Bacteria for the treatment of disorders

Abstract

To provide modified probiotics, pharmaceutical compositions thereof, and methods of modulating and treating disorders.SOLUTION: Provided herein are a bacterium comprising one or more gene(s) encoding a non-native phenylalanine metabolizing enzyme (PME) and further comprising one or more phage genome(s), wherein one or more phage genes in the one or more phage genome(s) comprise one or more mutations. Also provided are pharmaceutical compositions thereof, and methods of modulating and treating disorders.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application incorporates by reference U.S. Provisional Application No. 62 / 523,225, filed June 21, 2017, U.S. Provisional Application No. 62 / 552,785, filed August 31, 2017, U.S. Provisional Application No. 62 / 552,829, filed August 31, 2017, U.S. Provisional Application No. 62 / 614,213, filed January 5, 2018, U.S. Provisional Application No. 62 / 624,299, filed January 31, 2018, and U.S. Provisional Application No. 62 / 523,202, filed June 21, 2017, the entire contents of each of which are expressly incorporated herein by reference in their respective entireties. [Background technology]

[0002] Increasing scientific evidence suggests that probiotic bacteria may be useful in treating or preventing a variety of diseases or disorders associated with the digestive tract, including gastrointestinal disorders such as Crohn's disease and inflammatory bowel syndrome. More recently, genetically engineered bacteria have emerged as a potential new therapeutic treatment modality for gastrointestinal diseases and have also opened the field of bacterial therapy to numerous other indications, including metabolic disorders, inflammatory diseases, and cancer. One advantage of genetically engineered bacteria is their ability to specifically target one or more disease mechanisms. For example, in the case of gastrointestinal disorders, bacteria can be engineered to contain genes for the expression of anti-inflammatory agents or agents that help heal a disrupted gastrointestinal barrier, such as the short-chain fatty acid butyrate, as described, for example, in International Publication No. 2016141108. Genetically engineered bacteria can also be considered a treatment modality for various metabolic disorders, including, but not limited to, rare metabolic disorders resulting from inborn errors in metabolism or IEM. For example, as described in WO2016090343, bacteria have been genetically modified to treat phenylketonuria (PKU) by expressing one or more enzymes that metabolize phenylalanine, thereby consuming excess phenylalanine in the gastrointestinal tract.

[0003] Bacteriophages are the most common biological entities worldwide, and it is well documented that the majority of bacterial species, both Gram-positive and Gram-negative, contain one or more DNA bacteriophages that are integrated into the bacterial chromosome as so-called prophage (Clokie et al., Phages in Nature, Bacteriophage. 2011 January-February;1(1):31-45).

[0004] DNA phages can be either lytic or temperate. Lytic phages infect bacterial cells and then program the synthesis of progeny phages that are released from the lysed cells. Conversely, temperate fDNA phages establish a stable relationship with their host bacteria, in which the integrated phage DNA (i.e., prophage) replicates in concert with the host genome, and phage genes that are damaging to the host are not expressed. However, bacteriophage particles can be released from cells containing intact prophage by a process called induction, during which prophage genes necessary for lytic growth are turned on, progeny phage particles are produced, and they are released from the cell upon lysis (reviewed in Casjens, Prophages and bacterial genomics: what have we learned so far?; Mol Microbiol. July 2003;49(2):277-300). In some cases, induction may occur spontaneously and randomly in a small or large fraction of bacteria harboring the prophage, whereas in other cases, specific, often undefined, environmental signals may cause the simultaneous induction of a particular prophage in many cells, leading to bacterial cell death.

[0005] Not all prophages have the ability to undergo the lytic cycle: non-functional, i.e., defective or cryptic, prophage can reach high levels of abundance in many bacteria as a result of mutational decay and / or loss of one or more genes essential for the lytic cycle over thousands of bacterial replication cycles (Bobay et al., Pervasive domestication of defective prophages by bacteria, Proc Natl Acad Sci USA. 2014 Aug. 19; 111(33):12127-12132, and references therein). Summary of the Invention

[0006] In some embodiments, the disclosure provides a bacterium comprising one or more phage genomes, wherein one or more of the phage genomes are defective. In some embodiments, the disclosure provides a bacterium comprising one or more phage genomes, wherein one or more of the phage genomes are defective such that lytic phage is not produced. In some embodiments, the disclosure provides a bacterium comprising one or more phage genomes, wherein one or more of the phage genomes are defective such that one or more phage genes are not expressed. In some embodiments, the disclosure provides a bacterium comprising one or more phage genomes, wherein one or more phage genes in the one or more phage genomes comprise one or more mutations. In some embodiments, the one or more phage genomes are present in the natural state of the probiotic bacterium. In some embodiments, the bacterium encodes one or more lysogenic phages. In some embodiments, the bacterium encodes one or more defective phages, cryptic phages, or satellite phages. In some embodiments, the bacterium encodes one or more tailiocins or gene transfer agents.

[0007] In some embodiments of the present disclosure, one or more of the phage genomes are mutated. Such mutations may include one or more deletions of a partial or complete sequence of one or more phage genes. Alternatively, the mutations may include one or more insertions of one or more nucleotides into one or more phage genes. In another embodiment, the mutations may include one or more substitutions of a partial or complete sequence of one or more phage genes. In another embodiment, the mutations include one or more inversions of a partial or complete sequence of one or more phage genes in the phage genome. Furthermore, the mutations may include any combination of one or more deletions, insertions, substitutions, or inversions. In certain embodiments, the one or more mutations reduce or prevent the production and release of phage particles from the bacterium compared to the same bacterium without the one or more targeted mutations in the one or more phage genomes. In some embodiments, the bacterium is a probiotic bacterium. Non-limiting examples of such probiotic bacteria include Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and Lactococcus. In some embodiments, the bacterium is Escherichia coli strain Nissle. In some embodiments, the mutated phage genome is the E. coli Nissle phage 1 genome, the E. coli Nissle phage 2 genome, and / or the E. coli Nissle phage 3 genome. In one embodiment, the mutated phage genome is the E. coli Nissle phage 3 genome. In one embodiment, the mutation is located in or comprises one or more genes selected from the following: ECOLIN_09965, ECOLIN_09970, ECOLIN_09975, ECOLIN_09980, ECOLIN_09985, ECOLIN_09990, ECOLIN_09995, ECOLIN_10000, ECOLIN_10005, ECOLIN_10010、ECOLIN_10015、ECOLIN_10020、ECOLIN_10025、ECOLIN_10030、ECOLIN_10035、ECOLIN_10040、ECOLIN_10045、ECOLIN_10050、ECOLIN_10055、ECOLIN_10065、ECOLIN_10070、ECOLIN_10075、ECOLIN_10080、ECOLIN_10085、ECOLIN_10090、ECOLIN_10095、ECOLIN_10100、ECOLIN_10105、ECOLIN_10110、ECOLIN_10115、ECOLIN_10120、ECOLIN_10125、ECOLIN_10130、ECOLIN_10135、ECOLIN_10140、ECOLIN_10145、ECOLIN_10150、ECOLIN_10160、ECOLIN_10165、ECOLIN_10170、ECOLIN_10175、ECOLIN_10180、ECOLIN_10185、ECOLIN_10190、ECOLIN_10195、ECOLIN_10200、ECOLIN_10205、ECOLIN_10210、ECOLIN_10220、ECOLIN_10225、ECOLIN_10230、ECOLIN_10235、ECOLIN_10240、ECOLIN_10245、ECOLIN_10250、ECOLIN_10255、ECOLIN_10260、ECOLIN_10265、ECOLIN_10270、ECOLIN_10275、ECOLIN_10280、ECOLIN_10290、ECOLIN_10295、ECOLIN_10300、ECOLIN_10305、ECOLIN_10310、ECOLIN_10315、ECOLIN_10320、ECOLIN_10325、ECOLIN_10330、ECOLIN_10335、ECOLIN_10340、およびECOLIN_10345。In one embodiment, the mutations, e.g., one or more deletions, are located in or comprise one or more genes selected from the following: ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175. A pharmaceutically acceptable composition comprising the bacterium disclosed herein and a pharmaceutically acceptable carrier.

[0008] In some embodiments, the bacterium further comprises one or more circuits for expression of one or more effector molecules.

[0009] In some embodiments, the present disclosure relates to compositions and therapeutic methods for alleviating hyperphenylalaninemia. In some embodiments, the compositions comprise genetically engineered bacteria capable of expressing a phenylalanine metabolic enzyme (PME). See, for example, WO2017087580A1, the entire contents of which are incorporated herein by reference. Phenylanine is an essential amino acid found primarily in food proteins. Typically, a small amount is utilized for protein synthesis, and the remainder is hydroxylated to tyrosine in an enzymatic pathway requiring phenylalanine hydroxylase (PAH) and the cofactor tetrahydrobiopterin. Hyperphenylalaninemia is a group of diseases associated with excessive levels of phenylalanine, which can be toxic and cause brain damage. Primary hyperphenylalaninemia is caused by a deficiency in PAH activity due to mutations in the PAH gene and / or blockage of cofactor metabolism.

[0010] Phenylketonuria (PKU) is a severe form of hyperphenylalaninemia caused by mutations in the PAH gene. PKU is an autosomal recessive genetic disease that ranks as the most common inborn error of metabolism worldwide (1 in 3,000 births), affecting approximately 13,000 patients in the United States. Over 400 different PAH gene mutations have been identified (Hoeks et al., 2009). The accumulation of phenylalanine (phe) in the blood can cause severe damage to the central nervous system in children and adults. If left untreated in newborns, PKU can cause irreversible brain damage. Treatment for PKU currently involves completely eliminating phenylalanine from the diet. Most natural sources of protein contain phenylalanine, an essential amino acid required for growth. This means that patients with PKU rely on medical foods and phenylalanine-free protein supplements, along with amino acid supplements that provide just enough phenylalanine for growth. This diet is difficult for patients and affects their quality of life.

[0011] Current PKU therapy requires a significantly modified diet consisting of protein restriction. Treatment from birth generally reduces brain damage and mental retardation (Hoeks et al., 2009; Sarkissian et al., 1999). However, protein-restricted diets must be carefully monitored, and essential amino acids and vitamins must be supplemented in the diet. Furthermore, access to low-protein foods is challenging because they are more costly than high-protein, unmodified foods (Vockley et al., 2014). Growth retardation is common in children with PKU who continue a low-phenylalanine diet (Dobbelaere et al., 2003). In adults, additional problems such as osteoporosis, maternal PKU, and vitamin deficiencies may develop (Hoeks et al., 2009). Excessive levels of phenylalanine in the blood, which can freely cross the blood-brain barrier, can also cause neurological disorders, behavioral problems (e.g., irritability, fatigue), and / or physical symptoms (e.g., seizures, skin rashes, musty body odor). Although international guidelines recommend lifelong dietary phenylalanine restriction, this is widely regarded as difficult and unrealistic ( Sarkissian et al., 1999 ), and “continued efforts are required to overcome the greatest challenge to living with PKU: lifelong adherence to a low-phenylalanine diet” ( Macleod et al., 2010 ).

[0012] In a subset of patients with residual PAH activity, oral administration of the cofactor tetrahydrobiopterin (also known as THB, BH4, Kuvan, or sapropterin) can be used in conjunction with dietary restriction to lower blood phenylalanine levels. However, cofactor therapy is costly and is only suitable for mild forms of phenylketonuria. The annual cost of Kuvan can be as high as $57,000 per patient, for example. Furthermore, side effects of Kuvan can include gastritis and severe allergic reactions (e.g., wheezing, dizziness, nausea, and flushing of the skin).

[0013] The enzyme phenylalanine ammonia lyase (PAL) can metabolize phenylalanine to nontoxic levels of ammonia and trans-cinnamic acid. Unlike PAH, PAL does not require THB cofactor activity to metabolize phenylalanine. Oral enzyme therapy using PAL has been studied, but "human and even animal studies have not continued because PAL is not available in sufficient quantities at an affordable cost" (Sarkissian et al., 1999). A pegylated form of recombinant PAL (PEG-PAL) is also under development as an injectable treatment. However, most subjects administered PEG-PAL suffer from injection site reactions and / or develop antibodies to this therapeutic enzyme (Longo et al., 2014). Therefore, there is a significant unmet need for effective, reliable, and / or long-term treatments for diseases associated with hyperphenylalaninemia, including PKU. There is also an unmet need for treatments that control patients' blood Phe levels while allowing for the consumption of more natural protein.

[0014] In some embodiments, the present disclosure provides genetically engineered bacteria that encode and express phenylalanine ammonia lyase and / or phenylalanine hydroxylase and / or L-amino acid deaminase and are capable of alleviating hyperphenylalaninemia. The phenylalanine ammonia-lyase (PAL) enzyme can metabolize phenylalanine to nontoxic levels of ammonia and trans-cinnamic acid. Unlike PAHs, PAL does not require THB cofactor activity to metabolize phenylalanine. L-amino acid deaminase (LAAD) catalyzes the oxidative deamination of phenylalanine to produce phenylpyruvic acid and trace amounts of ammonia and hydrogen peroxide. Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries, and PPA is the starting material for synthesizing D-phenylalanine, a raw material intermediate in the production of many chiral drugs and food additives. Therefore, LAAD has been studied from the perspective of industrial PPA production (Hou et al., 2015, Appl Microbiol Biotechnol. 2015 Oct;99(20):8391-402; "Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches"). Phenypyruvic acid cannot cross the blood-brain barrier (Steele, Fed Proc. 1986 Jun;45(7):2060-4; "Blood-brain barrier transport of the alpha-keto acid analogs of amino acids.” This conversion has shown utility in controlling the neurological phenotype of PKU.

[0015] In certain aspects, the present disclosure relates to genetically engineered bacteria capable of alleviating hyperphenylalaninemia in mammals. In certain aspects, the compositions and methods disclosed herein can be used to treat diseases associated with hyperphenylalaninemia (e.g., phenylketonuria). In certain embodiments, the genetically engineered bacteria are non-pathogenic and can be introduced into the gastrointestinal tract to reduce toxic levels of phenylalanine. In certain embodiments, phenylalanine ammonia-lyase and / or phenylalanine hydroxylase and / or L-amino acid deaminase are stably produced by the genetically engineered bacteria, and / or the genetically engineered bacteria are capable of in vivo and / or in vivo expression. The genetically engineered bacteria are stably maintained in vitro. In certain embodiments, the genetically engineered bacteria further comprise a phenylalanine transporter gene for increasing phenylalanine uptake. The present invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, as well as methods for modulating and treating disorders associated with hyperphenylalaninemia.

[0016] The genetically engineered bacteria may also include one or more gene sequences associated with biosafety and / or biocontainment, e.g., a death switch, a gene guard system, and / or an auxotrophy. In some embodiments, the engineered bacteria may include an antibiotic resistance gene. Expression of these gene sequences may be regulated using various promoter systems, such as any of the promoter systems disclosed herein, which may include the use of the same promoter to regulate one or more different genes, may include the use of different copies of the same promoter to regulate different genes, and / or may include the use of different promoters used in combination to regulate the expression of different genes. The use of different regulatory or promoter systems to control gene expression provides flexibility (e.g., the ability to differentially regulate gene expression under different environmental conditions and / or the ability to temporally control gene expression) and the ability to "fine-tune" gene expression, any or all of which may be useful for optimizing gene expression and / or bacterial growth.

