e.g., as discussed in Chan et al., Nat. Chem. Biol., 12, 82-86, 2016, each of which is incorporated herein by reference in its entirety).

[0346] Thus, in one embodiment, the kill switch comprises a toxin gene, the expression of which is induced in response to environmental conditions and / or signals.

[0347] The expression of the toxin gene can be provided by an inducible promoter. Such promoters are well known to those skilled in the art and can include oxygen level dependent promoters (e.g., FNR [fumarate and nitrate reductase regulator] inducible promoter, ANR [anaerobic arginine deiminase and nitrate reductase regulator] inducible promoter and DNR [dissimilatory nitrate respiration regulator] inducible promoter).

[0348] An inducible promoter can be a promoter that is induced by inflammation or an inflammatory response (eg, an RNS and / or ROS inducible promoter).

[0349] The inducible promoter can be a temperature-sensitive promoter (e.g., as described elsewhere herein). Temperature-sensitive promoters include TlpA, TcI, TetR (and A89D and I193N mutants thereof), LacI (and A241T and G265D mutants thereof), GrpE, HtpG, Lon, RpoH, Clp, and DnaK (described in more detail in Piraner et al., Nature Chemical Biology, 13, 75-80, 2016, doi: 10.1038 / nchembio.2233; the reference is incorporated herein in its entirety).

[0350] In one embodiment, the kill switch comprises a toxin gene whose expression is induced by a change in temperature. In one embodiment, the kill switch comprises a toxin gene whose expression is induced by an increase in oxygen levels. In one embodiment, the kill switch comprises a toxin gene whose expression is induced by the addition of a molecule that is not normally present in the local environment, such as the gut (e.g., arabinose, sugar alcohols (e.g., sorbitol), tetracycline, IPTG, rhamnose, and non-naturally occurring amino acids).

[0351] Inducible promoters can be promoters that are induced by substances that may or may not be naturally present in the local environment (i.e., exogenously added to the local environment). Examples include, but are not limited to, arabinose inducible promoters (e.g., pBAD promoter), tetracycline inducible promoters, IPTG inducible promoters, rhamnose inducible promoters, xylitol inducible promoters, sorbitol inducible promoters, and nutrient inducible promoters.

[0352] Examples of such toxins that can be used in kill switches include, but are not limited to, bacteriocins, lysins, and other molecules that promote cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination.

[0353] In one embodiment, the toxin gene is a CRISPR / Cas system that targets and cuts the chromosome of the bacteria, or a bacterial or plasmid DNA (e.g., conjugative plasmid DNA) containing a plasmid (i.e., a recipient bacterium when the plasmid is a conjugative plasmid). In one embodiment, the toxin gene is a CRISPR / Cas system that targets and cuts essential genes in the chromosome of the bacteria or recipient bacteria or on the conjugative plasmid. Essential genes are well known to those skilled in the art. Examples include, but are not limited to, DNA synthesis genes such as thyA), cell wall synthesis genes (e.g., dapA), and amino acid synthesis genes (e.g., serA or metA).

[0354] In the case of bacteria for producing and secreting IAA, the kill switch may be contained within the chromosome of the bacteria. Alternatively, it may be on a plasmid contained within the bacteria for producing and secreting IAA.

[0355] Nutritional deficiency

[0356] In one embodiment regarding the modification of the bacterium, the bacterium may further comprise a mutation that causes the bacterium to become an auxotroph. In one embodiment, the bacterium comprises an auxotroph.

[0357] In one embodiment, the bacterium comprises an auxotrophy, such as a deletion of thyA.

[0358] In one embodiment, the bacterium is an auxotrophic type selected from cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB and thil auxotrophic types. In one embodiment, the bacterium or recipient bacteria has more than one auxotrophic type, for example, it can be ΔthyA and ΔdapA auxotrophic types.

[0359] In one embodiment, the bacteria is auxotrophic and further comprises a kill switch.

[0360] In the case of bacteria for producing and secreting IAA, the auxotrophy may be contained within the chromosome of the bacteria. Alternatively, the auxotrophy may be on a plasmid contained within the bacteria for producing and secreting IAA.

[0361] Preparations and compositions comprising bacteria, conjugative plasmids or host bacteria

[0362] Pharmaceutical compositions are provided which comprise a bacterium, a plasmid (eg, a conjugative plasmid) or a host (eg, a donor) bacterium as described herein, and a pharmaceutically acceptable excipient or carrier.

[0363] Bacteria, plasmids (e.g., conjugative plasmids), or host (e.g., donor) bacteria can be formulated in a pharmaceutical composition comprising a diluent, excipient, or carrier. The formulation can be contained in a medical device (e.g., an ampoule, a syringe, or an inhaler), or formulated as a tincture, a capsule, or a sustained release formulation. The formulation can be an oral tablet contained in a blister pack.

[0364] In one embodiment, the pharmaceutical composition comprising a bacterium, a plasmid (e.g., a conjugative plasmid), or a host (e.g., a donor) bacterium as described herein is formulated for oral or rectal administration. In one embodiment, the pharmaceutical composition is formulated for oral administration. In one embodiment, the pharmaceutical composition is formulated as a capsule or a coated tablet.

[0365] Preparations comprising bacteria, plasmids (e.g., conjugative plasmids) or host (e.g., donor) bacteria can be freeze-dried before encapsulation. Therefore, preparations can include freeze-dried modified bacteria, plasmids (e.g., conjugative plasmids) or host (e.g., donor) bacteria. In one embodiment, pharmaceutical compositions comprising bacteria as described herein, plasmids (e.g., conjugative plasmids) or host (e.g., donor) bacteria are freeze-dried preparations. In one embodiment, pharmaceutical compositions comprising bacteria as described herein, plasmids (e.g., conjugative plasmids) or host (e.g., donor) bacteria are encapsulated preparations to be released in the lower intestinal tract of a subject. In one embodiment, pharmaceutical compositions comprising bacteria as described herein, plasmids (e.g., conjugative plasmids) or host (e.g., donor) bacteria are encapsulated preparations to be released in the large intestine of a subject. In a specific embodiment, pharmaceutical compositions comprising bacteria as described herein, plasmids (e.g., conjugative plasmids) or host (e.g., donor) bacteria are encapsulated preparations to be released in the small intestine of a subject.

[0366] Acceptable carriers, excipients, or stabilizers are nontoxic to patients 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, gelatin, 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 cases, due to the stability of the carrier, the formulation may include a preservative (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). The skilled formulationist is aware of the agents that are compatible with the different modes of delivery of the bacteria, conjugative plasmids or host (e.g., donor) bacteria described herein.

[0367] Bacteria, plasmids (e.g., conjugative plasmids), or host (e.g., donor) bacteria can also be formulated in liposomes. Liposomes containing bacteria, plasmids (e.g., conjugative plasmids), or host (e.g., donor) bacteria are prepared by methods known in the art, such as Epstein et al. (1985) Proc. Natl. Acad. Sci. USA 82: 3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. USA 77: 4030; and described in 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, which is incorporated herein by reference.

[0368] Bacteria, plasmids (e.g., conjugative plasmids), or host (e.g., donor) bacteria described herein can also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.

[0369] Sustained-release preparations can also be prepared.Sustained-release preparations include semipermeable matrixes of solid hydrophobic polymers containing antagonists, and the matrix is ​​the form of formed articles such as films or microcapsules.Examples of sustained-release matrixes include polyesters, hydrogels (for example, poly-(methacrylic acid-2-hydroxyethyl ester) or poly-(vinyl alcohol)), polylactic acid (U.S. Patent number 3,773,919), copolymers of L-glutamic acid and L-glutamic acid ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM (injectable microspheres consisting of lactic acid-glycolic acid copolymers and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules exceeding 100 days, some hydrogels release proteins in shorter time periods.

[0370] The formulation may be contained in a medical device such as an ampoule, a syringe or an inhaler.

[0371] Suitable dosage amounts for genetically modified bacteria may range from about 10 5 Up to 10 12 bacteria, for example, about 10 5 bacteria, about 10 6 bacteria, about 107 bacteria, about 10 8 bacteria, about 10 9 bacteria, about 10 10 bacteria, about 10 11 bacteria or about 10 12 The composition can be administered once a day, once a week, or once a month. It can be administered multiple times / day (e.g., twice or three times / day).

[0372] Suitable dosage amounts for genetically modified bacteria may range from about 1×10 4 Up to 1×10 12 Colony forming units (CFU) / gram, for example, about 1×10 4 CFU / g, about 1×10 5 CFU / g, about 1×10 6 CFU / g, about 1×10 7 CFU / g, about 1×10 8 CFU / g, about 1×10 9 CFU / g, about 1×10 10 CFU / g, about 1×10 11 CFU / g, or approximately 1×10 12 CFU / g. In particular, the formulation contains about 1×10 5 Up to 1×10 12 Colony forming units (CFU) / g. The preparation may contain about 1×10 6 Up to 1×10 11 Colony forming units (CFU) / g. The preparation may contain about 1×10 7 Up to 1×10 11 Colony forming units (CFU) / g. The preparation may contain about 1×10 8 Up to 1×10 10 Colony forming units (CFU) / g. In particular, the formulation contains (approximately) 1×10 9Colony forming units (CFU) / gram of modified bacteria or host cells as described herein. Compositions can be applied once a day, once a week or once a month. It can be applied multiple times / day (for example, twice or three times / day). In some embodiments, genetically engineered bacteria are coated with enteric coatings for release into a specific region of the intestinal tract or intestinal tract, such as in the small intestine or large intestine. The usual pH curve 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 curve may be changed. In some embodiments, the coating is degraded in a specific pH environment to specify a release site. In some embodiments, at least two coatings are used. In some embodiments, the outer coating and the inner coating are degraded at different pH levels. In a specific embodiment, modified bacteria or host cells are formulated as enteric delayed release capsules.

[0373] Uses of the methods, bacteria, plasmids (e.g., conjugative plasmids), and host bacteria disclosed herein

[0374] The methods described herein can be performed ex vivo. The methods described herein can be performed in vitro. The methods described herein can be performed in vivo.

[0375] Provided are bacteria, plasmids (eg, conjugative plasmids), host (eg, donor) bacteria, or pharmaceutical preparations as described herein for use in a method of treating or preventing a disease or condition mediated by a deficiency or insufficient amount of IAA in a patient.

[0376] Provided are methods of producing IAA in the intestine of a subject, comprising administering to the subject a bacterium, a plasmid (eg, a conjugative plasmid), a host (eg, a donor) bacterium, or a pharmaceutical preparation as described herein.

[0377] The patient can be a human or animal subject. The patient can be a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey and a human). The patient can be a rodent, a mouse, or a rat. The patient can be a vertebrate, a reptile, a bird, or a fish. In particular, the patient is a human.

[0378] Provided are bacteria, plasmids (eg, conjugative plasmids), host (eg, donor) bacteria or pharmaceutical preparations as described herein for use as a medicament or in therapy.

[0379] Provided are methods of treating a metabolic disease comprising administering to a subject in need thereof a bacterium, a plasmid (eg, a conjugative plasmid), a host (eg, a donor) bacterium, or a pharmaceutical preparation as described herein.

[0380] Provided are methods of treating a cardiometabolic disease comprising administering to a subject in need thereof a bacterium, a plasmid (eg, a conjugative plasmid), a host (eg, a donor) bacterium, or a pharmaceutical preparation as described herein.