[0017] In some embodiments, the bacteria inhibit the production of proteins associated with inflammation or inflammatory responses or immunosuppression, liver damage, metabolic diseases, and the like, in hypoxic conditions and in the presence of disease- or tissue-specific molecules or metabolites. Any one or more effector molecules can be expressed in the presence of a molecule or metabolite present in the gut or tumor microenvironment, or in the presence of some other metabolite that may or may not be present in the gut or tumor microenvironment. In some embodiments, any one or more circuits are present on one or more plasmids (e.g., high copy or low copy) or integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the engineered bacterium further comprises one or more of the following: (1) one or more auxotrophies, such as any auxotrophy known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more death switch circuits, such as any of the death switches described herein or known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such as any of the transporters described herein or known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein or otherwise known in the art, and (6) one or more combinations of such additional circuits. Compositions of the bacteria and methods for the treatment, prevention, or management of one or more diseases or disorders are also provided. [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1A shows a schematic of the predicted phage locations on the EcN genome (CP007799.1) from the PHAST analysis. Three high-scoring intact phages are labeled phages 1–3. Phage 1 (PHAST score 110) is 18.8 kb long and spans coordinates 241,563–260,441 within the EcN genome. Phage 2 (PHAST score 150) is 52.4 kb long and spans coordinates 1,325,883–1,378,287. Phage 3 (PHAST score 150) is 59 kb long and spans coordinates 3,405,101–3,418,180. Several low-scoring phages, designated "incomplete" or "doubtful" by the PHAST algorithm, were also identified; they do not contain all of the major components of the phage and may therefore represent partial phages or false-positive predictions. Abbreviations: EcN = E. coli Nissle 1917; PHAST = Phage Search Tool software; kb = kilobases. Figure 1B shows a table illustrating five putative prophages in the Nissle genome (three intact prophages, one incomplete prophage, and one suspected prophage) according to PHASTER scoring. [Figure 2] Figure 1 is a schematic diagram showing one of three high-scoring phages in Nissle, referred to herein as "Phage 1," containing all major components of the phage, using the Phast tool. Putative genes are labeled: Hyp = hypothetical, PLP = other phage-like protein, Oth = other, RNA = tRNA, TRA = transposase. [Figure 3] Figure 1 is a schematic diagram showing one of three high-scoring phages in Nissle using the Phast tool, referred to herein as "phage 2," containing all major components of the phage. Putative genes are labeled: Hyp = hypothetical, PLP = other phage-like protein, Oth = other, RNA = tRNA, TRA = transposase, Lys = lytic, Ter = terminase, Coa = coat, Sha = tail shaft, Fib = tail fiber. [Figure 4] Figure 1 is a schematic diagram showing one of three high-scoring phages in Nissle using the Phast tool, referred to herein as "phage 3," containing all major components of a phage. Putative genes are labeled: Hyp = hypothetical, PLP = other phage-like protein, Oth = other, RNA = tRNA, TRA = transposase, Lys = lytic, Ter = terminase, Coa = coat, Sha = tail shaft, Fib = tail fiber. [Figure 5] Figure 5A shows the first of two lower-scoring "failed" or "suspect" phages identified using the Phast tool. Putative genes are labeled: Hyp = hypothetical, PLP = other phage-like proteins, Oth = other, RNA = tRNA, TRA = transposase, int = integrase, Att = attachment site. Figure 5B shows the second of two lower-scoring "failed" or "suspect" phages identified using the Phast tool. Putative genes are labeled: Hyp = hypothetical, PLP = other phage-like proteins, Oth = other. [Figure 6] FIG. 1 is a schematic representation of the predicted Nissle phage 3 sequence (59,056 bp). [Figure 7] Schematic representation of the phage 3 deletion within the phage 3 genome used in SYN-PKU-2002. [Figure 8] FIG. 1 is a schematic diagram showing a 49,496 bp phage 3 sequence containing a knockout deletion, such as that contained in SYN-PKU-2002. [Figure 9] FIG. 1 is a schematic diagram of sections of a phage that can be deleted to inactivate the phage, for example, as deleted in SYN-PKU-2002. [Figure 10]This is a schematic diagram showing subregions within the 43 kb phage 3 sequence aligning to sequences from other E. coli strains. The sequence identified as phage 3 was compared to 5691 E. coli and Shigella genome assemblies downloaded from NCBI. The left column lists the accession numbers of the E. coli genomes that were positive in the analysis. At the top of the diagram are the coordinates of the DNA sequence according to the number of kb from the start of the sequence. Lines indicate the sequence from each specific E. coli genome that aligns with the phage 3 DNA sequence. Abbreviations: E. coli = Escherichia coli; kb = kilobases; NCBI = National Center for Biotechnology Information; DNA = deoxyribonucleic acid. [Figure 11] Figure 1 is a schematic diagram showing partial regions within the 43 kb phage 3 sequence aligned to sequences of other E. coli strains. The region within the shaded box was selected as the site for deletion in the phage 3 knockout strategy. [Figure 12] Figure 1 is a bar graph showing the distribution of the number of predicted "intact" phages across a set of 287 E. coli genomes derived from Refseq. The Refseq database was analyzed for the number of intact phages present in published complete E. coli genomes. Although the histogram shows a non-normal distribution, it is clear that almost all E. coli genomes contain intact prophages, and the majority of published complete E. coli genomes contain more intact prophages than EcN. Abbreviations: E. coli = E. coli; EcN = E. coli Nissle 1917; Refseq = Reference Sequence. [Figure 13] Figure 1 shows a DNA gel electrophoresis experiment validating phage-specific PCR primers. Performance of PCR primer pairs for phages 1, 2, and 3 and rpoB on EcN genomic DNA is shown. Abbreviations: bp = base pairs; EcN = E. coli Nissle; rpoB = β subunit of bacterial RNA polymerase. [Figure 14]Figure 1 shows a DNA gel electrophoresis experiment showing the EcN prophage region amplified from an ATCC13706 plaque plug. Abbreviations: bp = base pairs; EcN = E. coli Nissle1917; rpoB = β subunit of bacterial RNA polymerase. [Figure 15] Comparison of blood phenylalanine concentrations relative to baseline 4 hours after subcutaneous phenylalanine injection between SYN-PKU710 and SYN-PKU708 strains. Mice received a single dose of phenylalanine at 0.1 mg / g body weight via subcutaneous injection. At 1, 2, and 3 hours after Phe challenge, bacteria (or water) were administered to mice via oral gavage (300 μl / dose, total 3 × e10 cfu / mouse). The percent reduction in ΔPhe in SYN-PKU710 and SYN-PKU708 was calculated to be 29% and 40%, respectively. [Figure 16] Figure 16A shows line graphs depicting TCA production (Figure 16A) and phenylpyruvate production (Figure 16B) in SYN-PKU-2002, both measures of in vitro phenylalanine degradation by SYN-PKU-2002. SYN-PKU-2002 was prepared by growing it in lysogeny broth (LB) aerobically (uninduced) or anaerobically with the addition of IPTG and arabinose (induced). In vitro, incubation of activated SYN-PKU-2002 in the presence of phenylalanine resulted in the production of TCA and PP over time, demonstrating that SYN-PKU-2002 can metabolize phenylalanine. [Figure 17]Figures 17A and 17B show the changes in phenylalanine levels and hippuric acid recovery in mice gavaged with either streptomycin-resistant Nissle or the phage-free strain SYN-PKU-2002 (phage-free SYN-PKU-710). Mice received a single subcutaneous dose of phenylalanine (0.1 mg / g body weight). At 1, 2, and 3 hours after Phe challenge, mice received bacteria (or water, not shown) via oral gavage (3 x 250 μl). Whole blood was collected via submandibular bleeding at each time point and analyzed for phenylalanine levels. Urine collection in metabolic cages began immediately after the first bacterial challenge and continued throughout the study and was analyzed for hippuric acid levels. [Figure 18] Figure 18A shows the change in phenylalanine levels after Phe challenge (Figure 18A) and the recovery of hippuric acid from urine collected from animals treated with the indicated doses of SYN-PKU-2002 (Figure 18B). Briefly, animals were placed in metabolic cages (three mice per cage, two cages per group) and administered a single dose of phenylalanine (0.1 mg / gram body weight) via subcutaneous injection. At 1, 2, and 3 hours after Phe challenge, mice were administered bacteria by oral gavage at doses of 1 × 10 11 , 5 × 10 10 , 2.5 × 10 10 , 1.25 × 10 10 , 6.25 × 10 9 , or 3.13 × 10 9 cells. SYN-PKU901 was administered orally to the control group (n = 9) at the highest dose of 1 × 10 11 cells. Urine was collected from all animals until 4 hours after Phe challenge. To determine changes in serum Phe, blood was collected by submandibular bleeding at T = 0 and T = 4 hours in both SYN-PKU-2002- and SYN-PKU901-treated mice in the highest dose group (1 × 10 cells). [Figure 19]Figure 1 shows the results of an in vivo competition experiment between the phage-containing strain SYN-PKU-713 and the phage-free strain SYN-PKU-2001. Mice were administered equal doses (approximately 3 x 10 cells) daily for three days. As described in the Examples, fecal pellets were collected daily, and CFUs were determined by plating assay based on the differential antibiotic resistance of the two strains. The results show no significant differences in transport or colonization between Nissle's phage-free PKU strains SYN-PKU-713 and SYN-PKU-2001. [Figure 20] 1 is a graph showing gastric phenylpyruvic acid measurements in two pigs at various time points before and after administration of SYN-PKU-2001. [Figure 21] This graph shows the conversion efficiency of oral trans-cinnamic acid to urinary hippuric acid in non-human primates. NHPs (n=6) were orally administered 13C-trans-cinnamic acid (13C-TCA) and urine was collected over 6 hours. 13C-hippuric acid (13C-HA) was measured by mass spectrometry. The percentage of urinary 13C-HA recovered as a function of administered 13C-TCA was calculated and used as a normalization factor for HA recovery in subsequent experiments. This factor accounts for TCA that is not converted to HA or is lost due to incomplete urine collection, allowing for a more accurate description of strain activity. [Figure 22]Figures 22A, B, C, D, and E are graphs showing profiling and efficacy in non-human primates (NHPs). In Figure 22A, fasted NHPs (n=6) were administered 5 g of peptide (left) or mock challenge (right) alone (black bars), or 5 g of peptide (left) or mock challenge (right) together with 5 x 10 cells of SYN-PKU-2002 (striped bars), and urine was collected for 6 hours. Normalized HA recovery is shown as the mean ± standard deviation. Animals administered SYN-PKU during the study performed in Figure 22A also received 13C-phenylalanine intravenously (IV) 1 hour after peptide or mock challenge (Figure 22B). Normalized urinary 13C-HA (which can only be derived from intravenously administered 13C-Phe) found in animals administered peptide challenge is shown as black bars. Urinary 13C-HA was not recovered in animals that remained fasted. In Figure 22C, fasted NHPs were orally administered d5-phenylalanine (d5-Phe) with or without SYN-PKU-2002. The dashed line represents the amount of d5-Phe administered. d5-Hippuric acid (d5-HA) was found only in animals administered SYN-PKU-2002 (strip bar). Data represent the mean ± standard deviation (n = 6) of normalized d5-HA recovery. Serum d5-Phe was measured in NHPs receiving SYN-PKU-2002 (light gray line) or mock-treated (dark gray line) (Figure 22D). Data represent the mean ± standard deviation (n = 6) of d5-Phe concentrations. In Figure 22E, NHPs received d5-Phe alone or d5-Phe with 5 x 10 cells of SYN-PKU-2002. Blood was collected over 6 hours, and the area under the curve for serum d5-Phe was calculated. Data show AUC ± upper and lower limits of the 90% confidence level, respectively. [Figure 23]Figures 23A and 23B show the detection of SYN-PKU-2002-specific metabolites in the serum of non-human primates. Using LC-MS / MS, serum concentrations of d5-HA (Figure 23A) and d5-TCA (Figure 23B) were measured in non-human primates orally administered d5-Phe and SYN-PKU-2002. When d5-Phe was administered in the absence of SYN-PKU-2002, no detectable d5-HA or d5-TCA was detected (data not shown). The presence of these metabolites demonstrates SYN-PKU-2002-specific activity in these animals. [Figure 24] 24A and 24B show the conversion of trans-cinnamic acid to urinary hippuric acid in NHPs. [Figure 25] 1 is a graph showing that SYN-PKU-2002 metabolizes Phe when orally administered in healthy non-human primates (NHPs). Oral gavage administration of SYN-PKU-2002 reduces the spike in blood Phe levels observed when a protein challenge is administered with radiolabeled Phe. [Figure 26]Figures 26A, 26B, 26C, and 26D are graphs showing SYN-PKU-2002 dose-dependent Phe conversion and plasma biomarker production in non-human primates following a single administration of SYN-PKU-2002 and protein meal, demonstrating the significant activity and efficacy of SYN-PKU-2002 in the NHP model. Fasted NHPs (n=5 per dose group) were administered a 5g peptide bolus along with the indicated dose (CFU) of SYN-PKU-2002. Urine was collected over 6 hours, and serum was collected at 0, 0.5, 1, 2, 4, and 6 hours. Figure 26A is a graph showing normalized urinary HA recovery from dose groups, presented as mean ± standard deviation. Figures 26B and 26C are graphs showing the calculated AUC for serum HA and serum TCA concentrations. White bars represent mean AUC ± standard deviation. Figure 26D is a graph showing serum Phe concentrations determined at the indicated time points. The three highest doses administered in a dose-response manner are shown compared to the no-cell control, as these three doses showed a significant decrease in the AUC of serum Phe (p<0.05). [Figure 27] 27A-C are graphs showing SYN-PKU-2002 dose-dependent conversion of Phe from casein (FIG. 27A), TCA levels (FIG. 27B), and hippuric acid (FIG. 27C) in NHPs. Blood metabolites were collected for 6 hours. [Figure 28] FIG. 1 is a graph showing SYN-PKU-2002 conversion of Phe in NHPs resulting in increased protein intake, which corresponds to a 2.5-fold increase in protein intake in PKU patients. [Figure 29]1 is a graph showing the in vitro activity of SYN-PKU-2002. 1 x 10 active cells were analyzed for PAL activity (dark blue bars, left y-axis) and LAAD activity (light blue bars, right y-axis) in 50 mM Phe assay buffer. Cells were pre-induced with L-arabinose (+ara), IPTG (+IPTG), or in an anaerobic chamber (-O), and TCA and PP rates were calculated by linear regression of TCA and PP production over time. Graphs show the mean and standard deviation of three biological replicates. [Figure 30] This graph shows the effect of dapA deletion on SYN-PKU-2002 growth in vitro. To characterize the growth of SYN-PKU-2002 containing mutations in the E. coli Nissle (EcN) and dapA genes, both strains were incubated in LB medium with (+) or without (-) diaminopimelic acid (DAP; 100 μg / mL) at 37°C for 960 minutes under constant shaking. OD was measured every 10 minutes to assess cell growth over time. The average of three biological replicates and two technical replicates is plotted for each time point. The data demonstrate that SYN-PKU-2002 cannot grow without the addition of exogenous DAP to the growth medium. [Figure 31]Figures 31A and 31B show the PAL activity of SYN-PKU-2002 toward peptides. SYN-PKU-2002 was grown in a bioreactor and induced for PAL and LAAD activity. Activated cells were incubated at 37°C for 60 minutes in Phe assay medium containing 50 mM Phe in the form of free Phe, Phe-Pro, Phe-Gly-Gly, Phe-Val, or Gly-Phe, or in 5 g / L peptone or tryptone. The total concentration of trans-cinnamic acid (Figure 31A) or phenylpyruvate (Figure 31B) produced was determined over time by LC-MS / MS, and the production rates of TCA and PP were calculated by linear regression. Significant PAL activity was observed with all substrates. LAAD activity was observed only when free Phe was used as a substrate; activity was reduced when the complex substrates peptone or tryptone were used. The graph shows the mean and standard deviation of three biological replicates. [Figure 32] 1 is a graph showing the growth characteristics of SYN766, a diaminopimelic acid auxotroph, at various concentrations of diaminopimelic acid. SYN766 (E. coli Nissle1917, ΔdapA) was incubated in growth medium containing decreasing concentrations of DAP for 960 minutes at 37°C under constant shaking. OD was measured every 10 minutes to assess cell growth over time. The average of three biological replicates is plotted for each time point. [Figure 33] Figure 1 shows the growth characteristics of various strains of E. coli Nissle in LB growth medium without diaminopimelic acid. To characterize the growth characteristics of various modified strains of EcN in the absence of DAP, cultures were incubated in LB without DAP at 37°C for 960 minutes under constant shaking. OD was measured every 10 minutes to assess cell growth over time. The average of three biological replicates and two technical replicates is plotted for each time point. [Figure 34]1 is a graph showing the growth characteristics of various strains of EcN in LB growth medium containing 100 μg / mL diaminopimelic acid. To characterize the growth characteristics of various modified strains of EcN in the presence of DAP, they were incubated in growth medium containing 100 μg / mL DAP at 37° C. for 960 minutes with constant shaking. OD was measured every 10 minutes to assess cell growth over time. The average of three biological replicates is plotted for each time point. [Figure 35] Figures 35A, B, and C show the effects of DAP auxotrophy and Phe degradation activity on EcN survival and transport in C57BL / 6 mice. Figure 35A shows the effect of DAP auxotrophy on fecal clearance in Group 1 mice (SYN-PKU901 / SYN766). Figure 35B shows the effect of genetic manipulation for Phe degradation on fecal clearance in Group 2 mice (SYN-PKU901 / SYN3282). Figure 35C shows the effect of genetic manipulation for Phe degradation and DAP auxotrophy on fecal clearance in Group 3 mice (SYN-PKU-2001 / SYN3282). Mixed doses of bacteria were orally administered to C57BL / 6 mice (n=5). To determine the number of administered bacteria, doses were plated in quadruplicate for CFU counts. At each time point, feces were collected, homogenized, and plated for bacterial CFU determination on antibiotic-selective media. For each time point, data represent the mean CFU / mg count of five fecal samples ± standard deviation and are normalized for each strain as a percentage of the initial CFU administered, unless otherwise noted in the Appendix. This normalization allows for direct comparison of survival / clearance between two strains within a group, despite variations in the actual CFU administered for each strain at T=0. [Figure 36]These figures show the effects of DAP auxotrophy and Phe-degrading activity on EcN transport and clearance in C57BL / 6 mice (Figures 36A-F). SYN-PKU901 or SYN-PKU-2001 was orally administered to C57BL / 6 mice (9 × 10 CFU / dose, n = 3 / time point). At the indicated times, effluent from the stomach (A), upper small intestine (B), middle small intestine (C), lower small intestine (D), cecum (E), and colon (F) was collected and plated for CFU counts. CFU determination was performed by microdilution in antibiotic-selective medium. For each time point, data represent CFU determined from triplicate effluent samples ± standard deviation. At 48 h post-administration, no CFUs were measured in any sample, indicating complete bacterial clearance. [Figure 37] Schematic diagram of the SYN-PKU-2002 genome. The locations of the genomic modification sites in SYN-PKU-2002 are indicated, with kbp designations indicating chromosomal locations relative to the 0 / 5.4 Mb reference marker. The chromosomal origin of replication is indicated by a red line. The green text box indicates the PheP gene insertion, the purple text box indicates the PAL gene insertion, the orange text box indicates the LAAD gene insertion, and the gray text box with a Δ symbol indicates the location of the dapA and Φ deletions. Italicized gene names in parentheses refer to the upstream and downstream genes surrounding the inserted gene. [Figure 38] Figure 1 shows SYN-PKU-2002 dose-dependent Phe conversion and plasma biomarker production in non-human primates following a single administration of SYN-PKU-2002 and protein meal, demonstrating significant activity and efficacy of SYN-PKU-2002 in the NHP model. Fasted NHPs (n=5 per dose group) were administered a 5g peptide bolus along with the indicated dose (CFU) of SYN-PKU-2002. Plasma was collected at 0, 0.5, 1, 2, 4, and 6 hours after administration at time 0. Each point represents the HA concentration measured in plasma at that time point after administration. Standard deviations are shown as vertical bars at each point. [Figure 39]Figure 1 shows the characterization of SYN-PKU-2001 in the gastrointestinal tract of non-human primates. Cynomolgus monkeys were administered 5.5 grams of peptone, 5 mL of 0.36 M sodium bicarbonate, 25 mg / kg of D5-phenylalanine, and SYN-PKU-2001 and euthanized 0.5 or 2 hours after administration. After euthanasia, tissue samples were collected from various sections of the gastrointestinal tract and analyzed to determine the concentrations of Phe and SYN-PKU-2001 in each section. DETAILED DESCRIPTION OF THE INVENTION

[0019] In one aspect, the present disclosure provides a bacterium that contains an endogenous phage and includes one or more modifications to the phage sequence. In some embodiments, the modifications alter the properties of the prophage sequence. Such mutations include one or more partial or complete deletions of one or more phage genes, one or more insertions of one or more nucleotides into one or more phage genes, one or more partial or complete substitutions of one or more phage genes in the phage genome, one or more inversions of one or more phage genes, or a combination thereof.

[0020] The present disclosure provides compositions comprising novel bacteria for the treatment of disorders, the bacteria comprising one or more bacteriophages or prophages in their natural state. In some embodiments, the bacteria comprise one or more modifications to the genome of one or more phages. In some embodiments, the one or more modifications render the phage or prophage inactive. In some embodiments, the bacteria are modified by expression of one or more effector molecules or The bacteria are further genetically modified to contain one or more genes for the production of erythropoietin, a soluble form ... and a soluble form of erythropoietin. Methods are provided for the production and use of these genetically engineered bacteria in novel therapies for the treatment of disorders.

[0021] In one embodiment, E. coli Nissle is used as the starting point, parent strain, or "chassis" for the genetically engineered bacterium. In one embodiment, the modified bacteriophage is a phage that is endogenous to E. coli Nissle in that it exists in the bacterium in its natural state.

[0022] In some embodiments, the genetically engineered bacterium comprises one or more genes encoding one or more effectors (e.g., PMEs). In some embodiments, the genetically engineered bacterium comprises one or more genes encoding PAL. In some embodiments, the genetically engineered bacterium comprises one or more genes encoding an LAAD. In some embodiments, the genetically engineered bacterium comprises one or more genes encoding PAL and one or more genes encoding an LAAD. In some embodiments, the genetically engineered bacterium comprises one or more genes encoding a transporter (e.g., PheP). In some embodiments, the genetically engineered bacterium comprises one or more genes encoding a transporter (e.g., PheP) and one or more genes encoding PAL. In some embodiments, the genetically engineered bacterium comprises one or more genes encoding a transporter (e.g., PheP) and one or more genes encoding an LAAD. In some embodiments, the genetically engineered bacterium comprises one or more genes encoding a transporter (e.g., PheP), one or more genes encoding an LAAD, and one or more genes encoding PAL. In any of the foregoing embodiments, the genetically engineered bacterium for phenylalanine consumption further comprises one or more of the following relative to its original state. In some embodiments, the endogenous bacteriophage genome. In some embodiments, the bacteriophage is mutated in one or more genes within the bacteriophage genome. Such mutations include deletions, insertions, substitutions, and inversions, and are located within or involve one or more bacteriophage genes.

[0023] Bacteriophages are the most common biological entities worldwide, and it is well documented that the majority of bacterial species, both Gram-positive and Gram-negative, contain one or more DNA bacteriophages integrated into the bacterial chromosome as so-called prophage (Clokie et al., Phages in Nature, Bacteriophage. January-February 2011; 1(1):31-45). For example, two separate studies on E. coli strains showed that 51 different functional phages were released from 27 E. coli strains analyzed, and that 83 of 107 E. coli strains tested released at least one functional phage type (Casjens, Prophages and bacterial genomics: what have we learned so far?; Mol Microbiol. 2003 July;49(2):277-300; Osawa et al., Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157:H7 isolates; J Med Microbiol (2001) 49:565-574; and Schicklmaier et al., A comparative study on the frequency of prophages among natural isolates of Salmonella and Escherichia coli with emphasis on generalized transducers. Antonie Van Leeuwenhoek (1998) 73: pp. 49-54).

[0024] As shown in Figure 12, almost all E. coli genomes contain intact prophages, and the majority of published complete E. coli genomes contain more intact prophages than EcN. Abbreviations: E. coli = E. coli; EcN = E. coli Nissle 1917; Refseq = Reference Sequence.

[0025] Among Gram-positive bacteria, the genomes of B. subtilis, Clostridium acetobutylicum, Lactococcus lactis, and many others have been shown to contain mostly intact prophages (Kunst et al., 1997; Bolotin et al., The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature (2001) 390: 249-256; Nolling et al., Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol (2001) 183: 4823-4838; Bolotin et al., The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res (2001) 11: pp. 731-753).

[0026] DNA phages can be lytic or temperate. Lytic phages infect bacterial cells and then program the synthesis of progeny phages, which are subsequently released from the lysed cell. Conversely, temperate DNA phages establish a stable relationship with the host bacterium. In this relationship, the integrated phage DNA (i.e., the prophage) replicates in concert with the host genome, and phage genes that are damaging to the host are not expressed. However, bacteriophage particles can be released from cells containing intact prophage by a process called induction, during which prophage genes necessary for lytic growth are turned on, progeny phage particles are produced, and they are released from the cell upon lysis (reviewed in Casjens, Prophages and bacterial genomics: what have we learned so far?; Mol Microbiol. 2003 July;49(2):277-300). Induction can occur spontaneously and randomly in a small fraction or even most of the bacteria containing the prophage, or specific, often undefined, environmental signals can cause the simultaneous induction of a particular prophage in many cells, resulting in the death of the bacterial cell. In some cases, the presence of prophage sequences can also enable some bacteria to possess properties that they would not have without the phage, such as antibiotic resistance, the ability to exist in a variety of environmental conditions, improved adhesion, virulence, or facilitated horizontal gene transfer (Casjens et al., 2001).

[0027] Not all prophages have the ability to undergo the lytic cycle. Non-functional prophages, i.e., defective or cryptic prophages, can reach high levels of abundance in many bacteria as a result of mutational decay and / or loss of one or more genes essential for the lytic cycle over thousands of bacterial replication cycles (Bobay et al., Pervasive domestication of defective prophages by bacteria, Proc Natl Acad Sci U S A. 2014 Aug. 19; 111(33): 12127-12132, and references therein). Notably, defective prophages also often lack genes encoding proteins with homologous recombination functions, such as those required to prevent or reverse further infection. contains many genes that can provide advantageous functions to the host, including bacteriocins, which may help in competition for nutrients, for example, through growth suppression of other neighboring bacterial species.

[0028] Phages can positively influence gene expression and fitness in E. coli in many ways. Cryptic, temperate, and lytic phages have been shown to confer multiple benefits to the host that promote survival under adverse environmental conditions. For example, gene sequences introduced into bacteria by phages have been associated with adaptation to different nutrients or niches, or an increased ability to eliminate competing strains. Dormant prophage has also been shown to prevent superinfection by other phages (e.g., lytic phages).

[0029] Several studies have shown that endogenous phages affect the ability of bacteria to grow on specific carbon sources. In addition to lambda, active Mu, P1, and P2 prophage and the cryptic prophage CP4-57 increase growth under glucose-limited and other growth conditions (Edlin, G., Lin, L. & Bitner, R. Reproductive fitness of P1, P2, and Mu lysogens of Escherichia coli. J. Virol. 21, pp. 560-564 (1977); Edlin, G., Lin, L. & Kudmar, R. λ Lysogens of E. coli reproduce more rapidly than non-lysogens. Nature 255, pp. 735-737 (1975); Wang, X., Kim, Y. & Wood, T. K. Control and benefits of CP4-57 prophage excision in Escherichia coli biofilms. ISME J. 3, pp. 1164-1179 (2009). Another study showed that when λ is integrated into the E. coli genome, it halts the cells' ability to grow on poor carbon sources. In this case, metabolic limitation may confer a survival advantage to the bacteria. Slowing bacterial growth in glucose-poor environments may help the bacteria evade detection by the immune system, increasing their chances of survival.

[0030] Other survival characteristics may be affected as well. Wang et al. generated a single E. coli strain lacking all nine cryptic prophage genes. This study showed that these prophages are beneficial for withstanding osmotic, oxidative, and acid stresses, increasing growth under various conditions, enhancing phosphorus and nitrogen utilization, and influencing biofilm formation (Wang et al., Cryptic prophages help bacteria cope with adverse environments; DOI: 10.1038 / ncomms1146). In pathogenic bacterial prophages, several studies have suggested that their acquisition is associated with changes in the virulence of pathogenic bacteria.

[0031] Thus, one skilled in the art might expect that modifications, such as mutation or deletion of part or all of an endogenous prophage, could alter (e.g., negatively affect) the fitness of a bacterium. Furthermore, one might expect that an endogenous prophage could alter (e.g., negatively affect) effector activity in engineered bacteria capable of producing this effector. This is particularly true when the endogenous prophage is present in all specimens of a particular strain subtype—indicating that bacteria containing the prophage sequence were able to evolutionarily outcompete bacterial forms lacking that prophage.

[0032] As further described in this disclosure, it can undergo dissolution under certain conditions and A prophage in E. coli Nissle was identified that was present in all Nissle specimens. Surprisingly, tests of bacterial fitness, residence time, and activity showed that bacteria containing mutations or deletions in the endogenous phage were essentially identical (e.g., at least of similar size).

[0033] Under similar assay conditions, there were no discernible differences in Phe degrading activity (in vitro or in vivo) between the strains. For example, under similar assay conditions, Phe consumption is of the same magnitude between the two strains (see, e.g., Figures 15 and 17A). In vivo competition studies between phage-containing and phage-free strains indicate no discernible differences in transport or colonization between Nissle's phage-free PKU strains (see, e.g., Figure 19).

[0034] Thus, in some embodiments, one or more modifications in the genome of a phage, e.g., mutations or deletions or other modifications described herein, do not alter the bacterial fitness of the modified or engineered bacterium. In some embodiments, engineered bacteria containing one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have essentially the same or at least similar bacterial fitness as the corresponding isogenic strain in the absence of the phage mutations. In further embodiments, one or more modifications in the genome of a phage, e.g., mutations or deletions or other modifications described herein, do not alter the strain activity (e.g., effector activity or metabolic activity) of the engineered bacterium capable of producing an effector compared to the corresponding isogenic strain without the phage mutations. In some embodiments, unmodified or engineered bacteria containing one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have essentially the same or at least similar bacterial strain activity (e.g., effector activity or metabolic activity) when compared to the corresponding isogenic strain without the phage mutations.

[0035] Furthermore, in some embodiments, one or more modifications, e.g., mutations or deletions or other modifications described herein, in the phage genome alter (e.g., increase or decrease) the bacterial fitness of the engineered bacterium. In some embodiments, engineered bacteria containing one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have altered (e.g., decreased or increased) bacterial fitness compared to a corresponding isogenic strain without the phage mutation(s). In some embodiments, one or more modifications, e.g., mutations or deletions or other modifications described herein, in the phage genome alter (e.g., decrease or increase) the strain activity (e.g., effector activity or metabolic activity) of the bacteria capable of producing an effector compared to a corresponding isogenic strain without the phage mutation(s). In some embodiments, unmodified or genetically engineered bacteria containing one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have altered (e.g., decreased or increased) bacterial strain activity (e.g., effector activity or metabolic activity) as a corresponding isogenic strain without the phage mutation(s).

[0036] In some embodiments, the genetically engineered bacteria comprise one or more E. coli Nissle bacteriophages (e.g., phage 1, phage 2, and phage 3). In some embodiments, the genetically engineered bacteria comprise one or more mutations in phage 3. Such mutations include deletions, insertions, substitutions, and inversions, and are located within or comprise one or more phage 3 genes. In some embodiments, the one or more insertions comprise an antibiotic cassette. In some embodiments, the mutation is a deletion. In some embodiments, the genetically engineered bacteria are located within or comprise one or more genes selected from the following: ECOLIN_09965, ECOLIN_09970, ECOLIN_09975 、ECOLIN_09980、ECOLIN_09985、ECOLIN_09990、ECOLIN_09995、ECOLIN_10000、ECOLIN_10005、ECOLIN_10010、ECOLIN_10015、ECOLIN_10020、ECOLIN_10025、ECOLIN_10030、ECOLIN_10035、ECOLIN_10040、ECOLIN_10045、ECOLIN_10050、ECOLIN_10055、ECOLIN_10065、ECOLIN_10070、ECOLIN_10075、ECOLIN_10080、ECOLIN_10085、ECOLIN_10090、ECOLIN_10095、ECOLIN_10100、ECOLIN_10105、ECOLIN_10110、ECOLIN_10115、ECOLIN_10120、ECOLIN_10125、ECOLIN_10130、ECOLIN_10135、ECOLIN_10140、ECOLIN_10145、ECOLIN_10150、ECOLIN_10160、ECOLIN_10165、ECOLIN_10170、ECOLIN_10175、ECOLIN_10180、ECOLIN_10185、ECOLIN_10190、ECOLIN_10195、ECOLIN_10200、ECOLIN_10205、ECOLIN_10210、ECOLIN_10220、ECOLIN_10225、ECOLIN_10230、ECOLIN_10235、ECOLIN_10240、ECOLIN_10245、ECOLIN_10250、ECOLIN_10255、ECOLIN_10260、ECOLIN_10265、ECOLIN_10270、ECOLIN_10275、ECOLIN_10280、ECOLIN_10290、ECOLIN_10295、ECOLIN_10300、ECOLIN_10305、ECOLIN_10310、ECOLIN_10315、ECOLIN_10320、ECOLIN_10325、ECOLIN_10330、ECOLIN_10335、ECOLIN_10340、およびECOLIN_10345。In one embodiment, the genetically engineered bacterium comprises a complete or partial deletion of one or more of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175. In one specific embodiment, the deletion is a complete deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, and ECOLIN_10170, and a partial deletion of ECOLIN_10175. In one embodiment, the sequence of SEQ ID NO: 130 is deleted from the phage 3 genome. In one embodiment, the sequence comprising SEQ ID NO: 130 is deleted from the phage 3 genome. In one embodiment, the genetically engineered bacterium comprises a modified phage genome sequence comprising SEQ ID NO: 281. In one embodiment, the genetically engineered bacterium comprises a modified phage genome sequence consisting of SEQ ID NO: 281.

[0037] In order to make this disclosure easier to understand, certain terms are first defined.These definitions should be read in light of the remainder of this disclosure and as understood by those skilled in the art.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Additional definitions are set forth throughout the detailed description.

[0038] "Hyperphenylalaninemia," "hyperphenylalaninemic," and "excess phenylalanine" are used interchangeably herein to refer to increased or abnormally high concentrations of phenylalanine in the body. In some embodiments, a diagnostic signal for hyperphenylalaninemia is at least 2 mg / dL, at least 4 mg / dL, and a blood phenylalanine level of at least 6 mg / dL, at least 8 mg / dL, at least 10 mg / dL, at least 12 mg / dL, at least 14 mg / dL, at least 16 mg / dL, at least 18 mg / dL, at least 20 mg / dL, or at least 25 mg / dL. As used herein, diseases associated with hyperphenylalaninemia include, but are not limited to, phenylketonuria, classic or typical phenylketonuria, atypical phenylketonuria, persistent mild hyperphenylalaninemia, non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase deficiency, and Segawa disease. Affected individuals may suffer from progressive and irreversible neurological deficits, mental retardation, encephalopathy, epilepsy, eczema, reduced growth, microcephaly, tremors, limb spasms, and / or hypopigmentation (Leonard 2006). Hyperphenylalaninemia may also occur secondary to other conditions, such as liver disease.

[0039] "Phenylalanine ammonia lyase" and "PAL" are used to refer to a phenylalanine metabolic enzyme (PME) that converts or processes phenylalanine to trans-cinnamic acid and ammonia. Trans-cinnamic acid is converted to hippuric acid, which is less toxic and is secreted in urine by liver enzymes in mammals. PAL can substitute for the enzyme PAH to metabolize excess phenylalanine. PAL enzyme activity does not require THB cofactor activity. In some embodiments, PAL is encoded by a PAL gene derived from a prokaryotic species. In alternative embodiments, PAL is encoded by a PAL gene derived from a eukaryotic species. In some embodiments, PAL is encoded by a PAL gene derived from a bacterial species, including, but not limited to, Achromobacter xylosoxidans, Pseudomonas aeruginosa, Photorhabdus luminescens, Anabaena variabilis, and Agrobacterium tumefaciens. In some embodiments, PAL is encoded by a PAL gene derived from Anabaena variabilis, referred to herein as "PAL1" (Moffitt et al., 2007). In some embodiments, PAL is encoded by a PAL gene derived from Photorhabdus luminescens, referred to herein as "PAL3" (Williams et al., 2005). In some embodiments, PAL is encoded by a PAL gene from a yeast species, such as Rhodosporidium toruloides (Gilbert et al., 1985). In some embodiments, PAL is encoded by a PAL gene from a plant species, such as Arabidopsis thaliana (Wanner et al., 1995). Any suitable nucleotide and amino acid sequence of PAL, or a functional fragment thereof, can be used.

[0040] The terms "phenylalanine hydroxylase" and "PAH" refer to enzymes that catalyze the hydroxylation of the aromatic side chain of phenylalanine to produce tyrosine in humans in conjunction with the cofactor tetrahydrobiopterin. The human gene encoding PAH is located on the long (q) arm of chromosome 12 between positions 22 and 24.2. The amino acid sequence of PAH is highly conserved among mammals. Nucleic acid sequences for human and mammalian PAHs are well known and widely available. The full-length human cDNA sequence for PAH was reported in 1985 (Kwok et al., 1985). Active fragments of PAHs are also well known (e.g., Kobe et al., 1997).

[0041] "L-amino acid deaminase" and "LAAD" are enzymes that decompose L-amino acids, The term LAAD is used to refer to enzymes that catalyze the stereospecific oxidative deamination of L-amino acids to produce ammonia and hydrogen peroxide. For example, LAAD catalyzes the conversion of phenylalanine to phenylpyruvic acid. Numerous LAAD enzymes are known in the art, many of which are derived from bacteria, such as Proteus, Providencia, and Morganella, or from venom. LAAD is characterized by a fast kinetics of phenylalanine degradation (Hou et al., Appl Microbiol Technol. 2015 Oct;99(20):8391-402; "Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid"). While most eukaryotic and prokaryotic L-amino acid deaminases are extracellular, Proteus species LAAD is localized to the plasma membrane (inner membrane) and faces outward toward the periplasmic space where enzymatic activity resides. As a result of this localization, phenylalanine transport through the inner membrane to the cytoplasm is not required for phenylalanine degradation mediated by Proteus species LAAD. Phenylanine is readily imported into the periplasm through the outer membrane without the need for a transporter, obviating the need for a transporter to improve substrate availability.

[0042] In some embodiments, the genetically engineered bacterium comprises a LAAD gene from a bacterial species, including, but not limited to, bacteria of the genera Proteus, Providencia, and Morganella. In some embodiments, the bacterial species is Proteus mirabilis. In some embodiments, the bacterial species is Proteus vulgaris. In some embodiments, the LAAD encoded by the genetically engineered bacterium is localized to the plasma membrane and faces the periplasmic space, where catalytic activity occurs.

[0043] "Phenylalanine metabolic enzyme" or "PME" refers to an enzyme capable of degrading phenylalanine. Any phenylalanine metabolic enzyme known in the art can be encoded by genetically engineered bacteria. PMEs include, but are not limited to, phenylalanine hydroxylase (PAH), phenylalanine ammonia-lyase (PAL), aminotransferase, L-amino acid deaminase (LAAD), and phenylalanine dehydrogenase.

[0044] While the reaction with phenylalanine hydroxylase, phenylalanine dehydrogenase, or aminotransferase requires a cofactor, LAAD and PAL do not require any additional cofactors. In some embodiments, the PME encoded by the genetically engineered bacterium requires a cofactor. In some embodiments, the cofactor is provided simultaneously or sequentially with administration of the genetically engineered bacterium. In other embodiments, the genetically engineered bacterium is capable of producing the cofactor. In some embodiments, the genetically engineered bacterium encodes a phenylalanine hydroxylase. In some embodiments, the genetically engineered bacterium encodes a phenylalanine dehydrogenase. In some embodiments, the genetically engineered bacterium encodes an aminotransferase. In some embodiments, the PME encoded by the genetically engineered bacterium does not require a cofactor. Without being bound by theory, the lack of a cofactor requirement means that the rate of phenylalanine degradation by the enzyme depends on the availability of the substrate and is not limited by the availability of a cofactor. In some embodiments, the PME produced by the genetically engineered bacterium is PAL. In some embodiments, the PME produced by the genetically engineered bacterium is LAAD. In some embodiments, the genetically engineered bacterium encodes a combination of PMEs.

[0045] In some embodiments, the catalytic activity of PME depends on oxygen levels. In some embodiments, PME is catalytically active under microaerobic conditions. As a non-limiting example, LAAD catalytic activity depends on oxygen. In some embodiments, LAAD is active under hypoxic conditions, such as microaerobic conditions. In some embodiments of the present invention, PME functions at very low levels of oxygen, such as those found in the colon, or in the absence of oxygen. As a non-limiting example, PAL activity does not depend on the presence of oxygen.

[0046] As used herein, "effector" or "effector molecule" can refer to a molecule, such as a metabolite or polypeptide, that exerts a desired function. An effector can be encoded by a single gene. For example, a single gene can encode a secreted or displayed polypeptide. Alternatively, an effector can be synthesized by a biosynthetic pathway requiring multiple genes (e.g., butyrate). For example, a polypeptide encoded by multiple genes in a biosynthetic pathway that synthesizes a metabolite with desired properties can also be referred to as an effector. Similarly, a polypeptide encoded by multiple genes in a catabolic pathway, for example, for the degradation of a toxic metabolite, can also be referred to as an effector. These effector molecules can also be referred to as "therapeutic metabolites," "therapeutic molecules," or "therapeutic polypeptides." Other terms used interchangeably herein with effector are "polypeptide of interest" or "polypeptides of interest," "protein of interest," and "proteins of interest."

[0047] As used herein, "payload" refers to one or more polynucleotides and / or polypeptides of interest produced by a genetically engineered microorganism, such as a bacterium. In some embodiments, the payload is encoded by one or more genes or operons. In some embodiments, the one or more genes and / or operons comprising the payload are endogenous to the microorganism. In some embodiments, one or more elements of the payload are derived from a different microorganism and / or organism. In some embodiments, the payload is a therapeutic payload. In some embodiments, the payload is encoded by a gene for the biosynthesis of a molecule. In some embodiments, the payload is encoded by a gene for the metabolism, catabolism, or degradation of a molecule. In some embodiments, the payload is encoded by a gene for the import of a molecule. In some embodiments, the payload is encoded by a gene for the export of a molecule. In some embodiments, the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or repressor. In some embodiments, payload expression is driven from an inducible promoter, such as FNRS. In some embodiments, payload expression is driven from a constitutive promoter. In some embodiments, the payload comprises a repressor element, such as a death switch. In alternative embodiments, the payload is produced by a biosynthetic or biochemical pathway, which may optionally be endogenous to the microorganism. In some embodiments, the engineered microorganism comprises two or more payloads.

[0048] The present invention includes, inter alia, genetically engineered bacteria, pharmaceutical compositions thereof, and methods for modulating and treating disorders associated with hyperphenylalaninemia. In some embodiments, the genetically engineered bacteria contain a gene encoding a non-naturally occurring phenylalanine ammonia-lyase (PAL), which can process and reduce phenylalanine in mammals. In some embodiments, the engineered bacteria further contain a gene encoding a phenylalanine transporter. In some embodiments, the engineered bacteria may also contain a gene encoding a LAAD. The engineered bacteria may also contain a gene encoding a LAAD. The engineered bacteria may also contain a gene encoding a LAAD for biosafety and / or biocontainment, e.g., a death switch, a gene guard system, etc. The expression of these gene sequences may be regulated using various promoter systems, such as any of the promoter systems disclosed herein, which may be the same promoter regulating one or more different genes, different copies of the same promoter regulating different genes, or different promoters used in combination to regulate the expression of different genes. The use of different regulatory or promoter systems to control gene expression provides flexibility (e.g., the ability to differentially regulate gene expression under different environmental conditions and / or the ability to differentially regulate gene expression in time) and also provides the ability to "fine-tune" gene expression, any or all of which may be useful for optimizing gene expression and / or bacterial growth. Genetically engineered bacteria and pharmaceutical compositions comprising these bacteria can be used to metabolize phenylalanine in the body into non-toxic molecules to treat and / or prevent conditions associated with hyperphenylalaninemia, including PKU. In certain embodiments, compositions comprising genetically engineered bacteria can be used in the methods of the present disclosure to treat and / or prevent disorders associated with hyperphenylalaninemia.

[0049] Effector molecules also include anti-cancer molecules. "Anti-cancer molecule" refers to one or more therapeutic substances or drugs of interest produced by a genetically engineered microorganism (e.g., an engineered bacterium or an engineered oncolytic virus), which can reduce and / or inhibit cell growth or replication. In some embodiments, an anti-cancer molecule is a therapeutic molecule useful for modulating or treating cancer. In some embodiments, an anti-cancer molecule is a therapeutic molecule encoded by a gene. In alternative embodiments, an anti-cancer molecule is a therapeutic molecule produced by a biochemical or biosynthetic pathway, which may optionally be endogenous to the microorganism. In some embodiments, a genetically engineered microorganism is capable of producing more than one anti-cancer molecule. Non-limiting examples of anti-cancer molecules include immune checkpoint inhibitors (e.g., CTLA-4 antibodies, PD-1 antibodies, PDL-1 antibodies), cytotoxic agents (e.g., Cly A, FASL, TRAIL, TNF-α), immunostimulatory cytokines and costimulatory molecules (e.g., OX40, CD28, ICOS, CCL21, IL-2, IL-18, IL-15, IL-12, IFN-γ, IL-21, TNF, GM-CSF), antigens and antibodies (e.g., tumor antigens, neoantigens, CtxB-PSA fusion protein, CPV-OmpA fusion protein, NY-ESO-1 tumor antigen, RAF1, antibodies against immunosuppressive molecules, anti-VEGF, anti-CXR4 / C Antibodies include antibodies against immune checkpoints (e.g., XCL12, anti-GLP1, anti-GLP2, anti-galectin 1, anti-galectin 3, anti-Tie2, anti-CD47, antibodies against immune checkpoints, antibodies against immunosuppressive cytokines and chemokines), DNA transfer vectors (e.g., endostatin, thrombospondin-1, TRAIL, SMAC, Stat3, Bcl2, FLT3L, GM-CSF, IL-12, AFP, VEGFR2), and enzymes (e.g., E. coli CD, HSV-TK). In some embodiments, anti-cancer molecules include nucleic acid molecules that mediate gene editing, such as RNA interference, microRNA response or inhibition, TLR response, antisense gene regulation, target protein binding (aptamers or decoy oligos), and CRISPR interference.In some embodiments, bacteria or viruses can be used as vectors to introduce DNA into mammalian cells, for example, by bactofection (Bernardes et al., 2013).

[0050] Non-limiting examples of effector molecules include "anti-inflammatory molecules" and / or "gut barrier function enhancer molecules." Anti-inflammatory molecules and / or gut barrier function enhancer molecules include short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL-10, IL-27, TGF-β1, TGF-β2, N-acylphosphatidylethanolamine (NAPE), elafin (peptidase inhibitor 3), and Examples of pro-inflammatory molecules include, but are not limited to, compounds that inhibit pro-inflammatory molecules, such as single-chain variable antibodies (scFvs), antisense RNAs, siRNAs, or shRNAs that neutralize TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-17, and / or chemokines (e.g., CXCL-8 and CCL2). These molecules also include AHR agonists (e.g., indole acetic acid, indole-3-aldehyde, and indole) and PXR agonists (e.g., IPA), as described herein, which, for example, result in IL-22 production. Such molecules also include HDAC inhibitors (e.g., butyrate), activators of GPR41 and / or GPR43 (e.g., butyrate and / or propionate and / or acetate), activators of GPR109A (e.g., butyrate), inhibitors of NF-kappaB signaling (e.g., butyrate), modulators of PPARγ (e.g., butyrate), activators of AMPK signaling (e.g., acetate), and modulators of GLP-1 secretion. Such molecules also include hydroxyl radical scavengers and antioxidants (e.g., IPA). A molecule may be primarily anti-inflammatory (e.g., IL-10) or primarily gastrointestinal barrier function enhancing (e.g., GLP-2). A molecule may be both anti-inflammatory and gastrointestinal barrier function enhancing. An anti-inflammatory and / or gastrointestinal barrier function enhancer molecule may be encoded by a single gene; for example, elafin is encoded by the PI3 gene. Alternatively, the anti-inflammatory and / or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes (e.g., butyrate).

[0051] Effector molecules also include metabolic effector molecules. "Metabolic effector molecules" and / or "satiety effector molecules" are also used. "Non-steroidal anti-inflammatory molecules" include, but are not limited to, n-acyl-phosphatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin-releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, short chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide ribosides (NRs), nicotinamide, and nicotinic acid (NA). Such molecules may also include compounds that inhibit molecules that promote metabolic disease (e.g., single-chain variable antibodies (scFv), antisense RNA, siRNA, or shRNA that inhibit dipeptidyl peptidase-4 (DPP4) or the ghrelin receptor). The metabolic and / or satiety effector molecule may be encoded by a single gene, e.g., glucagon-like peptide 1 is encoded by the GLP-1 gene. In some embodiments, the genetically engineered bacteria comprising gene sequences containing one or more circuits for the production or catabolism of tryptophan and / or one of its metabolites further comprise gene sequences for the expression of one or more metabolic and / or satiety effector molecules.