[0381] Provided are methods for treating a metabolic disease selected from the group consisting of leaky gut, type 1 diabetes, type 2 diabetes (including complications of type 1 and type 2 diabetes, such as insulin sensitivity in 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) deficiency, proprotein convertase subtilisin / kexin type 1 (PCSK1) deficiency, Src homolog 2B 1 (SH2B 1) deficiency, prohormone 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 ulna-mammary syndrome, the method comprising administering to a subject in need thereof a bacterium, a plasmid (e.g., a conjugative plasmid), a host (e.g., a donor) bacterium or a pharmaceutical preparation as described herein.

[0382] Provided are methods for treating a metabolic disease selected from the group consisting of metabolic syndrome, type 2 diabetes (including complications of type 2 diabetes, such as insulin sensitivity in type 2 diabetes), and non-alcoholic fatty liver disease, the method comprising administering to a subject in need thereof a bacterium, a plasmid (e.g., a conjugative plasmid), a host (e.g., a donor) bacterium, or a pharmaceutical preparation as described herein.

[0383] Provided are bacteria, plasmids (eg, conjugative plasmids), host (eg, donor) bacteria or pharmaceutical preparations as described herein for use in the treatment of metabolic diseases.

[0384] Provided are bacteria, plasmids (eg, conjugative plasmids), host (eg, donor) bacteria or pharmaceutical preparations as described herein for use in the treatment of a cardiovascular metabolic disease.

[0385] Provided are bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) bacteria, or pharmaceutical preparations as described herein for use in the treatment of a metabolic disease selected from the group consisting of: leaky gut, type 1 diabetes, type 2 diabetes (including complications of type 1 and type 2 diabetes, such as insulin sensitivity in type 2 diabetes), metabolic syndrome, Bard-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) deficiency, proprotein convertase subtilisin / kexin type 1 (PCSK1) deficiency, Src homolog 2B 1 (SH2B1) deficiency, prohormone 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, A1strom syndrome, Cohen syndrome, and ulna-mammary syndrome.

[0386] Provided are bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) bacteria or pharmaceutical preparations as described herein for use in the treatment of a metabolic disease selected from the group consisting of metabolic syndrome, type 2 diabetes (including complications of type 2 diabetes, such as insulin sensitivity in type 2 diabetes) and non-alcoholic fatty liver disease.

[0387] Metabolic syndrome affects approximately 20-30% of the middle-aged population and represents an increased risk of cardiovascular disease (the leading cause of death in the U.S.). Obesity, dyslipidemia, hypertension, and type 2 diabetes are described as metabolic syndrome. In some embodiments, the bacteria, conjugative plasmids, host (e.g., donor) cells, or pharmaceutical compositions described herein can be used in the treatment, prevention, and / or management of metabolic syndrome and / or obesity.

[0388] Metabolic syndrome is a clustering of at least three of the following five medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels.

[0389] 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, hypertension, hypogonadism, subfertility, infertility, abdominal obesity, pro-thrombotic conditions, and pro-inflammatory conditions.

[0390] Cardiovascular disease includes coronary artery disease (CAD), such as angina and myocardial infarction, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease and venous thrombosis. Coronary artery disease, stroke and peripheral arterial disease are related to atherosclerosis caused, inter alia, by high blood pressure, smoking, diabetes, lack of exercise, obesity, hypercholesterolemia, poor diet and excessive alcohol consumption.

[0391] Obesity is a common, fatal and expensive disease in developed countries, affecting all age groups, races and genders. Obesity can be classified as an inflammatory disease because it is associated with immune activation and chronic, low-grade systemic inflammation. Endotoxemia, a process resulting from the translocation of endotoxin compounds (lipopolysaccharide [LPS]) of gram-negative enteric bacteria. In the past decade, it has become apparent that insulin resistance and T2DM are characterized by low-grade inflammation. In this regard, LPS triggers a low-grade inflammatory response, and therefore the process of endotoxemia can lead to the development of insulin resistance and other metabolic disorders. IAA can also be used in the treatment of type 2 diabetes.

[0392] In certain embodiments, the bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) cells, or pharmaceutical compositions described herein reduce tryptophan levels in, e.g., serum and / or the intestine of a patient, e.g., for the prevention, treatment, and / or management of obesity.

[0393] Metabolic syndrome is an important risk factor for cardiovascular morbidity and mortality, as well as all-cause mortality. Therefore, detection, prevention, and treatment of potential risk factors for metabolic syndrome are key approaches to reducing cardiovascular morbidity in the general population.

[0394] Bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) cells, or pharmaceutical compositions as described herein can be administered to a patient in one or more doses. It should be understood that the precise dosage and duration of treatment depends on the disease being treated, and can be determined empirically using known test protocols or by inference from in vivo or in vitro test data, taking into account the age, weight, and sex of the patient. It should be noted that concentrations and dosage values ​​can also vary with the severity of the condition to be alleviated. It should be further understood that for any particular patient, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the individual administering the composition or supervising the administration of the composition.

[0395] Recently, it has been shown that bacterial IAA can enhance the efficacy of chemotherapy in pancreatic cancer patients, see Tintelnot et al., Nature, 615, 2023, 168-174, doi: https: / / do1.org / 10.1038 / s41586-023-05728-y and Seo & Wargo, Cell Reports Medicine, 4, 101039, May 16, 2023, doi: https: / / doi.org / 10.1016 / j.xcrm.2023.101039, each of which is incorporated herein by reference in its entirety.

[0396] Further, IAA has been shown to activate the Toll-like receptor 4 and c-Jun N-terminal kinase (TLR4-JNK) pathway, as well as reduce the expression of tumor necrosis factor alpha (TNF-α), which in turn inhibits the proliferation of colorectal cancer (CRC) cells, see Tomii et al., Bioscience, Biotechnology, and Biochemistry, 87(8), 2023, 839-849, which is incorporated herein by reference in its entirety. In this study, IAA did not induce cytotoxicity, suggesting that it regulates cell cycle progression by activating JNK and thereby inhibits cell proliferation.

[0397] Therefore, bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) bacteria or pharmaceutical preparations as described herein are provided for use in cancer, particularly pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC) treatment. Also provided is a method for treating cancer, particularly colorectal cancer (CRC) and / or pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC) by administering an effective amount of bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) bacteria or pharmaceutical preparations to a patient in need thereof. In this embodiment, the subject may be receiving chemotherapy. Chemotherapy can be provided simultaneously, before or after the administration of bacteria, plasmids (e.g., conjugative plasmids), host (e.g., donor) bacteria or pharmaceutical preparations. Chemotherapy is well known to those skilled in the art.

[0398] IAA has been shown to reduce liver lipogenesis and oxidative and inflammatory stress in mouse models, see Ji et al., Nutrients, 11, 2062, 2019, doi: 10.3390 / nu11092062, which is incorporated herein in its entirety. The authors showed that IAA may be useful for treating non-alcoholic fatty liver disease (NAFLD), as well as improving insulin resistance, lipid metabolism, oxidative stress, and inflammatory stress. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) subtypes. According to a review conducted at the end of 2023, non-alcoholic fatty liver disease (NAFLD) is increasingly referred to as metabolic dysfunction-associated fatty liver disease (MASLD), see Rinella et al., Hepatology 78(6), 1966-1986, 2023, doi: 10.1097 / HEP.0000000000000520.

[0399] Therefore, provide by having this patient in need of applying effective amount of bacterium as described herein, plasmid (such as conjugative plasmid), host (such as donor) bacterium or pharmaceutical preparation, for the treatment of non-alcoholic fatty liver disease (NAFLD) bacterium, plasmid (such as conjugative plasmid), host (such as donor) bacterium or pharmaceutical preparation. Also provide by having this patient in need of applying effective amount of bacterium as described herein, plasmid (such as conjugative plasmid), host (such as donor) bacterium or pharmaceutical preparation, for improving insulin resistance, lipid metabolism, oxidative stress and / or inflammatory stress bacterium, plasmid (such as conjugative plasmid), host (such as donor) bacterium or pharmaceutical preparation as described herein.

[0400] As used herein, with respect to methods of treatment, "prevention" includes a reduction in the risk of contracting a disease. "Treatment or prevention" can be complete or partial treatment or prevention, i.e., a reduction, but not a complete reduction, of a disease / condition or its symptoms; or a reduction in risk, but not a complete prevention of a disease / condition or its symptoms. Similarly, the method treats or prevents (i.e., reduces the risk of) a disease or condition or an undesirable symptom of a therapy.

[0401] The disease or condition may be metabolic syndrome or metabolic cardiovascular 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.

[0402] To improve the ability of any modified bacteria, host cells or plasmids described herein to effectively colonize and produce an IAA described herein, a course of antibiotics can be administered to a subject to provide a niche in the relevant microbiome that can be colonized within one month (e.g., within 2 weeks, within a week, e.g., within 5, 4 or 3 days, particularly within 34 hours) of receiving the first dose of the modified bacteria, host cell or pharmaceutical composition.

[0403] The antibiotic treatment may be an aminoglycoside (e.g. selected from amikacin, liposomal amikacin, gentamicin, prazomicin and tobramycin). The antibiotic may be a β-lactam inhibitor (e.g. selected from ceftolozane and cilastatin). The antibiotic may be a β-lactamase inhibitor (e.g. selected from avibactam, clavulanate, clavulanic acid, sulbactam, tazobactam, relebactam and vaborbactam). The antibiotic may be a carbapenem (e.g. selected from doripenem, ertapenem, imipenem and meropenem). The antibiotic can be a cephalosporin (e.g., selected from cefaclor, cefdroxil, cefazolin, cefdinir, cefditoren, cefepime, cefdiroxil, cefixime, cefotaxime, cefotetan, cefoxitin, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime and cefalexin). The antibiotic can be a fluoroquinolone (e.g., selected from ciprofloxacin, delafloxacin, gemifloxacin, levofloxacin and moxifloxacin). The antibiotic can be a folate pathway inhibitor (e.g., selected from sulfisoxazole, sulfamethoxazole and trimethoprim). The antibiotic can be fosfomycin (e.g., fosfomycin). The antibiotic can be a glycopeptide (e.g., selected from dalbavancin, oritavancin, telavancin and vancomycin). The antibiotic can be a glycine tetracycline (e.g., tigecycline). The antibiotic can be a ketolide (e.g., telithromycin). The antibiotic may be a lincosamide (e.g., clindamycin). The antibiotic may be a lipopeptide (e.g., daptomycin). The antibiotic may be a macrocycle (e.g., fidaxomicin). The antibiotic may be a macrolide (e.g., selected from azithromycin, clarithromycin, and erythromycin). The antibiotic may be a monoamine (e.g., aztreonam). The antibiotic may be a nitrofuran (e.g., nitrofurantoin). The antibiotic may be a nitroimidazole (e.g., selected from metronidazole and tinidazole). The antibiotic may be a nucleoside analog (e.g., selected from monupiravir and remdesivir). The antibiotic may be an oxazolidinone (e.g., selected from linezolid and tedizolid). The antibiotic may be a penicillin (e.g., selected from amoxicillin, ampicillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin). The antibiotic may be a phenylpropanol (e.g., chloramphenicol). The antibiotic may be a polyene (e.g., selected from amphotericin B, liposomal amphotericin B, and amphotericin B lipid complex). The antibiotic may be a polymerase acid endonuclease inhibitor (e.g., baloxavir marboxil). The antibiotic may be a polymyxin (e.g., selected from polymyxin mesylate, colistin, and polymyxin B). The antibiotic may be a pleuromutilin (e.g., lefamulin). The antibiotic may be a protease inhibitor (e.g., namatevir). The antibiotic may be rifampicin. The antibiotic may be a streprogramin (e.g., selected from quinupristin and dalfopristin). The antibiotic may be a tetracycline (e.g., selected from elacycline, minocycline, omadacycline, and tetracycline). The antibiotic may be any combination of the antibiotics listed above.