[0052] Other non-limiting examples of effector molecules are described in pending, co-owned international patent applications PCT / US2016 / 34200 (filed May 25, 2016), PCT / US2017 / 013072 (filed January 11, 2017), PCT / US2017 / 016603 (filed February 3, 2017), PCT / US2017 / 016609 (filed February 4, 2016), PCT / US2017 / 017563 (filed February 10, 2017), PCT Nos. 6,111,159, 6,161, 6,171, 6,181, 6,19 ...

[0053] In certain embodiments, new or improved effectors (e.g., PMEs) can be identified according to methods known in the art or described herein and are encoded by genetically engineered bacteria. In some embodiments, the enzymes encoded by genetically engineered bacteria are wild-type enzymes isolated from viruses, prokaryotes, or eukaryotes. In some embodiments, the enzyme sequences are further modified or mutated to increase one or more specific properties of the enzyme, such as stability or catalytic activity.

[0054] "Phenylalanine metabolite" refers to a metabolic product produced as a result of the breakdown of phenylalanine. The metabolic product can be produced directly from phenylalanine by an enzyme that uses phenylalanine as a substrate, or indirectly by a different enzyme downstream in the metabolic pathway that acts on the phenylalanine metabolite substrate. In some embodiments, the phenylalanine metabolite is produced by a genetically engineered bacterium that encodes a PME.

[0055] In some embodiments, the phenylalanine metabolite results directly or indirectly from PAH activity, for example, from PAH produced by genetically engineered bacteria. In some embodiments, the metabolite is tyrosine. In some embodiments, the phenylalanine metabolite accumulates in the blood or urine of PKU patients due to defective PAH activity. Non-limiting examples of such PKU metabolites include phenylpyruvate and phenyllactate. Other examples include phenylacetate, phenylethylamine, and phenylacetylglutamine.

[0056] In some embodiments, the phenylalanine metabolites are derived directly or indirectly from PAL activity, for example, from PAL produced by genetically engineered bacteria. Non-limiting examples of such PAL metabolites include trans-cinnamic acid and hippuric acid. In some embodiments, the phenylalanine metabolites are derived directly or indirectly from LAAD activity, for example, from LAAD produced by genetically engineered bacteria. Examples of such LAAD metabolites are phenylpyruvic acid and phenyllactic acid.

[0057] The term "phenylalanine transporter" is used to refer to a membrane transport protein capable of transporting phenylalanine into bacterial cells (see, e.g., Pi et al., 1991). In Escherichia coli, the pheP gene encodes a high-affinity phenylalanine-specific permease responsible for phenylalanine transport (Pi et al., 1998). In some embodiments, the phenylalanine transporter is encoded by the pheP gene from bacterial species, including, but not limited to, Acinetobacter calcoaceticus, Salmonella enterica, and E. coli. Other phenylalanine transporters include the ageneral amino acid permease, encoded by the aroP gene, which transports three aromatic amino acids, including phenylalanine, with high affinity and, together with PheP, is responsible for the largest portion of phenylalanine uptake. Additionally, low-level phenylalanine transport activity is also observed. The activity of the LIV-I / LS system is traced to the activity of the LIV-I / LS system, a branched-chain amino acid transporter composed of two periplasmic binding proteins, the LIV binding protein (LIV-I system) and the LS binding protein (LS system), and the membrane component LivHMGF. In some embodiments, the phenylalanine transporter is encoded by the aroP gene from a bacterial species. In some embodiments, the phenylalanine transporter is encoded by the LIV binding protein and the LS binding protein and the LivHMGF gene from a bacterial species. In some embodiments, the genetically engineered bacterium comprises more than one phenylalanine transporter selected from pheP, aroP, and the LIV-I / LS system.

[0058] "Phenylalanine" and "Phe" are used to refer to the amino acid having the formula CHCHCH(NH)COOH. Phenylalanine is a precursor to tyrosine, dopamine, norepinephrine, and epinephrine. L-phenylalanine is an essential amino acid and is the form of phenylalanine found primarily in food proteins. The stereoisomer D-phenylalanine is found in smaller amounts in food proteins, and DL-phenylalanine is a combination of both forms. Phenylalanine can refer to one or more of L-phenylalanine, D-phenylalanine, and DL-phenylalanine.

[0059] As used herein, the term "transporter" is meant to refer to a mechanism (e.g., a protein, protein, or protein complex) for importing molecules, such as amino acids, peptides (such as dipeptides, tripeptides, and polypeptides), toxins, metabolites, substrates, and other biomolecules from the extracellular environment into a microorganism.

[0060] "Operably linked" refers to a nucleic acid sequence, e.g., a gene encoding PAL, that is joined to a regulatory region sequence in a manner that allows expression of the nucleic acid sequence acting in cis. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and can include promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions, transcription initiation sites, termination sequences, polyadenylation sequences, and introns.

[0061] An "inducible promoter" refers to a regulatory region that is operably linked to one or more genes, such that expression of the genes is increased in the presence of an inducer of the regulatory region.

[0062] A "directly inducible promoter" refers to a regulatory region operably linked to a gene encoding an effector molecule (e.g., a phenylalanine metabolic enzyme such as PAL), such that in the presence of an inducer of the regulatory region, the effector molecule is expressed. An "indirectly inducible promoter" refers to a regulatory system comprising two or more regulatory regions, e.g., a first regulatory region operably linked to a gene encoding a first molecule (e.g., a transcriptional regulator capable of regulating a second regulatory region operably linked to a gene encoding an effector molecule). In the presence of an inducer of the first regulatory region, the second regulatory region can be activated or repressed, thereby activating or repressing expression of the effector molecule. Both directly inducible promoters and indirectly inducible promoters are encompassed by the term "inducible promoter."

[0063] "Exogenous environmental conditions" or "environmental conditions" refer to the setting or circumstances in which a promoter described herein is directly or indirectly induced. The phrase is meant to refer to environmental conditions that are external to the engineered microorganism, but endogenous or natural to the host subject environment. Thus, "exogenous" and "endogenous" refer to the environmental conditions that are external to the mammalian body. The terms "exogenous" and "exogenous" can be used interchangeably to refer to environmental conditions that are endogenous to the intact microbial cells but external or extrinsic to the intact microbial cells. In some embodiments, the exogenous environmental conditions are specific to the mammalian gastrointestinal tract. In some embodiments, the exogenous environmental conditions are specific to the mammalian upper gastrointestinal tract. In some embodiments, the exogenous environmental conditions are specific to the mammalian lower gastrointestinal tract. In some embodiments, the exogenous environmental conditions are specific to the mammalian small intestine. In some embodiments, the exogenous environmental conditions are hypoxic, microaerobic, or anaerobic conditions, such as the environment of the mammalian gastrointestinal tract. In some embodiments, the exogenous environmental conditions refer to the presence of molecules or metabolites, e.g., propionate, that are specific to the mammalian gastrointestinal tract in health or disease. In some embodiments, the exogenous environmental conditions are specific to the tumor microenvironment. In some embodiments, the exogenous environmental conditions are molecules or metabolites that are specific to the tumor microenvironment. In some embodiments, the exogenous environmental conditions are tissue- or disease-specific metabolites or molecules. In some embodiments, the exogenous environmental conditions are a low pH environment. In some embodiments, the genetically engineered microorganisms of the present disclosure comprise a pH-dependent promoter. In some embodiments, the genetically engineered microorganisms of the present disclosure comprise an oxygen level-dependent promoter. In some aspects, the bacteria have evolved transcription factors that can sense oxygen levels. Different signaling pathways can be triggered by different oxygen levels and occur with different kinetics.

[0064] As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to settings or circumstances or environmental conditions outside of an engineered microorganism and relate to in vitro culture conditions of the microorganism. "Exogenous environmental conditions" can also refer to conditions during growth, production, and manufacturing of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low-oxygen culture conditions, and other conditions under a set oxygen concentration. Such conditions also include the presence of chemical and / or nutritional inducers, such as tetracycline, arabinose, IPTG, and rhamnose, in the culture medium. Such conditions also include the temperature at which the microorganism is grown prior to in vivo administration. For example, with certain promoter systems, some temperatures allow expression of the payload, while others do not. Oxygen levels, temperature, and medium composition affect such exogenous environmental conditions. Such conditions affect the growth rate, the induction rate of the payload (e.g., a PME such as PAL or LAAD), the induction rate of a transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production.

[0065] An "oxygen level-dependent promoter" or "oxygen level-dependent regulatory region" refers to a nucleic acid sequence to which one or more oxygen level-sensitive transcription factors can bind, and binding and / or activation of the corresponding transcription factor activates downstream gene expression.

[0066] Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, for example, Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003). Non-limiting examples are shown in Table 1.

[0067] In a non-limiting example, the promoter (PfnrS) was derived from the E. coli Nissle fumarate and nitrate reductase gene S (fnrS), which is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al., 2010). The PfnrS promoter is activated under anaerobic and / or hypoxic conditions by the global transcriptional regulator FNR, which is naturally found in Nissle. Under anaerobic and / or hypoxic conditions, FNR dimerizes and binds to specific sequences in the promoters of specific genes under its control, thereby activating the PfnrS promoter. However, under aerobic conditions, oxygen reacts with the iron-sulfur clusters in the FNR dimers, converting them to an inactive form. Thus, the PfnrS inducible promoter can be used to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application with the FNRS, fnrS, FNR, P-FNRS promoter, and other such related names to refer to the promoter PfnrS.

[0068] [Table 1] Examples of transcription factors, responsive genes, and regulatory regions [Table 1]

[0069] As used herein, a "regulatable regulatory region" refers to a nucleic acid sequence that is under the direct or indirect control of a transcription factor and can activate, repress, derepress, or regulate gene expression in response to the level of an inducer. In some embodiments, the regulatable regulatory region comprises a promoter sequence. The inducer can be an RNS or other inducer described herein, and the regulatable regulatory region can be an RNS-responsive regulatory region or other responsive regulatory region described herein. The regulatable regulatory region can be operably linked to a gene sequence or gene cassette for the production of one or more payloads, such as a butyrogenic or other gene cassette or gene sequence. For example, in one specific embodiment, the regulatable regulatory region is an RNS-derepression regulatory region, such that when RNS is present, the RNS-sensing transcription factor no longer binds to and / or represses the regulatory region, thereby allowing expression of the operably linked gene or gene cassette. In this case, the regulatable regulatory region derepresses expression of the gene or gene cassette in response to RNS levels. Each gene or gene cassette may be operably linked to a regulatable regulatory region that is directly or indirectly controlled by at least one RNS-sensing transcription factor.

[0070] In some embodiments, the exogenous environmental condition is the presence or absence of reactive oxygen species. In other embodiments, the exogenous environmental condition is the presence or absence of reactive nitrogen species (RNS). In some embodiments, the exogenous environmental condition is a biological molecule involved in the inflammatory response, e.g., a molecule present in an inflammatory disorder of the gastrointestinal tract. In some embodiments, the exogenous environmental condition or signal is naturally present or absent in the environment in which the recombinant bacterial cells reside. In some embodiments, the exogenous environmental condition or signal is artificially generated, e.g., by creating or removing a biological state and / or administering or removing a biological molecule.

[0071] In some embodiments, exogenous environmental conditions and / or signals stimulate the activity of an inducible promoter. In some embodiments, the exogenous environmental conditions and / or signals that serve to activate an inducible promoter are not naturally present in the mammalian gastrointestinal tract. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite administered in combination with the pharmaceutical composition of the present invention, e.g., tetracycline, arabinose, or any biological molecule that serves to activate the inducible promoter. In some embodiments, exogenous environmental conditions and / or signals are added to culture medium containing recombinant bacterial cells of the present invention. In some embodiments, the exogenous environmental conditions that serve to activate the inducible promoter are naturally present in the mammalian gastrointestinal tract (e.g., hypoxic or anaerobic conditions, or biological molecules involved in inflammatory responses). In some embodiments, loss of exposure to the exogenous environmental conditions (e.g., in vivo) inhibits the activity of the inducible promoter because the exogenous environmental conditions are no longer present to induce the promoter (e.g., an aerobic environment outside the gastrointestinal tract). As used herein, a "non-naturally occurring" nucleic acid sequence refers to a nucleic acid sequence that is not normally present in bacteria, e.g., an additional copy of an endogenous sequence, or a heterologous sequence, such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and / or mutated compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, a non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, promoter, gene, and / or one or more genes in a gene cassette. In some embodiments, "non-native" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. Furthermore, multiple copies of any regulatory region, promoter, gene, and / or gene cassette may be present in the bacterium, and one or more copies of the regulatory region, promoter, gene, and / or gene cassette may be mutated or otherwise altered as described herein. In some embodiments, genetically engineered bacteria are engineered to contain multiple copies of the same regulatory region, promoter, gene, and / or gene cassette to increase copy number or to contain multiple different components of the gene cassette that perform multiple different functions.In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding an effector molecule (e.g., a PME) operably linked to a directly or indirectly inducible promoter not naturally associated with the gene, e.g., the FNR promoter operably linked to a gene encoding the effector molecule, or the ParaBAD promoter operably linked to a second effector molecule.

[0072] A "constitutive promoter" refers to a promoter that is capable of promoting the 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 include, but are not limited to, BBa_J23100, constitutive E. coli σ S Promoters (e.g., osmY promoter (International Genetically Engineered Machine (iGEM) Standard Biological Parts Registry names BBa_J45992; BBa_J45993)), constitutive E. coli σ 32 Promoter (e.g., htpG heat shock promoter (BBa_J45504)), constitutive E. coli σ 70 Promoters (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate-sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M131 02), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110), constitutive Bacillus subtilis σA Promoter (e.g., promoter veg(BBa_K143013), Promoter 43 (BBa_K143013), P liaG (BBa_K823000), P lepA (BBa_K823002), P veg (BBa_K823003)), constitutive Bacillus subtilis σ B promoters (e.g., promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)), Salmonella promoters (e.g., Pspv2 from Salmonella (BBa_K112706), Pspv from Salmonella (BBa_K112707)), bacteriophage T7 promoters (e.g., T7 promoters (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), bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof.

[0073] "Gastrointestinal tract" refers to the organs, glands, ducts, and systems responsible for the movement and digestion of food, the absorption of nutrients, and the excretion of waste products. In humans, the gastrointestinal (GI) tract begins at the mouth and ends at the anus, and includes the esophagus, stomach, small intestine, and large intestine. The GI tract also includes accessory organs and glands such as the spleen, liver, gallbladder, and pancreas. The upper GI tract includes the esophagus, stomach, and duodenum of the small intestine. The lower GI tract includes the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine (i.e., the cecum, colon, rectum, and anal canal). Bacteria can be found throughout the GI tract, for example, in the GI tract, particularly in the intestine.

[0074] In some embodiments, the engineered bacteria are active in the gastrointestinal tract (e.g., express one or more payloads, e.g., PMEs). In some embodiments, the engineered bacteria are active in the large intestine (e.g., express one or more payloads). In some embodiments, the engineered bacteria are active in the small intestine (e.g., express one or more payloads). In some embodiments, the engineered bacteria are active in the small and large intestines. Without wishing to be bound by theory, degradation of phenylalanine can be highly effective in the small intestine because amino acid absorption, e.g., phenylalanine absorption, occurs in the small intestine. Preventing or reducing phenylalanine uptake into the blood can avoid elevated Phe levels and the resulting Phe toxicity. Furthermore, extensive enteric recirculation of amino acids between the intestine and the body may allow for the removal of systemic phenylalanine in PKU (e.g., as described by Chang et al. in a rat model of PKU (Chang et al., "A new theory of enterorecirculation of amino acids and Its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria; Artificial Cells Blood Substit Immobil Biotechnol. 1995; 23(1):1-21). Blood-borne phenylalanine circulates in the small intestine (see, e.g., FIG. 15), where it can be removed by active bacteria. In some embodiments, the engineered bacteria pass through the small intestine. In some embodiments, the engineered bacteria have an extended residence time in the gastrointestinal tract. In some embodiments, the engineered bacteria colonize the small or large intestine. In some embodiments, the engineered bacteria colonize the colon. In some embodiments, the engineered bacteria have an extended residence time in the gastrointestinal tract. In some embodiments, the engineered bacteria do not colonize the gastrointestinal tract.

[0075] As used herein, the term "hypoxia" is meant to refer to a level, amount, or concentration of oxygen (O2) that is lower than the level, amount, or concentration of oxygen present in the atmosphere (e.g., For example, <21% O; <160 torr O). Thus, the term "hypoxic condition(s)" or "hypoxic environment" refers to conditions or environments containing oxygen levels lower than that present in the atmosphere. In some embodiments, the term "hypoxia" is meant to refer to the level, amount, or concentration of oxygen (O) found in the mammalian gastrointestinal tract, e.g., the lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term "hypoxia" refers to a level of O2 between 0 and 60 mmHg (0 torr O2) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg O2), "Hypoxia" refers to an amount, amount, or concentration of O2, including any and all decimal increments thereof (e.g., 0.2 mmHg, 0.5 mmHg O2, 0.75 mmHg O2, 1.25 mmHg O2, 2.175 mmHg O2, 3.45 mmHg O2, 3.75 mmHg O2, 4.5 mmHg O2, 6.8 mmHg O2, 11.35 mmHg O2, 46.3 mmHg O2, 58.75 mmHg, etc., although these exemplary decimal increments are provided herein for illustrative purposes and are not meant to be limiting in any way). In some embodiments, "hypoxia" refers to O2 below about 60 mmHg (e.g., 0 to about 60 mmHg). The term "hypoxia" can also refer to a range of levels, amounts, or concentrations of O2 between 0 and 60 mmHg inclusive, e.g., 0-5 mmHg O2, <1.5 mmHg O2, 6-10 mmHg, <8 mmHg, 47-60 mmHg, etc., although these exemplary ranges are provided herein for illustrative purposes and are not meant to be limiting in any way.See, for example, Albenberg et al., Gastroenterology, 147(5):1055-1063 (2014); Bergofsky et al., J Clin. Invest., 41(11):1971-1980 (1962); Crompton et al., J Exp. Biol., 43:473-478 (1965); He et al., PNAS (USA), 96:4586-4591 (1999); McKeown, Br. J. Radiol., 87:20130676 (2014) (doi:10.1259 / brj.20130676). Each of these documents discusses the oxygen levels found in the digestive tracts of various mammalian species, and each of these documents is incorporated herein by reference in its entirety. In some embodiments, the term "hypoxia" refers to the level, amount, or concentration of oxygen (O) found in organs or tissues other than the gastrointestinal tract of a mammal, such as the genitourinary tract, tumor tissue, etc., where oxygen is present at reduced levels, e.g., hypoxic or anoxic levels. In some embodiments, "hypoxia" refers to the level, amount, or concentration of oxygen (O) present in partially aerobic, semi-aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and / or anaerobic conditions. For example, Table A summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (O2) is expressed as the amount of dissolved oxygen ("DO"), which refers to the level of free, non-compound oxygen (O2) present in a liquid, and is typically reported in milligrams per liter (mg / L), parts per million (ppm; 1 mg / L = 1 ppm), or micromoles (1 μmol O2 = 0.022391 mg / L O2).Fondriest Environmental, Inc., "Dissolved Oxygen," Fundamentals of Environmental Measurements, November 19, 2013, www.fondriest.com / environmental-measurements / parameters / water-quality / dissolved-oxygen / . In some embodiments, the term "hypoxia" refers to a level, amount, or concentration of oxygen (O2) at or below about 6.0 mg / L DO. "DO" means a concentration of oxygen in a liquid or solution, such as 6.0 mg / L, 5.0 mg / L, 4.0 mg / L, 3.0 mg / L, 2.0 mg / L, 1.0 mg / L, or 0 mg / L, as well as decimal fractions thereof, such as 3.25 mg / L, 2.5 mg / L, 1.75 mg / L, 1.5 mg / L, 1.25 mg / L, 0.9 mg / L, 0.8 mg / L, 0.7 mg / L, 0.6 mg / L, 0.5 mg / L, 0.4 mg / L, 0.3 mg / L, 0.2 mg / L, and 0.1 mg / L DO. These exemplary decimal fractions are provided here for illustrative purposes and are not meant to be limiting in any way. Oxygen levels in a liquid or solution may be reported as a percentage of air saturation or a percentage of oxygen saturation (the ratio of the dissolved oxygen (O2) concentration in a solution to the maximum amount of dissolved oxygen for a solution at a constant temperature, pressure, and salt concentration under stable equilibrium). A well-oxygenated solution (e.g., a mixed and / or stirred solution) that does not contain oxygen producers or consumers is 100% air-saturated. In some embodiments, the term "hypoxic" refers to a solution that is 40% or less air-saturated, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 25%, 26%, 27%, 28%, 29 ... 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% are meant to refer to air saturation, and any and all decimal increments thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%) These include any range of oxygen saturation levels between 0 and 40%, inclusive (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.).The exemplary decimals and ranges listed herein are for illustrative purposes only and are not meant to be limiting in any way. In some embodiments, the term "hypoxia" is meant to refer to an oxygen saturation of 9% or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, O2 saturation, including any and all decimal increments therebetween (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%, 0.44%, 0.3%, 0.25%, 1.5%, 1.6%, 1.8%, 1.9%, 2.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%, 0.44%, 0.3%, 0.25%, 1.5%, 1.6%, 1.8 ... %, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%, 0.032%, 0.025%, 0.01%, etc.), as well as any range of oxygen saturation levels between 0 and 9%, inclusive (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-8%, 5-7%, 0.3-4.2% O2, etc.). The exemplary numbers and ranges listed herein are for illustrative purposes and are not meant to be limiting in any way.

[0076] [Table A] Intestinal oxygen tension [Table 2]

[0077] In some embodiments, the promoters described herein are induced, directly or indirectly, by conditions in the culture vessel (e.g., flask or fermentor or other suitable culture vessel) in which the strain is grown or maintained prior to in vivo administration. Non-limiting examples of such conditions provided during culture of the strain prior to administration in vivo include hypoxic, anaerobic, microaerobic, or aerobic conditions, other defined oxygen levels (e.g., those exemplified below), the presence of arabinose, IPTG, rhamnose, or other chemical and / or nutritional inducers described herein or known in the art. In some embodiments, conditions within the culture vessel are set at a constant oxygen level, e.g., 1%-10% oxygen, 10%-20% oxygen, 20%-30% oxygen, 30%-40% oxygen, 40%-50% oxygen, 60%-70% oxygen, 70%-80% oxygen, 80%-90% oxygen, 90%-100% oxygen, and other oxygen levels described herein, at which the promoter is induced directly or indirectly.

[0078] As used herein, the term "gene" or "gene sequence" is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. Gene, genetic sequence, or genetic sequence is meant to include a complete genetic sequence or a partial genetic sequence. Gene, genetic sequence, or genetic sequence is meant to include sequences that encode proteins or polypeptides, and also to include genetic sequences that do not encode proteins or polypeptides, such as regulatory sequences, leader sequences, signal sequences, or other non-protein-coding sequences.

[0079] "Microorganism" refers to an organism or microorganism of microscopic, submicroscopic, or ultramicroscopic size, typically consisting of a single cell. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, certain algae, and protozoa. In some aspects, a microorganism is engineered to produce one or more therapeutic molecules or proteins of interest (an "engineered microorganism"). In certain aspects, a microorganism is engineered to take up and catabolize specific metabolites or other compounds from its environment, e.g., the gastrointestinal tract. In certain aspects, a microorganism is engineered to synthesize specific beneficial metabolites or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus.

[0080] "Non-pathogenic bacteria" refers to bacteria that are unable to cause disease or adverse reactions in a host. In some embodiments, non-pathogenic bacteria are gram-negative bacteria. In some embodiments, non-pathogenic bacteria are gram-positive bacteria. In some embodiments, non-pathogenic bacteria are commensal bacteria present in the normal microflora of the gastrointestinal tract. Examples of non-pathogenic bacteria include, but are not limited to, species of the genera Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, such as Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, and Bacteroides subtilis. subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus caseiExamples of pathogenic bacteria include Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Patent Nos. 6,835,376; 6,203,797; 5,589,168; and 7,731,976). Naturally occurring pathogenic bacteria may also be genetically engineered to reduce or eliminate pathogenicity.

[0081] "Probiotics" is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, that can confer a health benefit to a host organism containing an adequate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Several species, strains, and / or subtypes of non-pathogenic bacteria are now recognized as probiotics. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria, Escherichia, Lactobacillus, and Saccharomyces, such as Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, E. coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. Patent No. 6,835,376). Probiotics may also be variants or mutant strains of bacteria (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006). Non-pathogenic microorganisms Bacteria may be genetically engineered to enhance or improve desired biological properties, such as survival rate. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.

[0082] As used herein, "stably maintained" or "stable" bacteria are used to refer to bacterial host cells that harbor non-native genetic material, e.g., genes encoding effector molecules, that are integrated into the host genome or propagated on a self-replicating extrachromosomal plasmid such that the non-native genetic material is maintained, expressed, and / or propagated. Stable bacteria can be maintained in vitro, e.g., in culture, and / or in The bacterium can survive and / or grow in vivo, for example, in the gastrointestinal tract. For example, a stable bacterium can be a genetically engineered bacterium containing a gene encoding an effector molecule (e.g., PAL) in which a plasmid or chromosome carrying the PAL gene is stably maintained in the host cell, such that the effector can be expressed in the host cell and the host cell can survive and / or grow in vitro and / or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material, e.g., the PAL gene. In some embodiments, copy number affects the expression level of the non-native genetic material, e.g., the PAL gene or the PAH gene.

[0083] As used herein, the terms "modulate" and "treat" and their cognates refer to the improvement of a disease, disorder, and / or condition, or at least one discernible symptom thereof. In another embodiment, "modulate" and "treat" refer to the improvement of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, "modulate" and "treat" refer to inhibiting the progression of a disease, disorder, and / or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to slowing or reversing the progression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition can include reducing or eliminating associated symptoms without necessarily including elimination of the underlying disease. For example, primary hyperphenylalaninemia is caused by a congenital genetic mutation for which there is no known cure. Hyperphenylalaninemia can also be secondary to other conditions, such as liver disease. Treating hyperphenylalaninemia can involve reducing or eliminating excess phenylalanine and / or associated symptoms, and does not necessarily encompass elimination of the underlying disease. As used herein, "prevent" and its cognates refer to delaying the onset of or reducing the risk of acquiring a given disease, disorder, and / or condition or symptoms associated with such disease, disorder, and / or condition.

[0084] Those in need of treatment may include individuals who already have a particular medical disease, as well as individuals who are at risk of having the disease or who may eventually contract the disease. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the occurrence of the disease, the presence or progression of the disease, or the likelihood that a subject with the disease will be amenable to treatment.

[0085] As used herein, a "pharmaceutical composition" refers to a preparation of the genetically engineered bacteria of the present invention together with other components, such as physiologically suitable carriers and / or excipients.

[0086] The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier," which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the bacterial compound being administered. Included in the phrase.

[0087] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.

[0088] The terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay onset, and / or reduce the risk of developing one or more symptoms of a disease or condition. Therapeutically effective amounts and therapeutically effective frequencies of administration are known in the art and can be determined by methods described below.

[0089] As used herein, the term "antibody" or "antibodies" is meant to encompass all variations of antibodies and fragments thereof having one or more particular binding specificities. Thus, the term "antibody" or "antibodies" includes full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelid), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single domain antibodies (sdAB, V), and the like. HThe term "antibody" is meant to include H-fragments, heavy-chain antibodies (HCAbs), nanobodies, diabodies, and minibodies. Antibodies can have two or more binding specificities, e.g., bispecifics. The term "antibody" is also meant to include so-called antibody mimetics. Antibody mimetics refer to small molecules (e.g., 3-30 kDa) that can specifically bind to an antigen but do not have antibody-related structure, and may be single amino acid chain molecules. Antibody mimetics include, but are not limited to, affibody molecules (Z domain of protein A), affilin (gamma-B crystallin), ubiquitin, affimer (cystatin), affitin (Sac7d) (from Sulfolobus acidocaldarius), alphabodies (triple helix coiled-coil), anticalins (lipocalins), avimers (domains of various membrane receptors), DARPins (ankyrin repeat motifs), Fynomers (SH3 domain of Fyn), Kunitz domain peptides (Kunitz domains of various protease inhibitors), ecallantide, and monobodies. In certain embodiments, the term "antibody" or "antibodies" is meant to refer to single-chain antibody(ies), single-domain antibody(ies), and camelid antibody(ies). The utility of antibodies in treating cancer and additional anti-cancer antibodies can be found, for example, in Scott et al., "Antibody Therapy for Cancer," Nature Reviews Cancer, April 2012, Vol. 12, which is incorporated by reference in its entirety.

[0090] A "single-chain antibody" or "single-chain antibody(ies)" typically refers to a peptide comprising an immunoglobulin heavy chain, an immunoglobulin light chain, and optionally a linker or bond, such as a disulfide bond. Single-chain antibodies lack the constant Fc region found in traditional antibodies. In some embodiments, a single-chain antibody is a naturally occurring single-chain antibody, such as a camelid antibody. In some embodiments, a single-chain antibody is a synthetic, engineered, or modified single-chain antibody. In some embodiments, a single-chain antibody can retain substantially the same antigen specificity as the original immunoglobulin despite the addition of a linker and removal of the constant region. In some aspects, a single-chain antibody can be an "scFv antibody," which is a fusion of the variable regions of an immunoglobulin heavy chain (VH) and a light chain (VL) (without the constant region), optionally linked by a short linker peptide of 10 to about 25 amino acids, as described, for example, in U.S. Pat. No. 4,946,778, the contents of which are incorporated herein by reference in their entirety. Fv fragments refer to a single-chain antibody fragment. An Fv fragment is the smallest fragment that retains the binding site of an antibody, which in some embodiments can maintain the specificity of the original antibody. Techniques for producing single-chain antibodies are described in U.S. Patent No. 4,946,778. The Vh and VL sequences of an scFv can be connected via the N-terminus of the VH connected to the C-terminus of the VL, or via the C-terminus of the VH connected to the N-terminus of the VL. ScFv fragments are independently folding entities that can be indistinguishably fused to other epitope tags or protein domains at either end. Linkers of various lengths can be used to link the Vh and VL sequences, and the linkers can be glycine-rich (providing flexibility) or serine- or threonine-rich (increasing solubility). Short linkers can prevent the association of the two domains, resulting in multimers (diabodies, tribodies, etc.). Long linkers can result in proteolysis or weak domain association (as described in Voelkel et al., 2011). Linkers of 15-20 or 18-20 amino acids in length are most frequently used. Further non-limiting examples of linkers, including other flexible linkers, are described in Chen et al., 2013 (Adv Drug Deliv Rev. 2013, Oct. 15; 65(10):1357-1369, Fusion Protein Linkers: Property, Design and Functionality), the contents of which are incorporated herein by reference in their entirety. Flexible linkers may also be rich in small or polar amino acids such as glycine and serine, but may contain additional amino acids such as threonine and alanine to maintain flexibility, and polar amino acids such as lysine and glutamic acid to improve solubility. Exemplary linkers include, but are not limited to, (Gly-Gly-Gly-Gly-Gly-Ser)n, KESGSSEQLAQFRSLD, and EGKSSGSESKST, (Gly)8, and the Gly- and Ser-rich flexible linker, GSAGSAAGSGEF.As used herein, "single-chain antibodies" also includes single-domain antibodies, including camelid antibodies and other heavy-chain antibodies, light-chain antibodies, including nanobodies, and single-domain VH or VL domains derived from humans, mice, or other species. Single-domain antibodies can be derived from any species, including, but not limited to, mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cows. Single-domain antibodies include domain-antigen-binding units with camelid scaffolds derived from camels, llamas, or alpacas. Camelids produce functional antibodies lacking light chains. The heavy-chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface contains only three CDRs, compared to six CDRs in classical antigen-binding molecules (Fabs) or single-chain variable fragments (scFvs). Camelid antibodies can achieve binding affinities comparable to those of conventional antibodies. Camelid scaffold-based antibodies can be produced using methods well known in the art. Cartilaginous fish also have heavy chain antibodies (IgNAR, "immunoglobulin novel antigen receptor") from which single domain antibodies called VNAR fragments can be derived. Alternatively, dimeric variable domains from IgG of human or mouse origin can be split into monomers. Nanobodies are single chain antibodies derived from light chains. The term "single chain antibody" also refers to antibody mimics.

[0091] In some embodiments, the antibodies expressed by the engineered microorganisms are bispecific. In certain embodiments, the bispecific antibody molecule comprises an scFv or fragment thereof, which has binding specificity for a first epitope and an scFv or fragment thereof, which has binding specificity for a second epitope. Antigen-binding fragments or antibody portions include bivalent scFvs (diabodies), bispecific scFv antibodies in which the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Monomeric single-chain diabodies (scDbs) are readily assembled in bacteria and mammalian cells and exhibit improved stability under physiological conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001) 14(10):815-823 (which describes optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies).

[0092] An "isolated" polypeptide, or a fragment, variant, or derivative thereof, refers to a polypeptide that is not in its natural environment. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including, but not limited to, bacterial or mammalian cells, are considered isolated for purposes of the present invention, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. A recombinant peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein produced by recombinant DNA technology, i.e., produced from cells, microorganisms, or mammals transformed with an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures typically do not contain glycans. Fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination thereof, are also included as polypeptides. The terms "fragment," "variant," "derivative," and "analog" include polypeptides having an amino acid sequence sufficiently similar to that of the original peptide and that retain at least one or more properties of the corresponding original polypeptide. Fragments of the polypeptides of the present invention include proteolytic fragments as well as deletion fragments. Fragments also include specific antibodies or biologically active or immunologically active fragments derived from any of the polypeptides described herein. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis methods known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0093] As used herein, the term "polypeptide" includes "polypeptide" and "polypeptides" and refers to a molecule consisting of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). The term "polypeptide" refers to any chain(s) of two or more amino acids and does not refer to a specific length of the product. Thus, "peptide," "dipeptide," "tripeptide," "oligopeptide," "protein," "amino acid chain," or any other term used to refer to a chain(s) of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "dipeptide" refers to a peptide of two linked amino acids. The term "tripeptide" refers to a peptide of three linked amino acids. The term "polypeptide" is also intended to refer to products of post-expression modification of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology. In other embodiments, polypeptides are produced by the genetically engineered bacteria or viruses of the present invention. Polypeptides of the present invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2,000 or more amino acids in size. Polypeptides may have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, while polypeptides that do not possess a defined three-dimensional structure but can adopt multiple different conformations are referred to as unfolded.The term "peptide" or "polypeptide" may refer to an amino acid sequence corresponding to a protein or portion of a protein, or may refer to an amino acid sequence corresponding to a non-protein sequence, e.g., a sequence selected from regulatory peptide sequences, leader peptide sequences, signal peptide sequences, linker peptide sequences, and other peptide sequences.

[0094] Polypeptides also include fusion proteins. The term "derivative" includes fusion proteins containing the sequence of the original peptide or a sequence sufficiently similar to the original peptide. As used herein, the term "fusion protein" refers to a chimeric protein containing the amino acid sequences of two or more different proteins. Typically, fusion proteins are generated through well-known in vitro recombinant techniques. A fusion protein may have similar structural function (but not necessarily to the same extent), regulatory function (but not necessarily to the same extent), biochemical function (but not necessarily to the same extent), and / or immunological activity (but not necessarily to the same extent) as the individual original proteins that are components of the fusion protein. "Derivatives" include, but are not limited to, peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. "Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide with the sequence of a second peptide. An amino acid in one peptide is similar to the corresponding amino acid in a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those set forth in Dayhoff, MO, ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, DC (1978), and Argos, EMBO J. 8 (1989), pp. 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Cys, Ser, Tyr, Thr; Val, Ile, Leu, Met, Ala, Phe; Lys, Arg, His; Phe, Tyr, Trp, His; and Asp, Glu.

[0095] As used herein, the term "sufficiently similar" refers to a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues compared to a second amino acid sequence so that the first and second amino acid sequences share a common structural domain and / or a common functional activity. For example, amino acid sequences that contain a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical are defined herein as being sufficiently similar. Preferably, variants are sufficiently similar to the amino acid sequence of the peptides of the present invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wild-type peptides by one or more amino acid deletions, additions, and / or substitutions, respectively, and may be naturally occurring variants or artificially designed variants.

[0096] As used herein, the term "linker," "linker peptide," or "peptide linker," or "linker" refers to a synthetic or non-natural or non-naturally occurring amino acid sequence that connects or links two polypeptide sequences, for example, connects two polypeptide domains.As used herein, the term "synthetic" refers to a non-naturally occurring amino acid sequence.Exemplary linkers are described herein.Further exemplary linkers are provided in U.S. Patent Application Publication No. 20140079701, the contents of which are incorporated herein by reference in their entirety.

[0097] As used herein, the term "codon-optimized sequence" refers to a sequence that has been modified from an existing coding sequence, or that has been modified to improve translation in an expression host cell or organism of, for example, a transcribed RNA molecule transcribed from the coding sequence, or to improve transcription of the coding sequence. Codon optimization refers to a sequence designed to improve the expression of a given coding sequence. Codon optimization includes, but is not limited to, processes involving selecting codons for a coding sequence to match the codon preferences of the expression host organism. The term "codon-optimized" refers to modifying codons in a gene or coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization can involve replacing at least one, or more than one, or a substantial number of codons with one or more codons that are more frequently used in the genes of the host organism. A "codon-optimized sequence" refers to a sequence modified from an existing coding sequence or designed to improve translation in an expression host cell or organism, for example, of a transcribed RNA molecule transcribed from the coding sequence, or to improve transcription of the coding sequence. In some embodiments, improved transcription and / or translation includes increasing the level of transcription and / or translation. In some embodiments, improved transcription and / or translation includes decreasing the level of transcription and / or translation. In some embodiments, codon optimization is used to fine-tune the expression level from a construct of interest. Codon optimization includes, but is not limited to, the process of selecting codons for a coding sequence to match the codon preferences of the expression host organism. Many organisms exhibit biases or preferences for using certain codons to encode the insertion of specific amino acids into growing polypeptide chains. Codon preference, or codon bias, differences in codon usage among organisms are permitted by the degeneracy of the genetic code and are well documented among many organisms. Codon bias often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis.Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0098] As used herein, the term "secretion system" or "secreted protein" refers to a natural or non-natural secretion mechanism that can secrete or export a protein of interest or a therapeutic protein from a microorganism, e.g., a bacterial cytoplasm. A secretion system may comprise a single protein or may comprise two or more proteins assembled into a complex, such as HlyBD. Non-limiting examples of secretion systems in Gram-negative bacteria include modified type III flagella, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multidrug efflux pumps, and various single-membrane secretion systems. Non-limiting examples of secretion systems in Gram-positive bacteria include the Sec and TAT secretion systems. In some embodiments, the protein of interest contains a "secretion tag" of RNA or peptide origin that directs the protein of interest or a therapeutic protein to a specific secretion system. In some embodiments, the secretion system is capable of removing this tag before secreting the protein of interest from the engineered bacteria. For example, in type V auto-secretion-mediated secretion, an N-terminal peptide secretion tag is removed upon translocation of the "passenger" peptide from the cytoplasm to the periplasmic compartment by the native Sec system. Furthermore, once the auto-secretor translocates across the outer membrane, the C-terminal secretion tag can be removed by autocatalytic or protease-catalyzed cleavage, thereby releasing the protein of interest into the extracellular environment.

[0099] As used herein, the term "transporter" is meant to refer to a mechanism, e.g., a protein(s), for importing molecules, e.g., amino acids, toxins, metabolites, substrates, etc., from the extracellular environment into a microorganism. For example, a phenylalanine transporter, such as PheP, imports phenylalanine into a microorganism.

[0100] Effectors also include immune checkpoint inhibitors. "Immune checkpoint inhibitor" or "immune checkpoint" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more immune checkpoint proteins. Immune checkpoint proteins regulate T cell activation or function and are known in the art. Non-limiting examples include CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2. Immune checkpoint proteins are involved in costimulatory or inhibitory interactions in T cell responses, regulating and maintaining self-tolerance and physiological immune responses. For example, systemic immunotherapy using CTLA-4 inhibitors can alter immune regulation, induce immune dysfunction, and lead to opportunistic autoimmune disorders (see, for example, Kong et al., 2014).

[0101] As used herein, a genetically engineered microorganism, e.g., an engineered bacterium or phage, or molecule that "inhibits" a biological molecule refers to a bacterium or virus or molecule that can reduce, decrease, or eliminate the biological activity, biological function, and / or number of that biological molecule compared to a control (e.g., an untreated control or an unmodified microorganism of the same subtype under the same conditions).

[0102] As used herein, a genetically engineered microorganism, e.g., an engineered bacterium or phage molecule that "activates" or "stimulates" a biological molecule, refers to a bacterium or phage molecule that can activate, increase, enhance, or promote the biological activity, biological function, and / or number of that biological molecule compared to a control (e.g., an untreated control under the same conditions or an unmodified microorganism of the same subtype).

[0103] The terms "phage" and "bacteriophage" are used interchangeably herein. Both terms refer to viruses that infect and replicate within bacteria. As used herein, "phage" or bacteriophage collectively refers to prophage, lysogenic phage, dormant phage, temperate phage, intact phage, defective phage, cryptic phage and satellite phage, phage tail bacteriocins, teriocins, and gene transfer agents.

[0104] As used herein, the term "prophage" refers to the genomic material of a bacteriophage, which integrates into a host cell replicon and replicates along with the host. A prophage can produce phage when specifically activated. In some cases, a prophage is unable to produce phage or has never produced phage (i.e., a defective or cryptic prophage). In some cases, prophage also refers to a satellite phage. The terms "prophage" and "endogenous phage" are used interchangeably herein.

[0105] As used herein, the terms "temperate phage" or "temperate bacteriophage" or "prophage" are used interchangeably to refer to a phage that resides within the DNA of a bacterial host and replicates along with the host during the bacterial replication cycle and cell division.

[0106] As used herein, the term "natural state" of a bacterium or organism or the "natural state" of a bacterium refers to an organism that has not been modified by genetic engineering. In some cases, the term "natural state" of a bacterium or organism or the "natural state" of a bacterium refers to an organism that has not been modified by genetic engineering, compared to an isogenic strain that has been modified with respect to a defined element. Thus, a bacterium may be in its natural state with respect to one defined element, but not with respect to another defined element. In some embodiments, In some embodiments, a bacterium may contain one or more of the same or different phages or prophages in its natural or native state. In some embodiments, a bacterium containing one or more of the same or different types of phages or prophages in its natural or native state serves as a progenitor strain for an engineered strain. As a result, the same one or more endogenous phages or prophages may also be present in the genetically engineered bacterium, for example, if the progenitor or parent strain contains such endogenous phages or prophages in its natural state. Thus, the genetically engineered bacterium also contains a prophage in its natural state (where a phage is a defined element in its natural state).

[0107] "Endogenous phage" or "endogenous prophage" also refers to a phage that is present in the natural state of the bacterium (and its parent strain).