[0404] Table 3: Example bacteria

[0405] Optionally, the modified bacterium or the bacterium into which the conjugative plasmid is hosted and / or transferred (ie, the recipient bacterium) is selected from this table.

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436] The invention is described in more detail in the following non-limiting examples.

[0437] Example

[0438] Example 1. IAA biosynthetic pathway on plasmid

[0439] 1.1 Cloning of the IAA biosynthetic pathway

[0440] A p15A-based plasmid (p1915, Seq ID No: 43) was constructed containing ipdC from Pantoea agglomerans (Seq ID No: 41), aspC from Escherichia coli (Seq ID No: 39), and iad1 from Ustilago maydis (Seq ID No: 42) under the control of a temperature-inducible promoter (Pts). The tnaB permease was also cloned in the vector under the control of the same promoter to improve tryptophan uptake (see Figure 2 As a control, plasmid p1138 carrying the purple pigment amilCP under the control of the same temperature-inducible promoter was constructed (see Figure 2 ).

[0441] 1.2 In vitro plasmid-based IAA production

[0442] E. coli MG1655 (b230) was transformed with plasmids p1915 (Seq ID No: 43) and p1138 to generate strains b5674 and b5673, respectively. IAA production was confirmed using the Salkowski colorimetric assay (see Example 3.3). Figure 3 As shown in , a significant increase (-300 μM) of IAA was detected in the bacterial overnight supernatant of strain b5674, in contrast to the empty control vector in strain b5673.

[0443] 1.3. Pathway stability for plasmid-based IAA production

[0444] In vivo study was performed in two groups of 5 mice, and the mice were administered with the Escherichia coli strain b5673 as described in Example 1.2, the control without IAA production, or the strain b5674 producing IAA as described in Example 1.2. Complete experimental details are provided in Example 3.2. Mice were orally administered with a single dose, and fecal samples were collected twice a day until 54 hours after administration. The bacterial level (CFU / g feces) of fecal samples was analyzed. At each time point, bacterium colonies were recovered from the flat plates of fecal samples-when possible, a single bacterium colony of the control strain b5673 and 3 bacterium colonies from the production strain b5674. Bacterium colonies were then grown in the M9 medium with the tryptophan added, and IAA production was analyzed using the Salkowski assay (see Example 3.3). This will confirm the ability of the strain to maintain its production capacity after colonization and gastrointestinal tract passage. Since animals were only administered once, the stability of the construct in the plasmid will be clarified until 54 hours after administration.

[0445] like Figure 4 As shown in , the results of the study showed similar colonization abilities of the two strains during the study period. It was further confirmed that colonies isolated from feces of animals dosed with the production strain b5674 produced IAA. Production by the colonies began to decline at 30 hours post-dose and had been halved by 54 hours post-dose, albeit with associated variability. Figure 4 As shown in, at the 54 hour time point for production strain b5674, two of the sampled colonies had IAA production levels comparable to those of control strain b5673. Therefore, these colonies have lost their production capacity. Therefore, without repeated dosing of the strain, the plasmid-borne IAA production pathway is unstable for continuous IAA production at high levels, as seen 6-24 hours after dosing. The use of antibiotic selection confirmed that even though IAA production was severely reduced, the plasmid was present in production strain b5674 at all time points. Based on the published data, we believe that this severe reduction in IAA production is due to mutations introduced into heterologous IAA biosynthetic genes.

[0446] 1.4 Cloning of the IAA biosynthetic pathway into a plasmid with export proteins

[0447] The IAA pathway was cloned together with the IAA exporter Pin2 from Arabidopsis thaliana (Seq ID No: 3) or aec from Pantoea agglomerans (Seq ID No: 2). Three new plasmids (p1880, p1881 and p1882) were cloned using plasmid p1915 (as described in Examples 1.1 and 1.2) as the backbone. The tnaB permease was removed and exporters were added to transport IAA out of the cell. The exporters used were pin2 from Arabidopsis thaliana (Seq ID No: 3) and aec from Pantoea agglomerans (Seq ID No: 2). p1880 (Seq ID No: 49), p1881 (Seq ID No: 50) and p1882 (Seq ID No: 51) each contain ipdC from Pantoea agglomerans (Seq ID No: 41), aspC from Escherichia coli (Seq ID No: 39) and iad1 from Ustilago maydis (Seq ID No: 42), and a chloramphenicol resistance gene under the control of a temperature-inducible promoter (Pts), and use the p15A origin. In addition, plasmids p1881 and 1882 carry the export proteins pin2 and aec, respectively, expressed from promoters driving expression of resistance markers. Cloning was performed as described in Materials and Methods (see Example 3 below), and plasmid maps are shown in Figure 5 middle.

[0448] 1.5 Plasmid-based IAA production with export proteins in vitro

[0449] E. coli MG1655 (b230) was transformed with plasmids p1880 (Seq ID No: 49), p1881 (Seq ID No: 50) and p1882 (Seq ID No: 51) to generate strains b5626, b5627 and b5629, respectively. In duplicate experiments, IAA production was confirmed using the Salkowski colorimetric assay (see Example 3.3). Figure 6 As shown in , the addition of aec increased the amount of IAA in the supernatant relative to a control that did not contain any export protein. The addition of pin2 had the same effect, although not to the same level as aec. Two further experiments were performed showing that the effect of pin2 was less significant, but the trend was still towards increased export with the pin2 export protein.

[0450] Example 2: Pathway engineering and stability in chromosomes

[0451] Due to the instability of IAA production in plasmid systems, the inventors continued to optimize IAA production in modified bacteria by integrating the gene into the chromosome and placing it under the control of a constitutive promoter. Since the plasmid-based IAA production pathway appears to increase loss-of-function mutations, it is reasonable to assume that overexpression of this pathway imposes a fitness cost on the cell. Therefore, the constitutive promoter used to drive systemic expression from the chromosome was optimized to produce high IAA levels without causing deleterious effects.

[0452] Optimization of IAA production and AEC protein export

[0453] Two chromosomal deletions were introduced into Symbioflor G6 / 7(b463) to further optimize IAA production in this strain. tnaA (tryptophanase) was deleted together with the leader peptide tnaC to constitutively express tnaB (tryptophan import protein) and prevent the conversion of tryptophan to indole by tnaA.

[0454] Deletion of the tryptophan transcriptional repressor (trpR) has three distinct effects: (i) derepressing the synthesis of chorismate (thereby increasing the amount of chorismate in the cell), (ii) derepressing trpEDCA, which converts chorismate to indole (thereby increasing the amount of indole in the cell), and (iii) derepressing the mtr proton symporter that transports tryptophan / indole into the cell (thereby increasing the amount of tryptophan / indole in the cell).

[0455] The deletion was performed using scarless lambda red recombination engineering (Datsenko & Wanner, PNAS, 97, 6640, 2000, incorporated herein by reference). The resulting strain was designated b5698. A streptomycin-resistant form of the strain was spontaneously obtained by plating an overnight culture on streptomycin plates and was stored as b6570.

[0456] To optimize IAA production, E. coli aspC was replaced by a different aminotransferase from Arabidopsis thaliana, taal (Seq ID No: 40), which is more specific for tryptophan and will increase IAA production while preventing the production of byproducts. In addition, the export protein aec from Pantoea agglomerans (Seq ID No: 2) expressed by the CmR promoter was added to transport IAA out of the cell. The construction was done in a cloDF 1-based plasmid using the medium-strength constitutive promoter Pc-aga (Seq ID No: 24).

[0457] The layout of the plasmid construct is shown in Figure 7Plasmid p2526 (Seq ID No: 45) contains ipdC from Pantoea agglomerans (Seq ID No: 41), aspC from Escherichia coli (Seq ID No: 39), and iadl from Ustilago maydis (Seq ID No: 42) under the control of the promoter Pc-aga (Seq ID No: 24). Plasmid p2527 (Seq ID No: 46) additionally contains the aec export protein from Pantoea agglomerans (Seq ID No: 2), which is expressed from a promoter driving expression of a resistance marker. Plasmid p2528 (Seq ID No: 47) contains ipdC from Pantoea agglomerans (Seq ID No: 41), taa1 from Arabidopsis thaliana (Seq ID No: 40), and iadl from Ustilago maydis (Seq ID No: 42) under the control of the promoter Pc-aga (Seq ID No: 24). Plasmid p2529 (Seq ID No: 48) additionally contains the aec export protein from Pantoea agglomerans (Seq ID No: 2), expressed from a promoter driving expression of the resistance marker.

[0458] Attempts to clone a plasmid containing the same gene as in plasmid p2526, but using the constitutive promoter pc-tga (Seq ID No: 1) were largely unsuccessful. Without being bound by theory, we believe this is because the conversion of tryptophan to IAA is unsustainably high, leading to accumulation of mutations in pathway genes due to high fitness costs.

[0459] Strain b5698 (ΔtnaAΔtrpR) was transformed with these plasmids to produce b7196 (containing plasmid p2526), ​​b7197 (containing plasmid p2527), b7198 (containing plasmid p2528), and b7199 (containing plasmid p2529), as described above. Figure 7 As shown in .

[0460] 2.2. In vitro IAA production

[0461] The four constructs, strain b7196 (aspC), strain b7197 (aspC, aec), strain b7198 (taa1), and strain b7199 (taal, aec), were subjected to Salkowski analysis (see Example 3.3) to determine the effects of two different aminotransferases (aspC vs. taa1) and aec exporters on IAA secretion. In contrast to the other constructs, strain b7199 (taa1, aec) showed a 3-4× increase in IAA production (see Figure 8), leading to the selection of the taa1 aminotransferase and the addition of the aec exportin as the optimized production pathway for all future modifications.

[0462] 2.3 Chromosomal integration of IAA biosynthetic genes

[0463] The optimized pathway was integrated into the chromosome of strain b5698 (ΔtnaAΔtrpR) to further increase genetic stability and IAA production. The heterologous pathway for IAA production from strain b7199 was integrated into the chromosome of strain b5698 (ΔtnaAΔtrpR) to generate strain b6089, which consisted of ipdC from Pantoea agglomerans (Seq ID No: 41), taa1 from Arabidopsis thaliana (Seq ID No: 40), iad1 from Ustilago maydis (Seq ID No: 42), and aec export protein from Pantoea agglomerans (Seq ID No: 2) under the control of the strong constitutive promoter Pc-tga (Seq ID No: 1). A streptomycin-resistant form of this strain was spontaneously obtained as described above and was maintained as b6131. ​​The layout of the chromosomal insertion is shown in Figure 7 middle.

[0464] IAA secretion was tested by comparing plasmid-based expression (from strain b7199) with the new lead construct (strain b6131) by the Salkowski assay (see Example 3.3). Fig. 9 The main strain (b6570, without heterologous genes) was added as a control.