[0108] As used herein, the term "phage knockout" or "inactivated phage" refers to a phage that has been modified so that it is no longer able to produce and / or package phage particles, or that it produces fewer phage particles than the wild-type phage sequence. In some embodiments, inactivated phage or phage knockout refers to the inactivation of a temperate phage in a lysogenic state, i.e., the inactivation of a prophage. Such modifications refer to mutations in the phage; such mutations include insertions, deletions (partial or complete deletion of the phage genome), substitutions, inversions at one or more locations within the phage genome, for example, within one or more genes within the phage genome.

[0109] As used herein, the term "isogenic" bacterial strain refers to a bacterial strain that is genetically identical or contains defined changes but is otherwise identical. For example, an isogenic mutant typically refers to two strains that are identical except that one contains defined mutations in one or more known genes or proteins. Thus, a phage-free or phageless strain has a corresponding isogenic strain that contains a prophage that can be induced to release phage particles from the bacterial cell.

[0110] As used herein, the adjectives "phage-free," "phage free," and "phageless" are used interchangeably to characterize bacteria or strains that contain one or more prophage, one or more of which have been modified. The modification may result in the loss of the ability of the prophage to be induced or to release phage particles. Alternatively, the modification may result in less efficient or less frequent induction or less efficient or less frequent phage release compared to an isogenic strain without the modification. The ability to induce and release phage may be measured using a plaque assay as described herein.

[0111] As used herein, the term "lysogen" refers to a bacterium containing a prophage in the lysogenic cycle in which the phage genes necessary for lysis are not expressed.

[0112] As used herein, phage induction refers to the part of the life cycle of a lysogenic prophage in which lytic phage genes are activated, phage particles are produced, and lysis occurs.

[0113] As used herein, the term induction refers to the conversion of a lysogenic infection to a productive infection, i.e., the induced prophage initiates the production and release of phage particles. Induction is often stimulated by damage to the bacterial DNA, resulting in the release of the prophage from the bacterial chromosome. This may or may not include excision of the graft.

[0114] In some embodiments, the genetically engineered bacteria are useful for treating, preventing, managing, reducing the severity of, ameliorating, or curing a disorder, disease, or condition. In some embodiments, the disorder is an autoimmune disorder. As used herein, "autoimmune disorder" includes acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune ovarian failure, and autoimmune ovarian failure. Focal inflammation, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and nerve neuropathy, Barrow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multiple myelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie's disease - Myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans' syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolysis anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere's disease, microscopic polyangiitis,Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (Streptococcal Infectious Pediatric Autoimmune Neuropsychiatric Disorders), P ediatric A Autoimmune N europsychiatric D isorders A associated with S streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polyglandular autoimmune syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progestational dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome In some embodiments, the disorder includes, but is not limited to, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, bullous dermatoses, vitiligo, and Wegener's granulomatosis.

[0115] In some embodiments, the disease is a metabolic disease. As used herein, "metabolic disease" includes, but is not limited to, type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedl syndrome; syndrome; Prader-Willi syndrome; nonalcoholic fatty liver disease; tuberous sclerosis complex; Albright syndrome; brain-derived neurotrophic factor (BDNF) deficiency; single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; proopiomelanocortin (POMC) deficiency; proprotein convertase subtilisin / kexin type 1 (PCSK1) deficiency; Src homology 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; Alström syndrome; Cohen syndrome; and ulno-mammary syndrome.

[0116] In some embodiments, the disorder is cancer. "Cancer" or "cancerous" is used to refer to a physiological condition characterized by unregulated cell growth. In some embodiments, cancer refers to a tumor. "Tumor" is used to refer to any neoplastic cell growth or proliferation, or any pre-cancerous or cancerous cell or tissue. Tumors may be malignant or benign. Cancer types include adrenal gland cancer, adrenocortical carcinoma, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytoma, brain stem glioma, craniopharyngioma, ependymoma), bronchial tumor, central nervous system tumor, breast cancer, Castleman's disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, gestational trophoblastic disease, cardiac cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, hypopharyngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia), liver cancer, lung cancer, lymphoma (e.g., AIDS-related lymphoma), , Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer (e.g., basal cell carcinoma, melanoma), small intestine cancer, gastric cancer, teratoma, testicular cancer, pharyngeal cancer, thymus cancer, thyroid cancer, rare childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. Side effects of cancer treatment may include, but are not limited to, opportunistic autoimmune disorders, systemic toxicity, anemia, loss of appetite, irritation of the bladder lining, bleeding and purpura (thrombocytopenia), changes in taste or smell, constipation, diarrhea, dry mouth, difficulty swallowing, edema, fatigue, hair loss (alopecia), infections, infertility, lymphedema, stomatitis, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infections, and / or problems with memory and concentration (National Cancer Institute). In some embodiments, the disorder is a hyperammonemic disorder.

[0117] In some embodiments, the disorder is a rare disease, including but not limited to hyperammonemia, urea cycle disorders, propionic acidemia, methylmalonic acidemia, maple syrup urine disease, isovaleric acidemia, hyperoxaluria, and phenylketonuria.

[0118] Exemplary circuits for treating, preventing, reducing the severity of, managing, ameliorating, or curing one or more of the above-mentioned disorders are disclosed in pending, commonly owned, international patent applications PCT / US2016 / 34200 (filed May 25, 2016), PCT / US2017 / 013072 (filed January 11, 2017), PCT / US2017 / 016603 (filed February 3, 2017), PCT / US2017 / 016609 (filed February 4, 2016), PCT / US2017 / 017563 (filed February 10, 2017), and PCT / US2017 / 017563 (filed February 10, 2017). ), PCT / US2017 / 017552 (filing date: February 10, 2017), PCT / US2016 / 044922 (filing date: July 29, 2016), PCT / US2016 / 049781 (filing date: August 31, 2016), PCT / US2016 / 37098 (filing date: June 10, 2016), PCT / US2016 / 069052 (filing date: December 28, 2016), PCT / US2016 / 32562 (filing date: May 13, 2016), PCT / US2016 / 062369 (filing date No. 6,2016, filed Nov. 16, 2016, and PCT / US2017 / 013072, the contents of which are incorporated herein by reference in their entireties.

[0119] As used herein, the articles "a" and "an" should be understood to mean "at least one" unless clearly indicated to the contrary.

[0120] The term "and / or," when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and / or C" indicates that the selection can be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The term "and / or" can be used interchangeably with "at least one" or "one or more" of the elements in the list.

[0121] (bacteria) In some embodiments, the bacteria disclosed herein comprise one or more mutations or modifications to an endogenous phage genome. In some embodiments, the bacterium comprises a bacteriophage in its native or natural state. In some embodiments, the phage is present in all isolates of a particular bacterium. In some embodiments, the phage is present in bacteria of the same species, strain, or substrain. In some embodiments, the phage is an intact prophage. In some embodiments, the phage is a defective prophage. In some embodiments, the one or more mutations render the phage unable to enter the lytic cycle. In some embodiments, the one or more mutations affect the ability of the phage to undergo the lytic cycle, e.g., reducing the frequency or number of bacteria in a given population that can undergo the lytic phase. In some embodiments, the one or more mutations prevent the phage from infecting other bacteria. In some embodiments, the one or more mutations alter (e.g., increase or decrease) the fitness of the bacterium. In some embodiments, the one or more mutations alter (e.g., increase or decrease) an effector function. In some embodiments, the one or more mutations do not alter the fitness of the bacterium. In some embodiments, the one or more mutations do not alter effector function. In some embodiments, the one or more mutations improve the process by which the bacteria is manufactured or produced, including large-scale manufacturing. In any of these embodiments, the bacteria may be in its otherwise natural state. Alternatively, in any of these embodiments, the bacteria may be further genetically engineered to include a gene sequence encoding one or more effector molecules.

[0122] In some embodiments, bacteria containing one or more mutant phages may be used as a bacterial chassis to which genetic circuits are added or modified.

[0123] In some embodiments, the bacteria are non-pathogenic bacteria. In some embodiments, the bacteria are commensal bacteria. In some embodiments, the bacteria are probiotic bacteria. In some embodiments, the bacteria are naturally occurring pathogenic bacteria that have been modified or mutated to reduce or eliminate pathogenicity. In some embodiments, the non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, the non-pathogenic bacteria are Gram-positive bacteria. Exemplary bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium spp. These include Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus lactis, and Saccharomyces boulardii. In certain embodiments, the bacterium is selected from the group consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis.

[0124] In some embodiments, the bacterium is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of the Enterobacteriaceae family that has evolved into one of the best-characterized probiotics (Ukena et al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008) and is GRAS (Generally Recognized as Safe). g enerally r ecognized a s s It has a virulence-resistant (safe) status (Reister et al., 2014; underlining added). Genome sequencing confirmed that E. coli Nissle lacks important virulence factors (e.g., E. coli α-hemolysin, P-fimbrial adhesin) (Schultz, 2008). Furthermore, E. coli Nissle has been shown to lack pathogenic adhesins, produce no enterotoxins or cytotoxins, and is not invasive or uropathogenic (Sonnenborn et al., 2009). As early as 1917, E. coli Nissle was packaged for therapeutic use in pharmaceutical capsules called Mutaflor. It is generally accepted that the therapeutic efficacy and safety of E. coli Nissle have been convincingly demonstrated (Ukena et al., 2007).

[0125] In some embodiments, the bacterium of the present disclosure is a tumor-targeting bacterium. Tumor-targeting bacteria are described in International Patent Application PCT / US2017 / 013072, filed January 11, 2017, published as WO2017 / 123675, the contents of which are incorporated herein by reference in their entirety.

[0126] Those skilled in the art will appreciate that the genetic modifications disclosed herein can be adapted to other species, strains, and subtypes of bacteria. Furthermore, genes from one or more different species can be transferred to each other, for example, the PAL gene from Rhodosporidium toruloides can be expressed in Escherichia coli (Sarkissian et al., 1999).

[0127] In any of these embodiments, any of the bacterial species disclosed herein or known in the art that can be used in accordance with the present disclosure comprises one or more mutations or modifications to one or more endogenous phage genomes. In some embodiments, the modification to the endogenous phage genome comprises one or more deletions, insertions, substitutions, or inversions, or combinations thereof, within the phage genome. In some embodiments, the modification is one or more deletions within the phage genome. In some embodiments, one or more phage genes are deleted. In some embodiments, one or more phage genes are partially deleted. In some embodiments, the modification is one or more insertions in the phage genome. In some embodiments, the insertion comprises a gene sequence encoding an antibiotic cassette described herein. In some embodiments, one or more genes within the phage genome are replaced with alternatives. In some embodiments, the replacement comprises a gene sequence encoding an antibiotic cassette. In some embodiments, the entire sequence of one or more phage genes is inverted. In some embodiments, a partial sequence of one or more phage genes is inverted.

[0128] Unmodified E. coli Nissle and the genetically engineered bacteria of the present invention can be destroyed, for example, by protective factors in the gastrointestinal tract or serum (Sonnenborn et al., 2009), or by activation of a kill switch several hours or days after administration. Thus, the genetically engineered bacteria may require continuous administration. In some embodiments, residence time is calculated for human subjects. In vivo residence time can be calculated for the genetically engineered bacteria of the present invention (see, for example, Figure 68 of WO2017087580, the entire contents of which are incorporated herein by reference).

[0129] In some embodiments, the genetically engineered bacterium comprises a gene encoding PAL, wherein the PAL gene is operably linked to a directly or indirectly inducible promoter. In some embodiments, the bacterium comprises a non-native PAL gene. In some embodiments, the bacterium comprises an additional copy of a native PAL gene. In some embodiments, the promoter is not naturally associated with the PAL gene. In some embodiments, the promoter is any one or more of the promoters disclosed herein.

[0130] In some embodiments, the genetically engineered bacterium comprises a gene encoding a PAH, wherein the PAH gene is operably linked to a directly or indirectly inducible promoter. In some embodiments, the bacterium comprises a non-native PAH gene. In some embodiments, the bacterium comprises an additional copy of a native PAH gene. In some embodiments, the promoter is not naturally associated with the PAH gene. In some embodiments, the promoter is any one or more of the promoters disclosed herein.

[0131] In some embodiments, the genetically engineered bacterium comprises a gene encoding an LAAD, wherein the LAAD gene is operably linked, either directly or indirectly, to an inducible promoter. In some embodiments, the bacterium comprises a non-native LAAD gene. In some embodiments, the bacterium comprises an additional copy of a native LAAD gene. In some embodiments, the promoter is not naturally associated with the LAAD gene. In some embodiments, the promoter is any one or more of the promoters disclosed herein.

[0132] In some embodiments, the genetically engineered bacterium further comprises a gene encoding a phenylalanine transporter (PheP). In certain embodiments, the bacterium comprises an additional copy of a native gene encoding a phenylalanine transporter, wherein the phenylalanine transporter gene is operably linked to a directly or indirectly inducible promoter. In alternative embodiments, the bacterium comprises a gene encoding a non-native phenylalanine transporter, wherein the phenylalanine transporter gene is operably linked to a directly or indirectly inducible promoter. Both embodiments are encompassed by the term "non-native" phenylalanine transporter. In some embodiments, the promoter is not naturally associated with the pheP gene. In some embodiments, the same promoter controls expression of PheP and PAL and / or PAH and / or LAAD. In some embodiments, the promoter controlling expression of PheP is different from the promoter controlling expression of PAL and / or PAH and / or LAAD. In some embodiments, the promoter controlling expression of PheP is any one or more of the promoters disclosed herein.

[0133] In some embodiments, the promoter operably linked to PAL, PAH, LAAD, and / or pheP is induced, directly or indirectly, by an exogenous environmental condition. In some embodiments, the promoter is induced, directly or indirectly, by an exogenous environmental condition specific to the mammalian gastrointestinal tract. In some embodiments, the promoter is induced, directly or indirectly, by an exogenous environmental condition specific to the mammalian small intestine. In some embodiments, the promoter is induced, directly or indirectly, by an exogenous environmental condition specific to the mammalian large intestine. In some embodiments, the promoter is induced, directly or indirectly, by hypoxic or anaerobic conditions and / or hypoxic conditions, such as the environment of the mammalian gastrointestinal tract. In some embodiments, the promoter is induced, directly or indirectly, by the presence of a molecule or metabolite specific to the mammalian gastrointestinal tract, such as propionate. In some embodiments, the promoter is induced, directly or indirectly, by exposure to tetracycline. In some embodiments, the promoter is induced, directly or indirectly, by exposure to arabinose. In some embodiments, the promoter is induced directly or indirectly by exposure to IPTG. In some embodiments, the promoter is induced directly or indirectly by exposure to rhamnose or other chemical and / or nutritional inducers known to those of skill in the art. In some embodiments, the promoter is directly or indirectly controlled by exogenous environmental temperature. In some embodiments, the promoter is induced directly or indirectly by exposure to IPTG or other LaC1-binding compounds. In some embodiments, the promoter is induced directly or indirectly by exposure to rhamnose. In some embodiments, the promoter is induced directly or indirectly by an increase in temperature. In some embodiments, the promoter is induced directly or indirectly by a decrease in temperature. In some embodiments, the promoter is induced directly or indirectly by a molecule co-administered with the genetically engineered bacteria of the present invention.Such a molecule can be tetracycline or IPTG or arabinose or other chemical and / or nutritional inducers known to those skilled in the art.

[0134] In some embodiments, the promoter is induced directly or indirectly prior to in vivo administration. Non-limiting examples of such conditions provided during culture of the strain prior to in vivo administration include hypoxic, anaerobic, microaerobic, or aerobic conditions, other defined oxygen levels (such as those exemplified below), the presence of arabinose, IPTG, rhamnose, or other chemical and / or nutritional inducers described herein or known in the art. In some embodiments, conditions in the culture vessel are set to a specific oxygen level, e.g., 1%-10% oxygen, 10%-20% oxygen, 20%-30% oxygen, 30%-40% oxygen, 40%-50% oxygen, 60%-70% oxygen, 70%-80% oxygen, 80-90% oxygen, 90%-100% oxygen, and other oxygen levels described herein, at which the promoter is induced directly or indirectly.

[0135] (bacteriophage) In some embodiments, the bacteria of the invention comprise one or more temperate, dormant, temperate, intact, defective, cryptic, or satellite phages, or bacteriocin / phage tails or gene transfer agents in their native state. In some embodiments, the prophage or bacteriophage is present in all isolates of a particular bacterium of interest. In some embodiments, the bacterium is a probiotic bacterium. In some embodiments, the bacterium is a genetically engineered derivative of a parent strain that comprises one or more such bacteriophages. Thus, such bacteria of the invention may be in their native state or may be further genetically modified to include circuitry for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacterium comprises one or more gene(s) within the prophage or bacteriophage genome that alter the properties or behavior of the bacteriophage. In some embodiments, the modification or mutation prevents the prophage from entering or completing the lytic process. In some embodiments, the modification or mutation prevents the phage from infecting other bacteria of the same or different type.

[0136] In some embodiments, the modification or mutation alters (e.g., decreases or increases) the fitness of the bacterial host. In some embodiments, the modification or mutation alters (e.g., decreases or increases) a desired effector function, e.g., of the genetically engineered bacterium. In some embodiments, the modification or mutation does not alter (e.g., decreases or increases) the fitness of the bacterial host. In some embodiments, the modification or mutation does not alter (e.g., decreases or increases) a desired effector function, e.g., of the genetically engineered bacterium.

[0137] Phage genome sizes vary widely, from the smallest Leuconostoc phage L5 (2,435 bp), ~11.5 kbp (e.g., Mycoplasma phage P1), ~21 kbp (e.g., Lactococcus phage c2), and ~30 kbp (e.g., Pasteurella phage F108), to the nearly 500 kbp genome of Bacillus megaterium phage G (Hatfull and Hendrix; Bacteriophages and Their Genomes, Curr Opin Virol. 2011 Oct. 1;1(4):298-303, and references therein. Phage genomes can encode fewer than 10 genes to several hundred. Although there are some examples of phages that integrate into bacterial plasmids, temperate phages or prophages typically integrate into the chromosome of their bacterial host (Waldor MK, Friedman DI, Adhya S, eds., Phages Their Role in Bacterial Pathogenesis and Biotechnology. Washington, DC: ASM Press; 2005, pp. 37-54). In some cases, the phage always resides in the same location within the bacterial host chromosome; this location is specific to each phage, i.e., different phages reside in different locations. Other phages are more permissive in that they can integrate at many different locations.

[0138] Accordingly, the bacteria of the present disclosure comprise one or more phage genomes, which may vary in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 1 bp to 10 kb in length. In one embodiment, the bacterium comprises a bacteriophage genome ranging from at least about 1 bp to 10 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 10 kb to 20 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 20 kb to 30 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 30 kb to 40 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 30 kb to 40 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 40 kb to 50 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 50 kb to 60 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 60 kb to 70 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 70 kb to 80 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 80 kb to 90 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 90 kb to 100 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 100 kb to 120 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 120 kb to 140 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 140 kb to 160 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 160 kb to 180 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 180 kb to 200 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 200 kb to 180 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 160 kb to 250 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 250 kb to 300 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 300 kb to 350 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 350 kb to 400 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 400 kb to 500 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome ranging from at least about 500 kb to 1000 kb in length. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome greater than 1000 kb in length.

[0139] In some embodiments, the bacteria of the invention comprise one or more phage genomes containing one or more genes encoding one or more polypeptides. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 1-5 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 5-10 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 10-15 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 15-20 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 20-25 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 25-30 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 30-35 genes. In one embodiment, the genetically engineered bacteria comprise a bacteriophage genome containing at least about 35-40 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 40-45 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 45-50 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 50-55 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 55-60 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 60-65 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 65-70 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 70-75 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 75-80 genes.In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 80-85 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 85-90 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 90-95 genes. In one embodiment, the genetic engineered bacterium comprises a bacteriophage genome containing at least about 85-90 genes. The engineered bacterium comprises a bacteriophage genome containing at least about 95-100 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 100-115 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 115-120 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 120-125 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 125-130 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 130-135 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 135-140 genes. In one embodiment, the engineered bacterium comprises a bacteriophage genome containing at least about 140-145 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 145-150 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 150-160 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 160-170 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 170-180 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 180-190 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 190-200 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing at least about 200-300 genes. In one embodiment, the genetically engineered bacterium comprises a bacteriophage genome containing more than about 300 genes.

[0140] In some embodiments, the phage is always or almost always in the same place or location within the bacterial host chromosome of a particular species. In some embodiments, the phage is found integrated into different locations within the host chromosome of a particular species. In some embodiments, the phage is on a plasmid.

[0141] The presence of prophage sequences can also confer specific properties to bacteria that are not present in isogenic strains without the phage. For example, in some cases, prophage may enable bacteria to acquire antibiotic resistance, reside in new environmental niches, improve adhesion, or become pathogenic. In addition, phages function as vehicles for gene transfer, as DNA from one bacterium can be picked up and released in another bacterium through the lytic process.

[0142] Thus, in some embodiments, the bacterium comprises a phage that confers antibiotic resistance to the bacterium, in some embodiments, the bacterium comprises a phage that confers additional fitness to the bacterium, in some embodiments, the bacterium comprises a phage that confers the bacterium the ability to grow in a new environment, or in some embodiments, the bacterium comprises a phage that confers the ability to transfer host genetic material to another bacterium of the same or a different species.