[0465] The results showed comparable IAA production levels between the genome-integrated strain b6131 and the plasmid-containing strain b7199. Therefore, we concluded that we could integrate the optimized pathway into the chromosome without deleterious effects on the bacteria.

[0466] IAA production was confirmed by liquid chromatography-mass spectrometry (LC-MS). Strain b6131 (genomically integrated) secreted 656.5 μM IAA (for methodology see Examples 3.2 and 3.4).

[0467] 2.3.1 LC-MS data for characterizing chassis effects

[0468] The secretion of other metabolites will be evaluated by LC-MS in addition. Wherein, the generation of indole-3-butyric acid (IBA) is expected. In addition, the IAA production from indole will also be tested. Since bacterial strain b6131 has been transformed to increase tryptophan and indole levels in the cell (by having the chromosome deletion of tnaA and trpR of the effect described in Section 2.1), we expect to observe the IAA production from indole.

[0469] Performed LC-MS analysis (A&M Labors, Germany, see Example 3.4 for methodology) to evaluate the impact of the framework optimization step (disappearance of tnaA and trpR) on substrate utilization (indole and tryptophan) and IAA and potential byproducts (indole-3-pyruvic acid, indole-3-butyric acid, indole-3-glycolic acid, indole-3-ethanol and skatole) produced. Tested indole utilization because indole is a precursor in tryptophan synthesis and is also very abundant in the human colon. Performed some transformation steps to allow indole synthesis, promote its uptake or effectively convert to tryptophan. Therefore, IAA production is expected in the presence of indole.

[0470] Bacterial overnight cultures in LB medium were refreshed in M9 medium containing Cas amino acids, 0.4% glucose and tryptophan (1 mM), indole (1 mM) or tryptophan and indole (1 mM each), followed by overnight culture at 37°C. After the bacterial cells formed pellets, the bacterial supernatant was transferred to a fresh Eppendorf tube containing acetonitrile at a ratio of 1:3. The sample was centrifuged at 2000 g for 10 minutes, and the supernatant was stored at -20°C and then shipped to A&MLabor (www.am-labor.de) for LCMS analysis.

[0471] First, the effects of tnaCA and trpR deletions were evaluated using the optimized IAA production / export pathway (ipdC-taa1-iad1-aec) carried on plasmid p2529 (see Fig. 12A and 12B ). A strain carrying the corresponding empty vector p2564 was used as a control. As expected, the deletion of tnaA abolished the conversion of tryptophan to indole ( Fig. 12A , b7208 relative to b7207), while the deletion of trpR resulted in a reduced turnover rate ( Fig. 12A , b7209 relative to b7207). The effect of the tnaA deletion was also reflected in the presence of the IAA production pathway. In the wild-type strain (b7201), part of the added tryptophan was converted to indole, while in the deletion variants, all tryptophan was converted to IAA or indole-3-ethanol ( Fig. 12A The presence of indole-3-ethanol (tryptophan) is not surprising since it is formed spontaneously from indole-3-acetaldehyde (see Figure 1 ), and it was also observed in a previous IAA production study (Romasi et al., J. Microbiol Biotechnol. 2013 Dec;23(12):1726-36).

[0472] In the presence of indole, trpR deletion allows for more efficient conversion of indole to tryptophan ( Fig. 12B , b7209 relative to b7207), while tnaA deletion had no effect. trpR deletion greatly enhanced IAA and indole-3-ethanol production from indole ( Fig. 12B , b7205 relative to b7201 and b7199 relative to b7203). This may be due to the derepression of the mtr transporter, which mainly promotes indole uptake.

[0473] 2.3.2 Performance of the Genome-Integrated IAA Production Pathway

[0474] IAA production by the lead construct b6131 (chromosomally integrated ipdC-taa1-iad1-aec operon, tnaA and trpR deleted) was evaluated in the presence of different substrate combinations (i.e., tryptophan, indole, or both) ( Fig. 12C The parent strain carrying deletions of tnaA and trpR but without the integrated operon was used as a control (b6570). The added tryptophan was completely converted to IAA and indole-3-ethanol by b6131, and a better IAA / indole-3-ethanol ratio was observed compared to the presence of the plasmid-borne system ( Fig. 12A , b7199). The added indole was also completely converted to IAA and indole-3-ethanol by b6131, with IAA being the major product. Fig. 12B This is a significant improvement compared to b7199 (where only partial conversion was achieved and most of the indole was converted to indole-3-ethanol). When the two substrates were added in an equimolar ratio to b6131, complete conversion of tryptophan was achieved, but only partial conversion of indole. However, the total IAA produced exceeded the levels obtained in the presence of a single substrate.

[0475] 2.3.3 Optimization of chromosome insertion site

[0476] The IAA production pathway (ipdC-taa1-iad1-aec) was inserted at two new locations to evaluate whether a chromosomal insertion point closer to the replication origin would increase IAA production. The strains were grown overnight in LB and then re-cultured in M91mM tryptophan and incubated at 37°C with shaking at 300rpm for an additional 24 hours. Samples were collected by spinning down the cells and harvesting the supernatant. The supernatant was collected and tested for IAA using the Salkowski assay (see Example 3.3). No increase in IAA production from b7848 or b7829 compared to b6131 was observed, see Fig.13 .

[0477] 2.3.4 IAA production under anaerobic conditions

[0478] The Salkowski assay (see Example 3.3) was used to quantify IAA production from b6570 (negative control) and b6131 (IAA producing strain) under anaerobic conditions. The strains were grown anaerobically overnight in LB, then re-cultured in reduced M91 mM tryptophan and incubated anaerobically for an additional 24 hours at 37°C with shaking at 300 rpm. Samples were collected by spinning down the cells and harvesting the supernatant. The supernatant was collected and tested for IAA. Fig.14 As shown in , the levels of IAA produced after overnight growth were significantly higher in cultures of b6131 compared to the control strain b6570.

[0479] 2.4. In vivo stability data of chromosomally integrated IAA biosynthetic genes

[0480] In vivo studies were performed in groups of 5 mice, which were orally administered with E. coli b6570 (control strain without IAA production) or pilot chromosome strain b6131 (IAA production, see Example 2.3). Mice were orally administered twice a day for 4 days (8 doses in total), and fecal samples were collected once a day until 4 days after the start of administration, and on the 7th day after administration. The bacterial levels (CFU / g feces) of fecal samples were analyzed. When possible, single colonies were recovered from the paving plates of fecal samples at 3 time points (1, 4 and 7 days after administration). The colonies were then grown in a culture medium with added tryptophan, and IAA production was analyzed using the Salkowski colorimetric assay (see Example 3.3). This will confirm the ability of the strain to maintain its production capacity after colonization and passage through the gastrointestinal tract.

[0481] The results of the study showed similar colonization abilities of both strains. It was further confirmed that colonies isolated from feces of animals dosed with the IAA-producing strain b6131 produced IAA. Production was stable throughout the study, further supporting the stability of the genomic inserted IAA pathway until 8 days after the start of dosing, see Fig.10 .

[0482] Ten colonies isolated from day 4 fecal samples were submitted for whole genome sequencing. No mutations were found in this pathway. This suggests that not only can high levels of IAA production be maintained for longer periods of time than the plasmid (as described in Examples 1.3 and Figure 4 ), and additionally the engineered bacteria were durable and the heterologous genes did not appear to impose a significant fitness cost.

[0483] 2.5. Food IAA production at 30°C and 37°C

[0484] The stability of the TAAL pathway in the lead candidate strain B6131 and its control strain B6570 was tested in vitro at temperatures of 30° C. and 37° C. The strains were grown overnight at these temperatures followed by OD measurements.

[0485] The Salkowski colorimetric assay (see Example 3.3) was used to measure the levels of IAA secreted by strains grown at 30°C and 37°C. The results show that IAA production ( Fig.11 ). Due to similar growth and similar production levels, it can be concluded that the optimized taa1-based pathway is stable at both 30°C and 37°C. This data shows that differences in production are not expected at physiological temperatures, in contrast to, for example, WO2021 / 242897 (Synlogic) Figure 3 This contrasts with the data shown in , where different IAA production pathways contained on plasmids carried by bacteria showed lower average production at 37°C compared to 30°C, suggesting suboptimal production under physiological conditions.

[0486] Example 3: Materials and Methods

[0487] 3.1 Transformation

[0488] Plasmids were constructed using in-Fusion cloning (Takara) according to the manufacturer's protocol (full sequences are provided in Table 7), and chromosomal manipulations were accomplished using scarless lambda red-mediated recombination (see, e.g., Datsenko & Wanner, supra; and Blank et al. 2011 PloS ONE, which are incorporated herein by reference in their entireties).

[0489] Strain b5698 was prepared using three rounds of recombineering (Δfix, ΔtrpR, ΔtnaCA) with the primers listed in Table 4. This strain was subjected to whole genome sequencing to verify the correct modification.

[0490] Strain b6089 was prepared by amplifying the IAA pathway from plasmid p2169 (see Table 5 and Seq ID No: 44) using primers oli1550xoli2893xoli9094 (see Table 7 and Seq ID No: 37, Seq ID No: 26 and Seq ID No: 38, respectively) and recombining it into the chromosome. The strain was sequenced for the whole genome to verify the correct modification.

[0491] Strain b6131 was prepared by plating an overnight culture of b6089 on LB plates supplemented with streptomycin and selecting spontaneous resisters. This strain was subjected to whole genome sequencing to verify the correct modification.

[0492] Strain b6570 was prepared by plating an overnight culture of b5698 on LB plates supplemented with streptomycin and selecting spontaneous resisters. This strain was subjected to whole genome sequencing to verify the correct modification.

[0493] Strains b7196, b7197, b7198 and b7199 were prepared by introducing plasmids p2526, p2527, p2528 and p2529 (see Table 5 and Seq ID No: 45, Seq ID No: 46, Seq ID No: 47 and Seq ID No: 48, respectively) into b5698 using standard electroporation.

[0494] Strains b5673 and b5674 were prepared by introducing plasmids p1138 and p1915 (Seq ID No: 43) (see Table 5), respectively, into b230 using standard electroporation.

[0495] Strains b5626, b5627 and b5629 were prepared by introducing plasmids p1880 (Seq ID No: 49), p1881 (Seq ID No: 50) and p1882 (Seq ID No: 51) (see Table 5) into b230, respectively, using standard electroporation.

[0496] Table 4: PCR products used to transform b5698

[0497]

[0498]

[0499] 3.2 In vivo work

[0500] 3.2.1 Study 1

[0501] In 10 female C57BL / 6JRj (Janvier Labs) mice, the research (as described in Example 1.3) about the IAA production based on plasmid in vivo was performed, and the mice were in about 6 weeks of age when they arrived. Mice were raised in IVC cages with two groups of 5, and adapted to at least 7 days before the study began. Throughout the study, mice had free access to food and drinking water. In order to ensure the availability of tryptophan, mice were fed with a high tryptophan diet (A22052401, L-Amino Acid Rodent Diet with 40 grams of L-tryptophan / kg, Research Diets Inc) that was changed three times per week. At the first 4 days, animals began to be treated by 5g / L streptomycin (Sigma-Aldrich, S6501) of drinking water, which was replaced by 500mg / L chloramphenicol (Sigma-Aldrich, C0378) treatment of drinking water from the first 1 day and until the termination at the 2nd day. Streptomycin treatment enabled the various E. coli strains to engraft, while chloramphenicol treatment ensured plasmid maintenance in the administered E. coli strains. On day 0, animals were dosed by oral gavage with approximately 1 x 10 7 Escherichia coli strains (b5673 (control) or b5674 (producing IAA)) of CFU. In the morning of the 4th day and the 0th day, fecal samples were collected from each animal to evaluate the eradication of Escherichia coli before inoculation. Then two fecal pellets were collected 6 hours after inoculation, and then in the morning and afternoon when the 1st day and the 2nd day. One fecal pellet in PBS was selected for CFU, and the other pellet was quickly frozen and sent to LC-MS for analysis with quantitative IAA. The fecal pellets in PBS (Biowest, L0615) were spotted on MacConkey agar with streptomycin and chloramphenicol to select the transformed bacterial strain. Then before selecting about Salkowski determination (see Example 3.3), bacterium colonies were counted. During the study, animals were weighed when arriving and at the first 4, -1, 0, 1 and 2 days. Animals were further clinically observed twice a day by trained personnel, and terminated on the 2nd day after the last sample.

[0502] 3.2.1 Study 2

[0503] In 30 male C57BL / 6JRj (Janvier Labs) mice, the research on in vivo stability as described in Example 2.4 was performed, and the mice were in about 5 weeks of age when arriving. Before the study began 40 weeks, these mice were always in 40% fat, 40% carbohydrate and 2% cholesterol diet (D09100310, SSNIFF, Germany). Mice were raised separately during the study. Throughout the study, mice had the random approach for food and drinking water. Before the study began 1 week, animals were randomly divided into 6 groups of 5 animals according to body weight. The 1st and 2nd groups were given common tap water as drinking water in the study from beginning to end, and the 3rd and 4th groups were given 1g / L streptomycin (Sigma-Aldrich, S6501) in drinking water for 24 hours from the first 2 days to the first 1 day, and the 5th and 6th groups were treated with 1g / L streptomycin (Sigma-Aldrich, S6501) in drinking water from the first 3 days until termination. Groups 1, 3, and 5 were dosed with the control strain b6570, while groups 2, 4, and 6 were dosed with the IAA-producing strain b6131. ​​Mice were dosed twice daily (AM+PM) with approximately 1×10 50 μL of 1% 1% 1% 2% 2% 1 ... 8 CFU.From the 1st day until termination (the 5th or 8th day), two fecal pellets were collected once a day from each animal in the morning.One fecal pellet in PBS was selected for CFU, and the other pellet was quickly frozen and sent to LC-MS for analysis with quantitative IAA.The fecal pellets in PBS were spotted on MacConkey agar with streptomycin, to select our strain.Then before selecting about Salkowski determination (see embodiment 3.3), bacterium colonies were counted.During the study, animals were weighed once a day.Animal was further clinically observed twice a day by trained personnel, and terminated on the 5th or 8th day after the last sample.

[0504] 3.2.2 Study 3

[0505] In vivo IAA production by our lead candidate (b6131) was tested. The study was performed in groups of 5 animals in C57B1 / 6 mice. Group 5 (control) and Group 6 (IAA producing) were pre-treated and treated with 1 g / L streptomycin in drinking water from day -3 until termination. Animals were treated with approximately 10 9CFU / dosage is inoculated, and twice a day is administered for 4 days. Two stool samples are collected at the 1st day (before the first dose), then once a day, until termination. One stool pellet is quickly frozen and sent to the IAA quantitatively analyzed by LC-MS (A&M Labor, Germany, see Example 3.4 below for methodology), and another stool pellet is put into PBS for internal CFU quantitative. There is pre-treatment and until the continuous streptomycin treatment of termination, the CFU count in the feces remains on 10 9 CFU / g feces at a stable level ( Fig.15 ).

[0506] LC-MS data showed clear differences in IAA levels between the groups. A 20-30 fold increase relative to the control was observed for the production strain ( Fig.16 The control (Group 5) maintained IAA levels steadily below 1 nmol / g feces, and the producer strain (Group 6) initially showed an increase in IAA to >30 nmol / g feces, stabilizing at around 20 nmol / g feces by day 8 (termination day).

[0507] Further analysis of the feces via LC-MS showed that most kynurenine and skatole levels were below the detection limit, so no further analysis could be performed on these data. However, we included tryptophan and indole for analysis because these can be converted to IAA by our strains. There was no significant or statistical variation in tryptophan between the groups, indicating that tryptophan levels were not affected by our strains and are likely not the main source of conversion to IAA ( Fig.17 ).

[0508] However, analysis of fecal indole levels revealed that Group 6 had consistently low levels close to 0 nmol / g feces throughout the study, while the control group (Group 5) did have indole levels around 20-50 nmol / g feces throughout the study. This suggests that indole use ( Fig.18 ) and IAA production ( Fig.16 ) have a negative correlation.

[0509] Based on these data, we have successfully engineered our strain to produce IAA in the mouse intestine.

[0510] Table 5: Plasmids used in this study

[0511]

[0512]

[0513] Table 6. Strains used in this study

[0514] Strain number describe b230 Escherichia coli K12 MG1655 b5673 b230(p1138) b5674 b230(p1915) b463 Symbioflor G6 / 7 isolated in SnipR Biome b5698 Symbioflor G6 / 7Δfix ΔtnaCA ΔtrpR b6089 b5698IAA+(Ptga-ipdC-taa1-iad1-aec) b6131 b5698IAA+(Ptga-ipdC-taa1-iad1-aec)strR b6570 b5698str b7196 b5698p2526 b7197 b5698p2527 b7198 b5698p2528 b7199 b5698p2529 b5626 b230 p1880 b5627 b230 p1881 b5629 b230 p1882

[0515] 3.3 Salkowski assay

[0516] The Salkowski assay is a spectrophotometric assay traditionally used to detect plant- or bacteria-derived IAA (Gang et al., Bio-Protocol., 9(9): e3230, 2019, which is incorporated herein by reference in its entirety). The assay is based on the detection of a pink color derived from the reaction of the Salkowski reagent (ferric chloride and perchloric acid) with IAA present in the bacterial supernatant. Briefly, an overnight culture of bacteria in LB medium is re-cultivated in M9 medium containing tryptophan (1 mM), Cas amino acids, 0.4% glucose, and the culture is induced after 1.5 hours if necessary (depending on whether the relevant promoter needs to be induced for expression), followed by an overnight culture at 37°C. The bacterial cells are pelleted and the bacterial supernatant is incubated in the presence of the Salkowski reagent for 45 minutes. The color change is evaluated by measuring the absorbance at 530 nm. A standard curve is used to calculate the IAA concentration of each bacterial supernatant. The method has been validated by LC / MS analysis.

[0517] 3.4 LC-MS analysis

[0518] The samples were prepared as follows: overnight cultures of bacteria in LB medium were re-cultured in M9 medium containing Cas amino acids, 0.4% glucose and tryptophan (1 mM), indole (1 mM), or tryptophan and indole (1 mM each), followed by overnight culture at 37°C. After the bacterial cells formed a pellet, the bacterial supernatant was transferred to a fresh Eppendorf tube containing acetonitrile at a ratio of 1:3. The samples were centrifuged at 2000 g for 10 minutes, and the supernatant was stored at -20°C and then shipped to A&M Labor for LCMS analysis.

[0519] Sample analysis was performed by A & M Labor using LC-MS essentially as follows. The analysis was performed using a ThermoScientific Vanquish LC connected to an OrbitrapExploris 240MS (ThermoFisher Scientific). An electrospray ionization interface was used as an ionization source. The analysis was performed in positive and negative ionization modes under polarity switching. UPLC was performed using a slightly modified version of the scheme described by Doneanu et al., 2011 (Water Application note 2011, 720004042en). Peak areas were extracted using Compound Discoverer 3.3 (ThermoScientific). The identification of compounds was performed at four levels; Level 1: by retention time (for internal true standards comparison), accurate mass (with an acceptable deviation of 3ppm) and MS / MS spectrum, Level 2a: by retention time (for internal true standards comparison), accurate mass (with an acceptable deviation of 3ppm) identification. Level 2b: identification by accurate mass (with an acceptable deviation of 3 ppm) and MS / MS spectra, Level 3: identification by accurate mass alone (with an acceptable deviation of 3 ppm).

[0520] Example 4: Conjugative plasmid expressing IAA

[0521] The IAA pathway, along with the AEC export proteins, can be moved into a conjugative plasmid that is able to move between bacterial cells belonging to certain species in the natural microbiome. This would allow natural strains to produce IAA, reducing the need for colonization of the administered host.

[0522] 4.1: Materials and methods

[0523] 4.1.1 Transformation

[0524] According to the manufacturer's protocol and chromosome manipulation, non-conjugative plasmids were constructed using in-Fusion cloning (Takara), and conjugative plasmid manipulation was completed using scarless lambda red-mediated recombination from plasmid p1910 (Datsenko & Wanner PNAS 97, 6640-45, 2000) (Blank et al. 2011 PLoS ONE). Primers used for chromosome modification are listed in Table 8, and sequences can be found in Table 7.

[0525] The sequences of the promoters and genes used are as follows: promoter Pc-tga (Seq ID No: 1), ipdC (Seq ID No: 41), taa1 (Seq ID No: 40), iad1 (Seq ID No: 42), aec (Seq ID No: 2), see Fig.19 .

[0526] Table 8

[0527]

[0528] The conjugative plasmid p2464 was prepared by amplifying the chloramphenicol cassette from pBAD33 using oli8116 x oli8117 and recombinantly engineering it into wt β10 in b3557. This resulted in strain b6584. This strain was subjected to whole genome sequencing to verify the correct modification.

[0529] Conjugative plasmid p2806 was prepared by amplifying the IAA pathway and chloramphenicol cassette from p2719 using primers oli8805 x oli8743 and recombinantly engineering it into wt β10 in b3557. This resulted in b7830. This strain was subjected to whole genome sequencing to verify the correct modification.

[0530] Strain b7951 was made based on b463 using three rounds of recombineering (Δp3, Δhly, and I-tev-clbS at csiR) with the primers listed in Table 1. This strain was subjected to whole genome sequencing to verify the correct modification.

[0531] Strain b8524 was prepared based on b7951 using one round of recombineering (ΔdapA) with the primers listed in Table 1. This strain was subjected to whole genome sequencing to verify the correct modification.

[0532] Strains B7848, b7919, and b7920 were prepared by joining p2806 into b5698, b7396, and b7399, respectively.

[0533] Strain B7962 was prepared by plating an overnight culture of b7919 on LB plates supplemented with streptomycin and selecting spontaneous resisters. This strain was subjected to whole genome sequencing to verify the correct modification.

[0534] 4.1.2 Conjugation assay

[0535] Donor and recipient are inoculated in LB with appropriate supplement, and grow to early exponential phase (OD0.4). Donor and recipient spotting are used for CFU, and are mixed at a ratio of 5:1. For each, 100 μL mixed cells are placed in the middle of LB flat board, and incubated ON at 37°C. Cells are scraped off from the flat board and resuspended in 300 μL LB. 10 times of dilutions are spotted onto non-selective LB flat board and on a flat board for donor and transconjugant selectivity. Flat board is incubated ON at 37°C and CFU is counted.