[0143] In some embodiments, the prophage may be a defective or cryptic prophage. A defective prophage can no longer undergo a lytic cycle. A cryptic prophage may not be able to undergo a lytic cycle or may never have undergone a lytic cycle. Functional studies of the entire repertoire of prophage in bacterial genomes suggest that the majority of prophage are defective at some level: excision, virion formation, lysis, or infectivity (Bobay et al., 2014). Defective or cryptic prophages may undergo mutational collapse and / or thousands of bacterial replications. High levels of abundance can be achieved in many bacteria as a result of the loss of one or more genes essential for the lytic cycle over time (Bobay et al., Pervasive domestication of defective prophages by bacteria, Proc Natl Acad Sci USA). Notably, defective prophages also often contain many genes that may confer adaptive or advantageous functions to the host, including genes encoding proteins with homologous recombination functions, mechanisms for preventing further infection, or bacteriocins (which may be useful in competition for nutrients, e.g., through the inhibition of growth of other neighboring bacterial species). For example, several defective prophages have been characterized in Escherichia coli K-12 (e.g., Rac, e14, DLP12, and QIN) and Bacillus subtilis (e.g., 186 and SKIN) (Casjens, 2001, and references therein). Each of these phages possesses several functional genes. For example, Rac encodes the RecE homologous recombination system.

[0144] Thus, in some embodiments, the bacterium comprises one or more defective or cryptic prophage. In some embodiments, the prophage gene confers homologous recombination function. In some embodiments, the prophage gene confers a function to prevent further infection. In some embodiments, the prophage gene confers a bacteriocin. In some embodiments, the phage gene promotes growth under adverse conditions by increasing carbon utilization, improving tolerance to osmotic, oxidative, and acid stress for increased growth under various conditions, enhancing phosphorus and nitrogen utilization, or affecting biofilm formation.

[0145] In some embodiments, the bacterium comprises one or more satellite phage genomes. Satellite phages are otherwise functional phages that lack their own structural protein genes, but have genomes that are configured for encapsulation by the structural proteins of other specific phages (Six and Klug, Bacteriophage P4: a satellite virus depending on a helper (e.g., prophage P2, Virology, Vol. 51, No. 2, February 1973, pp. 327-344). Thus, in some embodiments, the bacterium comprises a phage genome that lacks its own structural genes.

[0146] In some embodiments, the bacterium comprises one or more theriocins. Many bacteria, both Gram-positive and Gram-negative, produce a variety of particles resembling phage tails that are functional without the phage head (called theriocins), many of which have been shown to have bacteriocin properties (reviewed in Ghequire and Mot, The Tailocin Tale: Peeling off Phage; Trends in Microbiology, October 2015, Vol. 23, No. 10). Phage tail-like bacteriocins are classified into two distinct families: contractile phage tail-like (R-type) and non-contractile but flexible (F-type). Thus, in some embodiments, the bacterium comprises one or more theriocins that confer bacteriocins or other beneficial properties.

[0147] In some embodiments, the bacterium comprises one or more gene transfer agents. Gene transfer agents (GTAs) are phage-like elements encoded by some bacterial genomes. GTAs resemble phages but lack the characteristic abilities that define typical phages: they package random fragments of host cell DNA and transfer them horizontally to other bacteria of the same species (reviewed in Lang et al., Gene transfer agents: phage-like elements of genetic exchange, Nat Rev Microbiol. 2012 Jun 11;10(7):472-482). There, the DNA can replace the resident cognate chromosomal region by homologous recombination. However, because the majority of particles do not contain the gene encoding the GTA, these particles cannot propagate as viruses.

[0148] In some embodiments, the bacterium comprises one or more filamentous virions, which are integrated as dsDNA prophage (reviewed in Marvin DA et al., Structure and assembly of filamentous bacteriophages, Prog Biophys Mol Biol. 2014 Apr;114(2):80-122).

[0149] In any of the embodiments described herein, the genetically engineered bacteria described herein that express one or more enzymes and transporters (e.g., for phenylalanine consumption) contain one or more modifications or mutations in an endogenous prophage or bacteriophage genome. These modifications may alter the properties or behavior of the prophage. In some embodiments, the modifications or mutations do not substantially affect the fitness of the bacteria, and the bacterial fitness is substantially the same as that of an isogenic strain without the modifications or mutations. Prophages can be identified either experimentally or computationally. Experimental approaches involve inducing host bacteria to release phage particles by exposing the host bacteria to UV light or other DNA-damaging conditions. However, in some cases, the conditions under which prophage are induced are unknown, and therefore, the absence of plaques in a plaque assay does not necessarily prove the absence of prophage. Furthermore, this approach can only indicate the presence of viable phage, but does not reveal defective prophage. Therefore, computational identification of prophage from genome sequence data has become the most preferred route.

[0150] In some embodiments, the modification or mutation does not substantially affect the effector function, and the effector function is substantially the same as that of an isogenic strain without the modification or mutation. Table H provides a list of non-limiting examples of probiotic bacteria and the number of potential bacteriophages contained in the bacterial genome as determined by Phaster scoring. Table I provides a list of Clostridium strains and potential phage genomes. Phaster is a web server for bioinformatic identification of phage sequences in organisms (http: / / phaster.ca / ). Phaster scoring is based on the methodology described in phaster.ca and Zhou et al. ("PHAST: A Fast Phage Search tool," Nucl. Acids Res. (2011) 39(suppl 2):W347-W352) and Arndt et al. (Arndt et al. (2016) PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res., May 3, 2016). Briefly, three methods are applied with different criteria to score prophage regions (intact, suspect, or incomplete) in a given bacterial genome sequence. In the first method, if the number of specific phage organelles identified by Phaster is 100% or more of the total number of CDSs in that region, the region is marked with a total score of 150. If it is less than 100%, methods 2 and 3 are used. In method 2, if the number of a particular phage organism identified by Phaster in a given bacterial genome sequence exceeds 50% of the total number of CDSs in that region, that phage organism is considered the major potential phage for that region; the percentage of the total number of phage organisms in this table in the total number of proteins in that region is calculated and then multiplied by 100; the percentage of the length of that phage organism in the length of that region is calculated and then multiplied by 50 (taking into account the encapsulation ability of the phage head). In Method 3, if any of the specific phage-related keywords (such as "capsid," "head," "integrase," "base," "tail," "fiber," "coat," "transposase," "portal," "terminase," "protease," or "ricin") are present, the score is increased by 10 for each keyword found. If the region is larger than 30 Kb in size, the score is increased by 10. If at least 40 proteins are present in the region, the score is increased by 10. If all phage-related proteins and hypothetical proteins account for more than 70% of the total number of proteins in the region, the score is increased by 10. The total score from Method 2 is compared with the total score from Method 3, and the larger one is selected as the region's total score. If the region's total score is less than 70, it is marked as incomplete; if it is between 70 and 90, it is marked as suspicious; and if it is greater than 90, it is marked as intact.

[0151] [Table H] Compatible strains of common probiotics [Table 3]

[0152] Table I: Clostridium strains [Table 4]

[0153] In any of these embodiments, the bacteria described herein contain one or more modifications or mutations in an existing prophage or bacteriophage genome. These modifications alter the properties or behavior of the prophage. In some embodiments, the modifications or mutations prevent the prophage from entering or completing the lytic process. In some embodiments, the modifications or mutations prevent the phage from infecting other bacteria of the same or different type.

[0154] In some embodiments, the modification or mutation alters (e.g., decreases or increases) the fitness of the bacterial host. In some embodiments, the modification or mutation alters (e.g., decreases or increases) a desired effector function, e.g., of the genetically engineered bacterium. In some embodiments, the modification or mutation does not alter (e.g., decreases or increases) the fitness of the bacterial host. In some embodiments, the modification or mutation does not alter (e.g., decreases or increases) a desired effector function, e.g., of the genetically engineered bacterium. (If not, decrease or increase).

[0155] In some embodiments, the modification or mutation improves phenylalanine consumption. In some embodiments, phenylalanine consumption remains similar to levels observed in an isogenic strain containing the unmodified phage. In some embodiments, the modification or mutation does not substantially affect bacterial fitness, and bacterial fitness is substantially the same as the fitness of an isogenic strain without the modification or mutation.

[0156] In some embodiments, the bacterium amplifies at least about 1-2, at least about 2-3, at least about 3-4, at least about 4-5, at least about 5-6, at least about 6-7, at least about 7-8, at least about 8-9, at least about 9-10, at least about 10-11, at least about 11-12, at least about 12-13, at least about 13-14, at least about 14-15, at least about 15-16, at least about 16-17, at least about 17-18, or at least about Also about 18-19, at least about 19-20, at least about 20-21, at least about 21-22, at least about 22-23, at least about 23-24, at least about 24-25, at least about 25-26, at least about 26-27, at least about 27-28, at least about 28-29, at least about 29-30, at least about 30-31, at least about 31-32, at least about 32-33, at least about 33-34, at least about 34-35, at least about 35-36, at least about 36-37, at least about 38 7-38, at least about 38-39, at least about 39-40, at least about 40-41, at least about 41-42, at least about 42-43, at least about 43-44, at least about 44-45, at least about 45-46, at least about 46-47, at least about 47-48, at least about 48-49, at least about 49-50, at least about 50-51, at least about 51-52, at least about 52-53, at least about 53-54, at least about 54-55, at least about 55-56, at least about 56-57 7, at least about 57-58, at least about 58-59, at least about 59-60, at least about 60-61, at least about 61-62, at least about 62-63, at least about 63-64, at least about 64-65, at least about 65-66, at least about 66-67, at least about 67-68, at least about 68-69, at least about 69-70, at least about 70-71, at least about 71-72, at least about 72-73, at least about 73-74, at least about 74-75, at least about 75-76,At least about 76-77, at least about 77-78, at least about 78-79, at least about 79-80, at least about 80-81, at least about 81-82, at least about 82-83, at least about 83-84, at least about 84-85, at least about 85-86, at least about 86-87, at least about 87-88, at least about 88-89, at least about 89-90, at least about 90-91, at least about 91-92, at least about 92-93, at least about 93-94, at least about 94-95, at least about 95-96, at least about 96-97, at least about 97-98, at least about 98-99, at least about 99-100, or at least about 100 or more modifications or mutations.

[0157] In some embodiments, the modification or mutation improves effector function, e.g., phenylalanine consumption. In some embodiments, effector function, e.g., phenylalanine consumption, remains similar to that observed in an isogenic strain containing the unmodified phage. In some embodiments, the modification or mutation does not substantially affect bacterial fitness, and bacterial fitness is substantially the same as that of an isogenic strain without the modification or mutation.

[0158] In some embodiments, the modification or mutation reduces entry or completion of the prophage lytic process by at least about 1-2 fold, at least about 2-3 fold, at least about 3-4 fold, at least about 4-5 fold, at least about 5-10 fold, at least about 10-100 fold, or at least about 100-1000 fold, relative to an isogenic strain without the phage modification. In some embodiments, the modification or mutation completely prevents entry or completion of the prophage lytic process.

[0159] In some embodiments, the modification or mutation reduces entry or completion of the prophage lytic process by at least about 1% to 10%, at least about 10% to 20%, at least about 20% to 30%, at least about 30% to 40%, at least about 40% to 50%, at least about 50% to 60%, at least about 60% to 70%, at least about 70% to 80%, at least about 80% to 90%, or at least about 90% to 100% compared to an isogenic strain without the phage modification.

[0160] In some embodiments, the modification or mutation prevents the phage from infecting other bacteria of the same or different type by at least about 1-2 fold, at least about 2-3 fold, at least about 3-4 fold, at least about 4-5 fold, at least about 5-10 fold, at least about 10-100 fold, at least about 10-20 fold, at least about 20-30 fold, at least about 30-40 fold, at least about 40-50 fold, at least about 50-60 fold, at least about 60-70 fold, at least about 70-80 fold, at least about 80-90 fold, at least about 90-100 fold, or at least about 100-1000 fold, compared to an isogenic strain without the phage modification. In some embodiments, the modification or mutation completely prevents the phage from infecting other bacteria of the same or different type. In some embodiments, the modification or mutation prevents the phage from infecting other bacteria of the same or different type by at least about 1% to 10%, at least about 10% to 20%, at least about 20% to 30%, at least about 30% to 40%, at least about 40% to 50%, at least about 50% to 60%, at least about 60% to 70%, at least about 70% to 80%, at least about 80% to 90%, or at least about 90% to 100%.

[0161] In some embodiments, the modification or mutation alters (e.g., decreases or increases) the fitness of the bacterial host by at least about 1-2 fold, at least about 2-3 fold, at least about 3-4 fold, at least about 4-5 fold, at least about 5-10 fold, at least about 10-100 fold, or at least about 100-1000 fold, compared to an isogenic strain without the phage modification. In some embodiments, the modification or mutation alters (e.g., decreases or increases) the fitness of the bacterial host by, for example, at least about 1%-10%, at least about 10%-20%, at least about 20%-30%, at least about 30%-40%, at least about 40%-50%, at least about 50%-60%, at least about 60%-70%, at least about 70%-80%, at least about 80%-90%, or at least about 90%-100%, compared to an isogenic strain without the phage modification.

[0162] In some embodiments, the modification or mutation alters (e.g., decreases or increases) the desired effector function of, for example, the genetically engineered bacterium by at least about 1-2 fold, at least about 2-3 fold, at least about 3-4 fold, at least about 4-5 fold, at least about 5-10 fold, at least about 10-100 fold, or at least about 100-1000 fold. In some embodiments, the modification or mutation alters (e.g., decreases or increases) the desired effector function of, for example, the genetically engineered bacterium by at least about 1%-10%, at least about 10%-20%, at least about 20%-30%, at least about 30%-40%, at least about 40%-50%, at least about 50%-60%, at least about 60%-70%, at least about 70%-80%, at least about 80%-90%, or at least about 90%-100%, compared to an isogenic strain without phage modification.

[0163] In some embodiments, the mutation comprises one or more deletions within the phage genome sequence. As used herein, "deletion" refers to the removal of one or more nucleotides from a polynucleotide sequence. In some embodiments, the mutation comprises one or more insertions into the phage genome sequence. As used herein, "insertion" refers to the addition of one or more nucleotides to a polynucleotide sequence. In some embodiments, an antibiotic cassette may be inserted at one or more positions within the phage genome sequence. In some embodiments, the mutation comprises one or more substitutions within the phage genome sequence. As used herein, "substitution" refers to the replacement of one or more nucleotides in a polynucleotide sequence with the same number of nucleotides. In some embodiments, the mutation comprises one or more inversions within the phage genome sequence. As used herein, "inversion" refers to when a segment containing two or more nucleotides is reversed end to end within a polynucleotide sequence. In some embodiments, the inversion may be governed by a specific flippase. Exemplary circuits including multiple levels of control are illustrated herein and also described in commonly owned pending PCT application PCT / US2016 / 039434, the entire contents of which are incorporated herein by reference.

[0164] In some embodiments, the modification in the phage genome is a combination of two or more of an insertion, deletion, substitution, or inversion in one or more phage genome genes.

[0165] In any of the embodiments described herein, the modification may result in one or more frameshift mutations in one or more genes in the phage genome. As used herein, a frameshift mutation (also called a framing error or reading frameshift) refers to a genetic mutation caused by the insertion or deletion (insertion or deletion) of a number of nucleotides in a DNA sequence that is not divisible by three. The earlier the deletion or insertion occurs in the sequence, the more altered the protein will be. In any of the embodiments described herein, the modification may result in one or more missense mutations in one or more genes in the phage genome. As used herein, a missense mutation refers to a case where a single base pair change causes the substitution of a different amino acid in the resulting protein. This amino acid substitution may have no effect or may render the protein nonfunctional. In any of the embodiments described herein, the modification may result in one or more nonsense mutations in one or more genes in the phage genome. As used herein, a nonsense mutation refers to a mutation in which a sense codon corresponding to one of the 20 amino acids specified by the genetic code is changed to a chain-terminating codon, thereby truncating the polypeptide of interest.

[0166] In some embodiments, the modification in the phage genome is a combination of two or more frameshift, nonsense, or missense mutations in one or more phage genome genes. In some embodiments, the modified bacteriophage is located on a bacterial chromosome. In some embodiments, the modified bacteriophage is located on a bacterial plasmid. In some embodiments, the plasmid is modified. In some embodiments, the plasmid is completely eliminated. In some embodiments, the phage or prophage is present in all isolates of a particular species. In some embodiments, the prophage is present in all isolates of a particular phylum, order, suborder, family, class, subclass genus, species, subspecies, or clade.

[0167] (mutation) In some embodiments, the one or more mutations comprise at least about 1-500 base pairs of the phage genome. In some embodiments, the one or more mutations comprise at least about 500-1000 base pairs of the phage genome. ... The natural mutations comprise a phage genome of at least about 1,000-2,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 1,000-2,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 2,000-3,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 3,000-4,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 4,000-5,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 5,000-6,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 6,000-7,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 7,000-8,000 base pairs. In some embodiments, the one or more mutations comprise a phage genome of at least about 8,000-9,000 base pairs. In some embodiments, the one or more mutations comprise at least about 9,000-10,000 base pairs of the phage genome. In some embodiments, the one or more mutations comprise at least about 10,000-15,000 base pairs of the phage genome.In some embodiments, the one or more mutations are present in a phage genome of at least about 10,000-15,000 base pairs, a phage genome of at least about 15,000-20,000 base pairs, a phage genome of at least about 20,000-25,000 base pairs, a phage genome of at least about 25,000-30,000 base pairs, a phage genome of at least about 30,000-35,000 base pairs, a phage genome of at least about 35,000-40,000 base pairs, a phage genome of at least about 45,000-50,000 base pairs, a phage genome of at least about 50,000-55,000 base pairs, a phage genome of at least about 55,000-60,000 base pairs, a phage genome of at least about 60,000-65,000 base pairs, a phage genome of at least about 65,000-70,000 base pairs, a phage genome of at least about 70,000 base pairs, a phage genome of at least about 80,000 base pairs, a phage genome of at least about 85 ... The phage genome may be at least about 75,000 base pairs, at least about 75,000-80,000 base pairs, at least about 85,000-90,000 base pairs, at least about 90,000-95,000 base pairs, at least about 95,000-100,000 base pairs, at least about 100,000-110,000 base pairs, at least about 110,000-120,000 base pairs, at least about 120,000-130,000 base pairs, at least about 130,000-140,000 base pairs, at least about 140,000-150,000 base pairs, at least about 150,000-200,000 base pairs, or at least more than about 200,000 base pairs. In one particular embodiment, 9687 base pairs of the phage genome are mutated. In some embodiments, the mutated nucleotides are interspersed. In some embodiments, the mutated nucleotides are contiguous.In some embodiments, at least about 0.1-1%, at least about 1-2%, at least about 2-3%, at least about 3-4%, at least about 4-5%, at least about 5-6%, at least about 6-7%, at least about 7-8%, at least about 8-9%, at least about 9-10%, at least about 10-11%, at least about 11-12%, at least about 12-13%, at least about 13-14%, at least about 14-15%, or at least about 15-20%, of the phage genome. In some embodiments, at least about 15-16%, at least about 16-17%, at least about 17-18%, at least about 18-19%, at least about 19-20%, at least about 20-21%, at least about 21-22%, at least about 22-23%, at least about 23-24%, at least about 24-25%, at least about 25-26%, at least about 26-27%, at least about 27-28%, at least about 28-29%, or at least about 29-30% of the phage genomes are mutated. In some embodiments, at least about 30-40% of the phage genomes are mutated. In some embodiments, at least about 40-50% of the phage genomes are mutated. In some embodiments, at least about 50-60% of the phage genomes are mutated. In some embodiments, at least about 60-70% of the phage genomes are mutated. In some embodiments, at least about 70-80% of the phage genomes are mutated. In some embodiments, at least about 10% of the phage genomes are mutated. At least about 80-90% is mutated. In some embodiments, at least about 90-100% of the phage genome is mutated.

[0168] In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes are mutated. In some embodiments, at least about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 12 In some embodiments, 4, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 genes are mutated. In some embodiments, 13 genes are fully or partially mutated. In one embodiment, 74 genes are fully or partially mutated.

[0169] In some embodiments, at least about 1% to 2%, at least about 2% to 3%, at least about 3% to 4%, at least about 4% to 5%, at least about 5% to 6%, at least about 6% to 7%, at least about 7% to 8%, at least about 8% to 9%, at least about 9% to 10%, at least about 10% to 11%, at least about 11% to 12%, at least about 12% to 13%, at least about 13% to 14%, at least about 14% to 15%, at least about 15% to 16%, at least about 16% to 17%, at least about 17% to 18%, at least about 18% to 20%, at least about 20% to 22%, at least about 21% to 23%, at least about 22% to 24%, at least about 23% to 25%, at least about 24% to 26%, at least about 25% to 26%, at least about 26% to 27%, at least about 27% to 28%, at least about 28% to 30%, at least about 30% to 31%, at least about 31% to 32%, at least about 32% to 33%, at least about 33% to 34%, at least about 34% to 35%, at least about 35% to 36%, at least about 36% to 37%, at least about 37% to 38%, at least about 38% to 40%, at least about 40% to 41%, at least about 41% to 42%, at least about 42% to 43%, at least about 43% to 44%, at least about 44% to 45%, at least about 45% to 46%, at least about 45% to 47%, at least about 48% to at least about 18%-19%, at least about 19%-20%, at least about 20%-21%, at least about 21%-22%, at least about 22%-23%, at least about 23%-24%, at least about 24%-25%, at least about 25%-26%, at least about 26% to about 27%, at least about 27%-28%, at least about 28%-29%, at least about 29%-30%, at least about 30%-31%, at least about 31%-32%, at least about 32%-33%, at least about 33%-34%, at least about 34%-35%, at least about 35% ~36%, at least about 36%-37%, at least about 37%-38%, at least about 38%-39%, at least about 39%-40%, at least about 40%-41%, at least about 41%-42%, at least about 42%-43%, at least about 43%-44%, at least about 44%-45%, at least about 45%-46%, at least about 46%-47%, at least about 47%-48%, at least about 48%-49%, at least about 49%-50%, at least about 50%-51%, at least about 51%-52%, at least about 52%-53%, at least Also about 53% to 54%, at least about 54% to 55%, at least about 55% to 56%, at least about 56% to 57%, at least about 57% to 58%, at least about 58% to 59%, at least about 59% to 60%, at least about 60% to 61%, at least about 61% to 62%, at least about 62% to 63%, at least about 63% to 64%, at least about 64% to 65%, at least about 65% to 66%, at least about 66% to 67%, at least about 67% to 68%, at least about 68% to 69%, at least about 69% to 70%, at least about 70% to 71%,at least about 71% to 72%, at least about 72% to 73%, at least about 73% to 74%, at least about 74% to 75%, at least about 75% to 76%, at least about 76% to 77%, at least about 77% to 78%, at least about 78% to 79%, at least about 79% to 80%, at least about 80% to 81%, at least about 81% to 82%, at least about 82% to 83%, at least about 83% to 84%, at least about 84% to 85%, at least about 85% to 86%, at least about 86% to 87%, at least about 87% to 88%, at least about 88% to 89%, at least about 89% to 90%, at least about 90% to 91%, at least about 91% to 92%, at least about 92% to 93%, at least about 93% to 94%, at least about 94, % to 95%, at least about 95% to 96%, at least about 96% to 97%, at least about 97% to 98%, at least about 98% to 99%, at least about 99% to 100%, or at least about 100% are fully or partially mutated.