[0536] 4.1.3 In vivo studies

[0537] A 4-week mouse study was performed to test the ability of this strain to produce IAA in the intestine of mice. Mice were pretreated with 1 g / L streptomycin in drinking water for 24 hours from day 12 to day 11. Then, mice were treated with approximately 5×10 8 The dosage of CFU / dosage is orally administered twice a day for 4 weeks. The bacterial strain administered is b7962, which is expected to have a better chance of colonization in the mouse intestinal tract due to its origin in mice. Fecal samples are collected 3 times a week (Monday, Wednesday and Friday), and spotting is used to determine CFU / g feces. Further, fecal samples are collected at the 1st day (before administration), the 3rd day, the 10th day and the 29th / 30th day (before the end) for quantitative IAA in feces.

[0538] 4.2 Results

[0539] 4.2.1 IAA production from conjugative plasmids

[0540] A large 80 kb conjugative plasmid (β10) was isolated from wild-type E. coli isolate b3557. As described in Materials and Methods (see Section 4.1.1), a pathway for producing IAA was engineered into it. The pathway contains four genes (ipdC [Seq ID No: 41] -taa1 [Seq ID No: 40] -iad1 [Seq ID No: 42] -aec [Seq ID No: 2]) in this order expressed by the constitutive promoter Pc-tga (Seq ID No: 1). The chloramphenicol marker CAM was also inserted close to the pathway. The engineered β10IAA plasmid (p2806) has an overall size of 86 kb. A map of the engineered construct is shown in Fig.19 middle.

[0541] Move this plasmid into:

[0542] 1) Escherichia coli Symbioflor G6 / 7ΔtnaCAΔtrpR (b5698, producing b7848),

[0543] 2) a wild-type E. coli isolate (B7396, yielding B79190), and

[0544] 3) Wild-type E. hallii isolate (B7399, yielding b7919).

[0545] The wild-type strain was isolated from mouse feces. IAA production was compared between strains carrying a conjugative vector (CGV) and their wild-type counterparts. A strain previously developed to produce IAA (b6131, see Example 3.1 and Table 6) was included as a positive control, using the same approach but integrated into the chromosome along with other modifications. Fig. 20 As can be seen in Figure 3, all strains carrying CGV produced high levels of IAA.

[0546] 4.2.2 IAA production from conjugative vectors in vivo

[0547] A streptomycin-resistant version of b7919, b7962, carrying the IAA-producing CGV was introduced into mice (see Example 4.1.3). Mice were pretreated with 1 g / L streptomycin in drinking water before the start of twice daily oral dosing. Twice daily dosing resulted in 2×10 9 Initial colonization detected by CFU / g feces then slowly decreased to 4×10 5 CFU / g feces and remain at this level until termination (see Fig.21 ).

[0548] Initially, high levels of IAA production were observed ( Fig. 22), however, this declines over time.At the 3rd day, IAA levels rose to the mean value of 26nmol / g feces, which was reduced to 5.5nmol / g feces at the 10th day, and further reduced to 2.5nmol / g feces when terminating the same day (the 29th / 30th day).The reduction in IAA production follows the trend of the CFU of the decreasing number found in feces, which indicates the colonization of the decreasing level regardless of the dosage twice a day.The re-isolates from all time points are subjected to full genome sequencing, and it is found that the IAA pathway from CVG is lost or mutated.This suggests that the burden imposed on cells is too heavy, and the optimization of expression level is necessary.Transconjugants were separated from LB, EMB and MacConkey plates supplemented with chloramphenicol to explore whether CGV moves into the natural microbial group.Transconjugants of natural Escherichia coli and Klebsiella strains were observed from the 10th day, and found with increasing numbers during the research process.It is speculated that the delay caused by the start of transconjugation is due to research design. At the beginning of the study, streptomycin was used to kill most of the native microbiome in order to increase colonization by the donor strain, however, this also reduced the number of possible recipients until they had regrown.

[0549] 4.2.3 Conjugation efficiency of modified vector

[0550] Conjugation assays were performed to assess the effect of the IAA pathway on conjugation efficiency. The donor strain was b8524 (SBFG6 / 7ΔdapA) with p2464 (wild-type β10) or p2806 (β10 with the IAA pathway). These plasmids were conjugated with b5652 (recipient E. coli strain) following the solid conjugation protocol described in Materials and Methods, and the number of transconjugants and the total number of recipients ( Fig.23 ). Plasmid p2806 appeared to have the lowest level of transconjugants, indicating that the presence of the IAA pathway was a burden on the cells.

[0551] Example 5: Materials and Methods

[0552] 5.1 Transformation

[0553] Plasmids were constructed using in-Fusion cloning (Takara) according to the manufacturer's protocol (full sequences are provided in Table 7), and chromosomal manipulations were performed using scarless lambda red-mediated recombination (Datsenko & Wanner PNAS 97, 6640-45, 2000, Blank et al. 2011 PLoS ONE). Primers are listed in Table 9.

[0554] Table 9: Primers used for strain transformation

[0555]

[0556] *See Table 5

[0557] Bacterial strain b8344 is based on b463 (see Table 6 above) and is made using five rounds of recombination (Δp3, Δhly, ΔtnaCA, ΔtrpR and IAA to csiR) using the primers listed in Table 9. The resulting bacterial strain is prepared by plating overnight cultures on LB plates supplemented with streptomycin and selecting spontaneous resisters. Whole genome sequencing is performed on the bacterial strain to verify the correct modification. Therefore, b8344 is Symbioflor G6 / 7ΔtnaCA ΔtrpR Δp3Δhly IAA+(Ptga-IpdC-taal-iad1-aec)strR.

[0558] 5.2 In vivo work

[0559] 5.2.1 In vivo IAA production after a single oral dose

[0560] In vivo IAA production studies were performed using strain b8344. Studies were performed in five (5) C57B1 / 6 mice. In the study, mice were pretreated with 1 g / L streptomycin in drinking water for 24 hours from day 12 to day 1. On day 1 and until day 11, animals were dosed with a 5% prebiotic solution in drinking water. On day 1 (24 hours after streptomycin removal), approximately 1 x 10 8 CFU as a single oral dose to inoculate animals. From day 11 until day 16 (termination day), animals were supplied only with normal drinking water. In the study, the level of colonization was assessed by collecting fecal pellets every day (except days 6, 7 and 14 (further excluding day 8)). Fecal pellets were placed in PBS and then spotted for CFU measurement. Another fecal pellet was collected on days 1, 4, 11 and 16 and quickly frozen for IAA quantification by LC-MS analysis (A&M Labor, Germany, see Example 3.4 above for methods). The strain was independent of prebiotic treatment because it maintained a high level of colonization throughout the study, stabilizing at approximately 1×10 7 CFU / g feces level ( Fig.24 ).

[0561] LC-MS data showed that the animals had baseline levels below 1 nmol / g feces. IAA levels increased to a mean of 15 nmol / g feces on day 4 and further increased to a mean of 54 nmol / g feces on day 11, despite a 2 log CFU reduction in the b8344 strain on day 11. On the termination day, the animals had IAA levels of 42 nmol / g feces and approximately 1×10 7 The stable level of CFU / g feces ( Fig.25 ).

[0562] Based on these data, we have successfully engineered our strain to produce IAA in the mouse intestine and showed that it is able to produce IAA for up to 15 days after a single oral dose.

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[0606] Any part of the disclosure may be read in combination with any other part of the disclosure unless otherwise apparent from the context.

[0607] According to the present disclosure, all compositions and / or methods disclosed and claimed herein can be prepared and performed without undue experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it is obvious to those skilled in the art that changes can be applied to the compositions and / or methods described herein and the steps or step sequences of the methods without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A modified bacterium or plasmid (e.g., a conjugative plasmid) for producing and secreting indole-3-acetic acid (IAA), wherein the bacterium comprises one or more heterologous genes for the biosynthesis of IAA, and wherein: a. The bacterium further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium; or b. The plasmid further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium, and the bacterium comprises the plasmid and produces and secretes IAA.

2. The modified bacterium or plasmid (eg, conjugative plasmid) according to claim 1, wherein the one or more heterologous genes for the biosynthesis of IAA are selected from: A. A gene for converting tryptophan into tryptamine (optionally, a gene for tryptophan decarboxylase (tdc)), a gene for converting tryptamine into indole-3-acetaldehyde (IAAld) (optionally, a gene for monoamine oxidase (tynA)), a gene for converting IAA1d into IAA (optionally, a gene selected from indole-3-acetaldehyde dehydrogenase (iad1) and indole-3-acetaldehyde oxidase (aao1)); B. A gene for converting indole to tryptophan (optionally, a gene for trpB), a gene for converting tryptophan to tryptamine (optionally, a gene for tryptophan decarboxylase (tdc)), a gene for converting tryptamine to indole-3-acetaldehyde (IAAld) (optionally, a gene for monoamine oxidase (tynA)), a gene for converting IAA1 to IAA (optionally, a gene selected from indole-3-acetaldehyde dehydrogenase (iad1) and indole-3-acetaldehyde oxidase (aao1)); C. A gene for converting tryptophan into indole-3-pyruvate (IPyA) (optionally, a gene selected from L-tryptophan oxidase (staO), L-tryptophan aminotransferase (aro9), aspartate aminotransferase (aspC), L-tryptophan-pyruvate aminotransferase (taa1) and tryptophan dehydrogenase (trpDH)), a gene for converting IPyA into IAA1d (optionally, a gene for indole-3-pyruvate decarboxylase (ipdC)), a gene for converting IAA1d into IAA (optionally, a gene selected from iad1 and aao1); D. A gene for converting indole to tryptophan (optionally, a gene for trpB), a gene for converting tryptophan to indole-3-pyruvate (IPyA) (optionally, a gene selected from the group consisting of L-tryptophan oxidase (staO), L-tryptophan aminotransferase (aro9), aspartate aminotransferase (aspC), L-tryptophan-pyruvate aminotransferase (taa1), and tryptophan dehydrogenase (trpDH)), a gene for converting IPyA to IAA1d (optionally, a gene for indole-3-pyruvate decarboxylase (ipdC)), a gene for converting IAA1d to IAA (optionally, a gene selected from the group consisting of iad1 and aao1); E. A gene for converting tryptophan into indole-3-pyruvate (IPyA) (optionally, a gene selected from staO, aro9, aspC, taa1 and trpDH), a gene for converting IPyA into IAA (optionally, a gene for indole-3-pyruvate monooxygenase (e.g., a gene selected from YUC genes, yuc2 and yuc6)); F. A gene for converting indole to tryptophan (optionally, a gene for trpB), a gene for converting tryptophan to indole-3-pyruvate (IPyA) (optionally, a gene selected from staO, aro9, aspC, taa1, and trpDH), a gene for converting IPyA to IAA (optionally, a gene for indole-3-pyruvate monooxygenase (e.g., a gene selected from YUC genes, yuc2, and yuc6)); G. A gene for converting tryptophan into indole-3-acetamide (IAM) (optionally, it is a gene for tryptophan 2-monooxygenase (iaaM)), a gene for converting IAM into IAA (optionally, it is a gene for indoleacetamide hydrolase (iaaH)); H. A gene for converting indole to tryptophan (optionally, it is a gene for trpB), a gene for converting tryptophan to indole-3-acetamide (IAM) (optionally, it is a gene for tryptophan 2-monooxygenase (iaaM)), a gene for converting IAM to IAA (optionally, it is a gene for indoleacetamide hydrolase (iaaH)); I. A gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx) (optionally, a gene selected from tryptophan N-monooxygenase (CYP79B2) and tryptophan N-monooxygenase (CYP79B3)), a gene for converting IAOx to indole-3-acetonitrile (IAN) (optionally, a gene for indoleacetaldehyde oxime dehydratase (CYP71A13)), a gene for converting IAN to IAA (optionally, a gene for nitrilase (e.g., selected from nit1, nit2 and nit3)); J. A gene for converting indole to tryptophan (optionally, a gene for trpB), a gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx) (optionally, a gene selected from the group consisting of tryptophan N-monooxygenase (CYP79B2) and tryptophan N-monooxygenase (CYP79B3)), a gene for converting IAOx to indole-3-acetonitrile (IAN) (optionally, a gene for indoleacetaldehyde oxime dehydratase (CYP71A13)), a gene for converting IAN to IAA (optionally, a gene for nitrilase (e.g., selected from the group consisting of nit1, nit2, and nit3)); K. a gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx) (optionally, a gene selected from the group consisting of CYP79B2 and CYP79B3), a gene for converting IAOx to indole-3-acetonitrile (IAN) (optionally, a gene of CYP71A13), a gene for converting IAN to IAM (optionally, a gene of nitrile hydratase (nthAB)), a gene for converting IAM to IAA (optionally, a gene of iaaH); L. a gene for converting indole to tryptophan (optionally, a gene for trpB), a gene for converting tryptophan to indole-3-acetaldehyde oxime (IAOx) (optionally, a gene selected from the group consisting of CYP79B2 and CYP79B3), a gene for converting IAOx to indole-3-acetonitrile (IAN) (optionally, a gene for CYP71A13), a gene for converting IAN to IAM (optionally, a gene for nitrile hydratase (nthAB)), and a gene for converting IAM to IAA (optionally, a gene for iaaH); M. Any combination of pathways A to L; and N. Any part of pathways A to L.

3. The modified bacterium or plasmid (e.g., conjugative plasmid) according to claim 2, wherein the one or more heterologous genes for the biosynthesis of IAA comprise: i. indole-3-pyruvate decarboxylase (ipdC); ii. tryptophan-pyruvate aminotransferase 1 (taa1); and iii. Indole-3-acetaldehyde dehydrogenase (iad1).

4. The modified bacterium or plasmid (e.g., conjugative plasmid) according to claim 3, wherein: i. the ipdC is from a species of the genus Pantoea, for example from Pantoea agglomerans, and optionally has a nucleic acid sequence of SEQ ID No: 41; and / or ii. the taa1 is from an Arabidopsis species, such as Arabidopsis thaliana, and optionally has a nucleic acid sequence of SEQ ID No: 40; and / or iii. The iad1 is from a species of the genus Ustilago, for example from Ustilago maydis, and optionally has a nucleic acid sequence of SEQ ID No:

42.

5. The modified bacterium or plasmid (eg, conjugative plasmid) according to any preceding claim, wherein the one or more heterologous genes for the biosynthesis of IAA are each or all under the control of one or more constitutive promoters.

6. A modified bacterium or plasmid (e.g., a conjugative plasmid) for the biosynthesis of indole-3-acetic acid (IAA), comprising a heterologous gene for the biosynthesis of IAA, the heterologous gene encoding: i. indole-3-pyruvate decarboxylase (ipdC) from a Pantoea species, for example from Pantoea agglomerans, optionally having the nucleic acid sequence of SEQ ID No: 41; ii. a tryptophan-pyruvate aminotransferase 1 (taa1) from an Arabidopsis species, for example from Arabidopsis thaliana, optionally having the nucleic acid sequence of SEQ ID No: 40; and iii. indole-3-acetaldehyde dehydrogenase (iad1) from a species of the genus Ustilago, for example from Ustilago maydis, optionally having the nucleic acid sequence of SEQ ID No: 42; Each or all of the heterologous genes i. to iii. are under the control of one or more constitutive promoters.

7. The modified bacterium or plasmid (eg, conjugative plasmid) according to claim 6, wherein: a. The bacterium further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium; or b. The plasmid further comprises a heterologous gene encoding an export protein capable of exporting IAA outside the bacterium, and the bacterium comprises the plasmid and produces and secretes IAA.

8. The modified bacterium or plasmid (eg, conjugative plasmid) according to any preceding claim, wherein the heterologous gene encoding an exporter protein capable of exporting IAA is under the control of a constitutive promoter.

9. The modified bacterium or plasmid (eg, conjugative plasmid) according to any preceding claim, wherein the one or more heterologous genes for the biosynthesis of IAA are contained within an operon under the control of a single constitutive promoter.

10. The modified bacterium or plasmid (eg, conjugative plasmid) according to claim 9, wherein the operon comprises the following genes in downstream order: i. optionally from a Pantoea species, for example from Pantoea agglomerans, further optionally having an indole-3-pyruvate decarboxylase (ipdC) of the nucleic acid sequence of SEQ ID No: 41; ii. tryptophan-pyruvate aminotransferase 1 (taa1) optionally from Arabidopsis species, for example from Arabidopsis thaliana, further optionally having the nucleic acid sequence of SEQ ID No: 40; and iii. indole-3-acetaldehyde dehydrogenase (iad1) optionally from a Ustilago species, for example from Ustilago maydis, further optionally having the nucleic acid sequence of SEQ ID No:

42.

11. The modified bacterium or plasmid (e.g., conjugative plasmid) according to claim 9 or claim 10, wherein the one or more heterologous genes for the biosynthesis of IAA and the heterologous gene encoding an export protein capable of exporting IAA are contained within an operon under the control of a single constitutive promoter.

12. A modified bacterium or plasmid (eg, a conjugative plasmid) according to any one of claims 5 to 11, wherein the constitutive promoter is a strong constitutive promoter with an Anderson score >0.4 (eg, >0.5), such as a tac promoter, such as a Pc-tga promoter having a sequence of SEQ ID No:

1.

13. The modified bacterium or plasmid (e.g., conjugative plasmid) according to claim 12, wherein the promoter is a promoter selected from RelB, BolA, Hya, YiaG and RpoH promoters, such as a promoter selected from the following: RelB promoter sequence, σ70; BolA promoter sequence, σS, σ70; Hya promoter sequence, σS, σ70; YiaG promoter sequence, σS; RpoH promoter sequence P1, σ70; RpoH promoter sequence P2, σS; RpoH promoter sequence P3, σ24; RpoH promoter sequence P4, σ70; RpoH promoter sequence P5, σ70; and RpoH promoter sequence P6, σ54, in particular a promoter having a nucleotide sequence selected from any one of Seq ID No: 60 to 69, or a nucleotide sequence having 90% (or 95%) homology thereto.

14. The modified bacterium or plasmid (e.g., conjugative plasmid) according to any one of claims 11 to 13, wherein the one or more heterologous genes for the biosynthesis of IAA and the heterologous gene encoding an exporter capable of exporting IAA contained within an operon comprise the following genes in downstream order: i. optionally from a Pantoea species, for example from Pantoea agglomerans, further optionally having an indole-3-pyruvate decarboxylase (ipdC) of the nucleic acid sequence of SEQ ID No: 41; ii. tryptophan-pyruvate aminotransferase 1 (taa1) optionally from Arabidopsis species, for example from Arabidopsis thaliana, further optionally having the nucleic acid sequence of SEQ ID No: 40; and iii. an indole-3-acetaldehyde dehydrogenase (iad1) optionally from a species of the genus Ustilago, for example from Ustilago maydis, further optionally having the nucleic acid sequence of SEQ ID No: 42; and iv. a heterologous gene encoding an IAA export protein, Optionally wherein the operon is comprised by the chromosome of the modified bacterium.

15. The modified bacterium according to any preceding claim, wherein the bacterium further comprises a modification in endogenous tryptophanase (tnaA) and / or tnaC that reduces expression (or prevents expression) of the tnaA and / or tnaC, such as the bacterium further comprises a deletion of one or more nucleotides in endogenous tnaA and / or tnaC that prevents or reduces (e.g., prevents) transcription or expression of the tnaA and / or tnaC, such as comprising a deletion of nucleotides that express a TnaC peptide at least when transcribed.

16. The modified bacterium of any preceding claim, wherein the bacterium further comprises a modification in an endogenous tryptophan transcriptional repressor (trpR) gene that reduces expression of (or prevents expression of) the trpR, such as the bacterium further comprises a deletion of one or more nucleotides in an endogenous trpR gene that prevents or reduces (e.g., prevents) transcription or expression of the trpR.

17. The modified bacterium according to any preceding claim, wherein the one or more heterologous genes for the biosynthesis of IAA are all contained by the chromosome of the modified bacterium.

18. The modified bacterium according to any preceding claim, wherein the heterologous gene encoding an exporter protein capable of exporting IAA is comprised by the chromosome of the modified bacterium.

19. A modified bacterium or plasmid (eg, conjugative plasmid) according to any preceding claim, wherein: a. the bacteria are gram-negative bacteria; or b. The plasmid can be transferred by conjugation to a recipient bacterial cell, such as a Gram-negative recipient bacterial cell.

20. The modified bacterium according to any preceding claim, or the plasmid (eg, conjugative plasmid) according to claim 19, wherein the bacterium or bacterial cell is a strain selected from any of the strains in Table 3.

21. The modified bacterium according to any preceding claim, or the plasmid (e.g., conjugative plasmid) according to claim 19 or claim 20, wherein the bacterium or bacterial cell is an E. coli strain, such as E. coli from phylogroups A, B1 and / or E, or an E. coli strain present in a probiotic product, such as colinfant New Born (e.g., strain A034 / 86) or symbioflor2 (e.g., strains G1 / 2, G4 / 9, G5, G6 / 7 and G8, in particular strain G6 / 7), or Mutaflor (e.g., E. coli Nissle).

22. A modified bacterium or plasmid (e.g., a conjugative plasmid) according to any preceding claim, wherein the bacterium or bacterial cell strain has been modified to remove some or all (e.g., all) prophage genes present in the genome of the bacterium or bacterial cell, or wherein the bacterium or bacterial cell strain lacks some or all (e.g., all) prophage genes.

23. A modified bacterium or plasmid (e.g., a conjugative plasmid) according to any preceding claim, wherein the bacterium or bacterial cell strain has been engineered to remove any identified virulence factor (e.g., hlyA, hlyB, hlyC and / or hlyD or any combination thereof) present in the genome of the bacterium or bacterial cell, or wherein the bacterium or bacterial cell strain lacks a virulence factor (e.g., hlyA, hlyB, hlyC and / or hlyD or any combination thereof).

24. The modified bacterium or plasmid (eg, conjugative plasmid) according to any one of claims 20 to 23, wherein the bacterium or bacterial cell is a strain belonging to a genus selected from the group consisting of Bifidobacterium, Bacteroides, Lactobacillus, Lactobacillus casei, Plantarum lactis, Lactobacillus probiotics, Lactobacillus associating, Lactobacillus mucosus, and Lactococcus (eg, a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorphis, Bacteroides vulgaris, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lactobacillus paracasei, Plantarum lactis, Lactobacillus breve, Lactobacillus salivarius, Lactobacillus mucosus reuteri, and Lactococcus lactis), in particular a strain belonging to the genus Bifidobacterium or Bacteroides (eg, a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides monomorphis, Bacteroides vulgaris, and Bacteroides thetaiotaomicron).

25. The modified bacterium or plasmid (e.g., a conjugative plasmid) according to any preceding claim, wherein the bacterium or plasmid does not comprise any heterologous genes for the biosynthesis of tryptophan, such as genes encoding one or more genes selected from trpA, trpB, trpC, trpD, and trpE, such as all of trpA, trpC, trpD, and trpE.

26. The modified bacterium or plasmid (eg, conjugative plasmid) according to any preceding claim, wherein the bacterium or plasmid does not comprise other heterologous genes except: I. one or more heterologous genes for the biosynthesis of IAA; and / or II. a heterologous gene encoding an export protein capable of exporting IAA; III. Optional kill switch; and IV. Optionally any heterologous or modified auxotrophic gene.

27. The modified bacterium or plasmid (eg, conjugative plasmid) according to any one of claims 1 to 5 or 7 to 26, wherein the heterologous gene encoding an exporter capable of exporting IAA encodes an auxin efflux protein, such as an exporter selected from: (i). Auxin efflux carrier (AEC) family protein transporters (e.g. AEC family proteins from bacterial species); (ii). PIN family protein transporter (eg, a PIN family protein transporter from a plant species); and (iii). ABC family protein transporter, such as ABCD subfamily protein transporter or ABCB subfamily protein transporter, such as ABCB-PGP subfamily protein transporter (eg, wherein the ABC family protein transporter is from a plant species).

28. The modified bacterium or plasmid (eg, conjugative plasmid) according to claim 27, wherein the export protein is an AEC family protein transporter.

29. The modified bacterium or plasmid (eg, conjugative plasmid) according to claim 27 or 28, wherein the auxin efflux protein is from a Pantoea species such as Pantoea agglomerans.

30. The modified bacterium or plasmid (eg, conjugative plasmid) according to any one of claims 1 to 5 or 7 to 29, wherein the heterologous gene encoding an export protein comprises the nucleotide sequence of SEQ ID No:

2.

31. The modified bacterium or plasmid (eg, conjugative plasmid) according to any one of claims 1 to 5 or 7 to 27, wherein the exporter of IAA is a PIN transporter (eg, PIN2 or PIN7), optionally wherein the PIN transporter is from a plant species (eg, from an Arabidopsis species, such as from Arabidopsis thaliana), or wherein the heterologous gene encoding the exporter comprises the nucleotide sequence of SEQ ID No:

3.

32. The modified bacterium according to any preceding claim, wherein the bacterium comprises a kill switch, and / or wherein the bacterium comprises an auxotrophic trophy, such as a deletion of thyA.

33. The modified bacterium or plasmid (eg, conjugative plasmid) according to any preceding claim, wherein the production of IAA is comparable, such as statistically similar or within statistical error, in vitro at 30°C and 37°C.

34. The modified bacteria of any preceding claim, wherein the bacteria is capable of maintaining a stable level of colonization in a mouse model for at least 5 days (e.g., at least 6 days, or at least 7 days, or at least 8 days), optionally as measured by the number of colony forming units (CFU) in feces.

35. The modified bacterium of claim 34, wherein the colonization level is at least (approximately) 1×10 4 CFU / g feces, at least (approximately) 1×10 5 CFU / g feces, for example at least (approximately) 1×10 6 CFU / g feces, or at least (approximately) 1×10 7 CFU / g feces, or at least (approximately) 1×10 8 CFU / g feces.

36. The modified bacterium of any preceding claim, wherein the bacterium is capable of producing IAA for at least 24 hours (e.g., at least 48 hours, or at least 72 hours, or at least 96 hours) in a mouse model, optionally by measuring IAA levels in feces.

37. The modified bacterium of claim 36, wherein the IAA level is at least (about) 10 nmol / g feces, such as at least (about) 15 nmol / g feces, or at least (about) 20 nmol / g feces.

38. The modified bacterium of any one of claims 34 to 37, wherein the measuring is performed in an in vivo mouse model, e.g., as described in Example 4.2.2 for Group 6.

39. A host bacterial cell comprising a plasmid according to any one of claims 1 to 16 or claims 19 to 38, optionally wherein the plasmid is a conjugative plasmid, and optionally wherein the host bacterial cell is a bacterium according to any one of claims 19 to 26.

40. A pharmaceutical composition comprising the modified bacterium according to any one of claims 1 to 38 or comprising the host bacterial cell according to claim 39, and a pharmaceutically acceptable excipient or carrier.

41. The pharmaceutical composition according to claim 40, formulated for oral or rectal administration, preferably oral administration, for example as a capsule or a coated tablet.

42. A pharmaceutical composition according to claim 40 or claim 41 which is a lyophilised formulation or a packaged formulation for release in the lower intestinal tract of a subject, for example in the small intestine or large intestine of a subject.

43. The pharmaceutical composition of claim 42, formulated as an enteric-coated delayed-release capsule.

44. A pharmaceutical composition according to any one of claims 40 to 43, wherein the formulation comprises freeze-dried modified bacteria or host cells.

45. A pharmaceutical composition according to any one of claims 40 to 44, wherein the formulation comprises (approximately) 1×10 9 Colony forming units (CFU) per gram of the modified bacterium according to any one of claims 1 to 60 or the host bacterial cell according to claim 61.

46. ​​A method of producing IAA in the intestinal tract of a subject, comprising administering to the subject the modified bacterium of any one of claims 1 to 38, the host bacterial cell of claim 39, or the pharmaceutical composition of any one of claims 40 to 45.

47. A method of treating a metabolic disease, such as a cardiovascular metabolic disease, optionally selected from the group consisting of leaky gut, type 1 diabetes, type 2 diabetes (including complications of type 1 and type 2 diabetes, such as insulin sensitivity in type 2 diabetes), metabolic syndrome, Bard-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) deficiency, proprotein convertase subtilisin / kexin Type 1 (PCSK1) deficiency, Src homolog 2B1 (SH2B1) deficiency, prohormone 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 ulna-mammary syndrome (particularly selected from metabolic syndrome, type 2 diabetes (including complications of type 2 diabetes, such as insulin sensitivity in type 2 diabetes) and non-alcoholic fatty liver disease), the method comprising administering to a subject in need thereof a modified bacterium according to any one of claims 1 to 38, a host bacterial cell according to claim 39, or a pharmaceutical composition according to any one of claims 40 to 45.

48. The modified bacterium according to any one of claims 1 to 38, the host bacterial cell according to claim 39, or the pharmaceutical composition according to any one of claims 40 to 45 for use as a medicament.

49. The modified bacterium according to any one of claims 1 to 38, the host bacterial cell according to claim 39, or the pharmaceutical composition according to any one of claims 40 to 45, for use in the treatment of a metabolic disease, such as a cardiovascular metabolic disease, optionally selected from the group consisting of leaky gut, type 1 diabetes, type 2 diabetes (including complications of type 1 and type 2 diabetes, such as insulin sensitivity in type 2 diabetes), metabolic syndrome, Bard-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) deficiency, proprotein convertase subtilisin / kexin Type 1 (PCSK1) deficiency, Src homolog 2B1 (SH2B1) deficiency, prohormone 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 ulna-mammary syndrome (particularly selected from metabolic syndrome, type 2 diabetes (including complications of type 2 diabetes, such as insulin sensitivity in type 2 diabetes) and non-alcoholic fatty liver disease).

50. A method for treating or preventing cancer, in particular colorectal cancer (CRC) and / or pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), the method comprising administering to a subject in need thereof a modified bacterium according to any one of claims 1 to 38, a host bacterial cell according to claim 39, or a pharmaceutical composition according to any one of claims 40 to 45, thereby treating or preventing the cancer, And optionally wherein the subject is receiving chemotherapy (e.g., simultaneously with, before or after administration of the bacteria, plasmid (e.g., conjugative plasmid), host cell or pharmaceutical agent.

51. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD), the method comprising administering to a subject in need thereof the modified bacterium of any one of claims 1 to 38, the host bacterial cell of claim 39, or the pharmaceutical composition of any one of claims 40 to 45, thereby treating or preventing NAFLD.

52. A method for improving insulin resistance, lipid metabolism, oxidative stress and / or inflammatory stress, the method comprising administering to a subject in need thereof a modified bacterium according to any one of claims 1 to 38, a host bacterial cell according to claim 39, or a pharmaceutical composition according to any one of claims 40 to 45, thereby improving insulin resistance, lipid metabolism, oxidative stress and / or inflammatory stress.

53. according to the method or purposes described in any one of claims 46 to 52, wherein within one month (for example within 2 weeks, within one week, for example within 5, 4 or 3 days, particularly within 34 hours) of receiving the first dose of modified bacteria, host cell or pharmaceutical composition, a course of antibiotics is administered to the experimenter.

54. The method or use of claim 53, wherein the antibiotic treatment is selected from aminoglycosides (e.g., amikacin, liposomal amikacin, gentamicin, prazomicin, and tobramycin), β-lactam inhibitors (e.g., ceftolozane and cilastatin), β-lactamase inhibitors (e.g., avibactam, clavulanate, clavulanic acid, sulbactam, tazobactam, relebactam, and vaborbactam), carbapenems (e.g., doripenem, ertapenem, imipenem, and meropenem), cephalosporins (e.g., cefaclor, cefoperazone, cefazolin, cephalosporins), The drugs include oxadiazine, chlordiazepoxide ... cycline), ketolides (e.g., telithromycin), lincosamides (e.g., clindamycin), lipopeptides (e.g., daptomycin), macrocycles (e.g., fidaxomicin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), monoamines (e.g., aztreonam), nitrofurans (e.g., nitrofurantoin), nitroimidazoles (e.g., metronidazole and tinidazole), nucleoside analogs (e.g., monopiravir and remdesivir), oxazolidinones (e.g., linezolid and tedizolid), penicillins (e.g., amoxicillin, ampicillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin [00136] The invention also includes but is not limited to the following: inhibitors of the present invention, such as chloramphenicol, chloramphenicol, liposomal amphotericin B, and amphotericin B lipid complexes, polymerase acid endonuclease inhibitors (such as baloxavir maboxil), polymyxins (such as polymyxin mesylate, colistin, and polymyxin B), pleuromutilins (such as lefamulin), protease inhibitors (such as namatevir), rifampicin, streptogramins (such as quinupristin and dalfopristin), tetracyclines (such as eracycline, minocycline, omadacycline, and tetracycline), or a combination thereof.

55. A method for producing a modified bacterium according to any one of claims 1 to 38, wherein the heterologous gene is contained by the chromosome of the bacterium, the method comprising introducing the heterologous gene into the chromosome of the bacterium using recombinant engineering, Optionally wherein the one or more heterologous genes for biosynthesis of IAA are as defined in any one of claims 2 to 4, 6, 10 or 14; and / or Optionally wherein the heterologous gene encoding an exporter capable of exporting IAA is as defined in any one of claims 27 to 31; and / or Optionally wherein the promoter is as defined in claim 12 or claim 13, and growing the bacteria; and optionally formulating the bacteria into a pharmaceutical composition (optionally a pharmaceutical composition according to any one of claims 40 to 45); And optionally the pharmaceutical composition is packaged together with instructions for use (optionally for the treatment of any disease according to any one of claims 49 to 52).

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