[0170] In some embodiments, the one or more mutations are located at the beginning or 5' end of the phage genome. In some embodiments, the one or more mutations are located at the end or 3' end of the phage genome. In some embodiments, the one or more mutations are located in the middle of the phage genome. In some embodiments, the phage genes are dispersed within the bacterial genome and the mutations are located at one or more of the dispersed positions.

[0171] In some embodiments, the optimal region for mutation, i.e., the region for achieving the desired effect, can be determined by analyzing homology with other phages in other bacteria. Homologous conserved regions in phages may be suitable for mutation because they are conserved and may contain one or more essential genes. In some embodiments, regulatory elements such as promoters are mutated. In some embodiments, coding sequences are mutated. In some embodiments, one or more mutation regions include one or more genes essential for the lytic cycle.

[0172] In some embodiments, the mutations are located within or encompass one or more genes encoding lytic genes. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more proteases or lysins. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more toxins. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more antibiotic resistance-associated proteins. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more phage translation-associated proteins. In some embodiments, the one or more mutations are located within or encompass one or more genes encoding structural proteins. Such structural genes include genes encoding head, tail, collar, or coat polypeptides. In some embodiments, the one or more mutations are located within or encompass one or more genes encoding head structure polypeptides. In some embodiments, the one or more mutations are located within or encompass one or more genes encoding tail structure polypeptides. In some embodiments, the one or more mutations are located within or encompass one or more genes encoding collar structure polypeptides. In some embodiments, the one or more mutations are located within or encompass one or more genes encoding tail proteins. In some embodiments, the one or more mutations are located within or encompass one or more genes encoding coat structure polypeptides. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more platform proteins. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more proteins required for bacteriophage assembly. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more portal proteins.In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in recombination. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more integrases. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more invertases. In some embodiments, the mutation is located within or encompasses one or more transposases. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more primases. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more tRNA-associated proteins. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more polypeptides involved in phage insertion. In some embodiments, the mutations are located within or encompass one or more genes encoding attachment sites. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more terminases. In some embodiments, the mutations are located within or encompass one or more genes encoding one or more host genes.

[0173] In some embodiments, the mutations are located within or encompass genes encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, or host proteins, and combinations thereof.

[0174] In some embodiments, the mutations are located within or encompass genes encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.

[0175] In some embodiments, the mutations are located within or encompass one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass three genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass four genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass five genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass six genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass six genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass seven genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass nine genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass ten genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass eleven genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass 12 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass 13 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass 14 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.In some embodiments, the mutations are located within or encompass 15 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutations are located within or encompass at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 2 In some embodiments, the mutations are located within or encompass one or more of the following genes: 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more genes. In some embodiments, the mutations are located within or encompass one or more host proteins within the phage genome.

[0176] (deletion) In some embodiments, the one or more deletions comprise at least about 1-500 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 500-1000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 1000-2000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 1000-2000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 2000-3000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 3000-4000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 4000-5000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 5000-6000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 6,000-7,000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 7,000-8,000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 8,000-9,000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 9,000-10,000 base pairs of the phage genome. In some embodiments, the one or more deletions comprise at least about 10,000-15,000 base pairs of the phage genome.In some embodiments, the one or more deletions are in a phage genome of at least about 10,000-15,000 base pairs, a phage genome of at least about 15,000-20,000 base pairs, a phage genome of at least about 20,000-25,000 base pairs, a phage genome of at least about 25,000-30,000 base pairs, a phage genome of at least about 30,000-35,000 base pairs, a phage genome of at least about 35,000-40,000 base pairs. a phage genome of at least about 40,000 to 45,000 base pairs, a phage genome of at least about 45,000 to 50,000 base pairs, a phage genome of at least about 50,000 to 55,000 base pairs, a phage genome of at least about 55,000 to 60,000 base pairs, a phage genome of at least about 60,000 to 65,000 base pairs, a phage genome of at least about 65,000 to 70,000 base pairs, a phage genome of at least about 70 000 to 75,000 base pairs, a phage genome of at least about 75,000 to 80,000 base pairs, a phage genome of at least about 80,000 to 85,000 base pairs, a phage genome of at least about 85,000 to 90,000 base pairs, a phage genome of at least about 90,000 to 95,000 base pairs, a phage genome of at least about 95,000 to 100,000 base pairs, a phage genome of at least about 100,000 to 110,000 base pairs The phage genome may comprise a phage genome of at least about 110,000-120,000 base pairs, a phage genome of at least about 120,000-130,000 base pairs, a phage genome of at least about 130,000-140,000 base pairs, a phage genome of at least about 140,000-150,000 base pairs, a phage genome of at least about 150,000-200,000 base pairs, or a phage genome of more than 200,000 base pairs. In one particular embodiment, 9687 base pairs of the phage genome are deleted. In some embodiments, the deleted nucleotides are interspersed. In some embodiments, the deleted nucleotides are contiguous.

[0177] In some embodiments, at least about 0.1-1%, at least about 1-2%, at least about 2-3%, at least about 3-4%, at least about 4-5%, at least about 5-6%, at least about 6-7%, at least about 7-8%, at least about 8-9%, at least about 9-10%, at least about 10-11%, at least about 11-12%, at least about 12-13%, at least about 13-14%, at least about 14-15%, or at least about 15-20%, of the phage genome. In some embodiments, at least about 15-16%, at least about 16-17%, at least about 17-18%, at least about 18-19%, at least about 19-20%, at least about 20-21%, at least about 21-22%, at least about 22-23%, at least about 23-24%, at least about 24-25%, at least about 25-26%, at least about 26-27%, at least about 27-28%, at least about 28-29%, or at least about 29-30% of the phage genome is deleted. In some embodiments, at least about 30-40% of the phage genome is deleted. In some embodiments, at least about 40-50% of the phage genome is deleted. In some embodiments, at least about 50-60% of the phage genome is deleted. In some embodiments, at least about 60-70% of the phage genome is deleted. In some embodiments, at least about 70-80% of the phage genome is deleted. In some embodiments, at least about 80-90% of the phage genome is deleted, hi some embodiments, at least about 90-100% of the phage genome is deleted.

[0178] In some embodiments, one or more genes are partially or completely deleted in the phage genome. In some embodiments, one or more genes are completely deleted and one or more In one embodiment, there is a deletion in the phage genome, with one or two genes at either end of the deletion being partially deleted, and the remaining genes being completely deleted. In some embodiments, the deleted genes are adjacent to each other. In some embodiments, the deleted genes are not adjacent to each other.

[0179] In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes are deleted. In some embodiments, at least about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 genes are deleted. In some embodiments, 13 genes are completely or partially deleted. In one embodiment, 74 genes are completely or partially deleted. In some embodiments, at least about 1% to 2%, at least about 2% to 3%, at least about 3% to 4%, at least about 4% to 5%, at least about 5% to 6%, at least about 6% to 7%, at least about 7% to 8%, at least about 8% to 9%, at least about 9% to 10%, at least about 10% to 11%, at least about 11% to 12%, at least about 12% to 13%, at least about 13% to 14%, at least about 14% to 15%, at least about 15% to 16%, at least about 16% to 17%, at least about 17% to 18%, at least about 18% to 20%, at least about 20% to 21%, at least about 21% to 22%, at least about 22% to 23%, at least about 23% to 24%, at least about 24% to 25%, at least about 25% to 26%, at least about 26% to 27%, at least about 27% to 28%, at least about 28% to 30%, at least about 30% to 31%, at least about 31% to 32%, at least about 32% to 33%, at least about 33% to 34%, at least about 34% to 35%, at least about 35% to 36%, at least about 36% to 37%, at least about 37% to 38%, at least about 38% to 40%, at least about 40% to 41%, at least about 41% to 42%, at least about 42% to 43%, at least about 43% to 44%, at least about 44% to 45%, at least about 45% to 46%, at least about 47% to 48%, at least about 48% to 18%, at least about 18% to 19%, at least about 19% to 20%, at least about 20% to 21%, at least about 21% to 22%, at least about 22% to 23%, at least about 23% to 24%, at least about 24% to 25%, at least about 25% to 26%, at least about 26% to 27%, at least about 27% to 28%, at least about 28% to 29%, at least about 29% to 30%, at least about 30% to 31%, at least about 31% to 32%, at least about 32% to 33%, at least about 33% to 34%, at least about 34% to 35%,at least about 35%-36%, at least about 36%-37%, at least about 37%-38%, at least about 38%-39%, at least about 39%-40%, at least about 40%-41%, at least about 41%-42%, at least about 42%-43%, at least about 43%-44%, at least about 44%-45%, at least about 45%-46%, at least about 46%-47%, at least about 47%-48%, at least about 48%-49%, at least about 49%-50%, at least about 50%-51%, at least about 51%-52%, at least about 52%-53%, at least about 53%-54%, at least about 54%-55%, at least about 55%-56%, at least about 56%-57%, at least about 57%-58%, at least about 58%-59%, at least about 59%-60%, at least about 60%-61%, at least about 61%-62%, at least about 62%-63%, at least about 63%-64% %, at least about 64% to 65%, at least about 65% to 66%, at least about 66% to 67%, at least about 67% to 68%, at least about 68% to 69%, at least about 69% to 70%, at least about 70% to 71%, at least about 71% to 72%, at least about 72% to 73%, at least about 73% to 74%, at least about 74% to 75%, at least about 75% to 76%, at least about 76% to 77%, at least about 77% to 78%, at least Also about 78% to 79%, at least about 79% to 80%, at least about 80% to 81%, at least about 81% to 82%, at least about 82% to 83%, at least about 83% to 84%, at least about 84% to 85%, at least about 85% to 86%, at least about 86% to 87%, at least about 87% to 88%, at least about 88% to 89%, at least about 89% to 90%, at least about 90% to 91%, at least about 91% to 92%, at least about 92%, Up to 93%, at least about 93% to 94%, at least about 94% to 95%, at least about 95% to 96%, at least about 96% to 97%, at least about 97% to 98%, at least about 98% to 99%, at least about 99% to 100%, or at least about 100% are completely or partially deleted.

[0180] In some embodiments, the one or more deletions are located at the beginning or 5' end of the phage genome. In some embodiments, the one or more deletions are located at the end or 3' end of the phage genome. In some embodiments, the one or more deletions are located in the middle of the phage genome. In some embodiments, the phage genes are dispersed within the bacterial genome and the deletions are located at one or more of the dispersed positions.

[0181] In some embodiments, the optimal region for deletion, i.e., the region to achieve the desired effect, can be determined by analyzing homology with other phages in other bacteria. Homologous conserved regions in phages are conserved and may contain one or more essential genes, and therefore may be suitable for deletion. In some embodiments, regulatory elements such as promoters are deleted. In some embodiments, coding sequences are deleted. In some embodiments, one or more deleted regions include one or more genes essential for the lytic cycle.

[0182] In some embodiments, the deletions are located within or encompass one or more genes encoding lytic genes. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more proteases or lysins. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more toxins. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more antibiotic resistance-associated proteins. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more phage translation-associated proteins. In some embodiments, the one or more deletions are located within or encompass one or more genes encoding structural proteins. Such structural genes include genes encoding head, tail, collar, or coat polypeptides. In some embodiments, the one or more deletions are located within or encompass one or more genes encoding head structural polypeptides. In some embodiments, the one or more deletions are located within or encompass one or more genes encoding tail structural polypeptides. In some embodiments, the one or more deletions are located within or encompass one or more genes encoding collar structure polypeptides. In some embodiments, the one or more deletions are located within or encompass one or more genes encoding coat structure polypeptides. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more platform proteins. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more proteins required for bacteriophage assembly. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more portal proteins. In some embodiments, the deletions are located within or encompass one or more genes encoding one or more polypeptides involved in recombination.In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more integrases. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more invertases. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more transposases. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more primases. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more tRNA-associated proteins. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more polypeptides involved in phage insertion. In some embodiments, the deletion is located within or encompasses one or more genes encoding attachment sites. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more terminases. In some embodiments, the deletion is located within or encompasses one or more genes encoding one or more host genes.

[0183] In some embodiments, the deletions are located within or encompass genes encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, or host proteins, and combinations thereof.

[0184] In some embodiments, the deletions are located within or encompass genes encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.

[0185] In some embodiments, the deletions are located within or encompass one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass three genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass four genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass five genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass six genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass seven genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.In some embodiments, the deletions are located within or encompass eight genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. The deletions are located within or encompass 9 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass 10 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass 11 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass 12 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass 13 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the deletions are located within or encompass 14 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof, hi some embodiments, the deletions are located within or encompass 15 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.In some embodiments, the deletions are at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more genes. In some embodiments, the deletion is located within or encompasses one or more host proteins within the phage genome.

[0186] (insert) In some embodiments, the insertion is in a coding region of the phage genome. In some embodiments, the insertion is inserted into a regulatory region of the phage genome. In some embodiments, the insertion comprises one or more antibiotic cassettes. Suitable antibiotic cassettes are known in the art, and non-limiting examples of such antibiotic cassettes are described herein. In some embodiments, the antibiotic is chloramphenicol. In some embodiments, the antibiotic is kanamycin. In some embodiments, the antibiotic is ampicillin. In some embodiments, the antibiotic is chloramphenicol and kanamycin. In some embodiments, the one or more insertions comprise a length of at least about 1-500 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 500-1000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 1000-2000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 1000-2000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 2000-3000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 3000-4000 base pairs, hi some embodiments, the one or more insertions comprise a length of at least about 4000-5000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 5,000-6,000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 6,000-7,000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 7,000-8,000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 8,000-9,000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 9,000-10,000 base pairs. In some embodiments, the one or more insertions comprise a length of at least about 10,000-15,000 base pairs. In some embodiments, the one or more insertions are at least about 10,000-15,000 base pairs in length, at least about 15,000-20,000 base pairs in length, at least about 20,000-25,000 base pairs in length, at least about 25,000-30,000 base pairs in length, at least about 30,000-35,000 base pairs in length, at least about 35,000-40,000 base pairs in length, at least about 40,000-45,000 base pairs in length, at least about 45,000-50,000 base pairs in length, at least about 50,000-55,000 base pairs in length, at least about 55,000-60,000 base pairs in length, at least about 60,000-65,000 base pairs in length, at least about 65,000-70,000 base pairs in length, at least about 70,000-80,000 base pairs in length, at least about 80,000-90,000 base pairs in length, at least about 90,000-100,000 base pairs in length, at least about 100,000-110,000 base pairs in length, at least about 110,000-120,000 base pairs in length, at least about 120,000-130,000 base pairs in length, at least about 130,000-140,000 base pairs in length, at least about 140,000-150,000 base pairs in length, at least about 150,000-160,000 base pairs in length, at least about 160,000-170,000 base pairs in length, at The length of the inserted fragment may be between 00 and 75,000 base pairs, at least about 75,000 and 80,000 base pairs, at least about 80,000 and 85,000 base pairs, at least about 85,000 and 90,000 base pairs, at least about 90,000 and 95,000 base pairs, at least about 95,000 and 100,000 base pairs, at least about 100,000 and 110,000 base pairs, at least about 110,000 and 120,000 base pairs, at least about 120,000 and 130,000 base pairs, at least about 130,000 and 140,000 base pairs, at least about 140,000 and 150,000 base pairs, at least about 150,000 and 200,000 base pairs, or at least more than 200,000 base pairs. In one particular embodiment, a length of 9687 base pairs is inserted. In some embodiments, the inserted nucleotides are interspersed.In some embodiments, the inserted nucleotides are contiguous.

[0187] In some embodiments, the one or more insertions are located within 1-500 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 500-1000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 1000-2000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 1000-2000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 2000-3000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 3000-4000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 4000-5000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 5000-6000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 6000-7000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 7,000-8,000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 8,000-9,000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 9,000-10,000 base pairs of the phage genome. In some embodiments, the one or more insertions are located within at least about 10,000-15,000 base pairs of the phage genome. In some embodiments, the one or more insertions are within at least about 10,000-15,000 base pairs of the phage genome, within at least about 15,000-20,000 base pairs of the phage genome, within at least about 20,000-25,000 base pairs of the phage genome, within at least about 25,000-30,000 base pairs of the phage genome, within at least about 30,000-35,000 base pairs of the phage genome, within at least about 35,000-40,000 base pairs of the phage genome, within at least about 40,000-45,000 base pairs of the phage genome. 0 base pairs, within at least about 45,000 to 50,000 base pairs of the phage genome, within at least about 50,000 to 55,000 base pairs of the phage genome, within at least about 55,000 to 60,000 base pairs of the phage genome, within at least about 60,000 to 65,000 base pairs of the phage genome, within at least about 65,000 to 70,000 base pairs of the phage genome, within at least about 70,000 to 75,000 base pairs of the phage genome, within at least about 75,000 to 80,000 base pairs of the phage genome, within at least about 80,000 to 85,000 base pairs of the phage genome, within at least about 85,000 to 90,000 base pairs of the phage genome, phage genome The inserted nucleotides are located within at least about 90,000-95,000 base pairs of the phage genome, at least about 95,000-100,000 base pairs of the phage genome, at least about 100,000-110,000 base pairs of the phage genome, at least about 110,000-120,000 base pairs of the phage genome, at least about 120,000-130,000 base pairs of the phage genome, at least about 130,000-140,000 base pairs of the phage genome, at least about 140,000-150,000 base pairs of the phage genome, at least about 150,000-200,000 base pairs of the phage genome, or greater than at least about 200,000 base pairs of the phage genome. In one particular embodiment, 9,687 base pairs of the phage genome are inserted. In some embodiments, the inserted nucleotides are interspersed. In some embodiments, the inserted nucleotides are contiguous.

[0188] In some embodiments, the insertions comprise at least about 0.1-1%, at least about 1-2%, at least about 2-3%, at least about 3-4%, at least about 4-5%, at least about 5-6%, at least about 6-7%, at least about 7-8%, at least about 8-9%, at least about 9-10%, at least about 10-11%, at least about 11-12%, at least about 12-13%, at least about 13-14%, at least about 14-15% of the phage genome. , at least about 15-16%, at least about 16-17%, at least about 17-18%, at least about 18-19%, at least about 19-20%, at least about 20-21%, at least about 21-22%, at least about 22-23%, at least about 23-24%, at least about 24-25%, at least about 25-26%, at least about 26-27%, at least about 27-28%, at least about 28-29%, or at least about 29-30% of the phage genome. In some embodiments, at least about 30-40% of the phage genome is inserted. In some embodiments, the insertion is located within at least about 40-50% of the phage genome. In some embodiments, the insertion is located within at least about 50-60% of the phage genome. In some embodiments, the insertion is located within at least about 60-70% of the phage genome. In some embodiments, the insertion is located within at least about 70-80% of the phage genome. In some embodiments, the insertion is located within at least about 80-90% of the phage genome, hi some embodiments, the insertion is located within at least about 90-100% of the phage genome.

[0189] In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes contain insertions. In some embodiments, at least about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 genes contain insertions. In some embodiments, 13 genes contain insertions. In one embodiment, 74 genes contain insertions.

[0190] In some embodiments, the one or more insertions are located at the beginning or 5' end of the phage genome. In some embodiments, the one or more insertions are located at the end or 3' end of the phage genome. In some embodiments, the one or more insertions are located in the middle of the phage genome. In some embodiments, the phage genes are interspersed within the bacterial genome and the insertions are located at one or more of the interspersed positions.

[0191] In some embodiments, the optimal region for insertion, i.e., the region for achieving the desired effect, can be determined by analyzing homology with other phages in other bacteria. Homologous conserved regions in phages may be suitable for insertion because they are conserved and may contain one or more essential genes. In some embodiments, a regulatory element such as a promoter is inserted. In some embodiments, a coding sequence is inserted. In some embodiments, one or more insertion regions include one or more genes essential for the lytic cycle.

[0192] In some embodiments, the insertion is located within one or more genes encoding lytic genes. In some embodiments, the insertion is located within one or more genes encoding one or more proteases or lysins. In some embodiments, the insertion is located within one or more genes encoding one or more toxins. In some embodiments, the insertion is located within one or more genes encoding one or more antibiotic resistance-associated proteins. In some embodiments, the insertion is located within one or more genes encoding one or more phage translation-associated proteins. In some embodiments, the one or more insertions are located within one or more genes encoding structural proteins. Such structural genes include genes encoding head, tail, collar, or coat polypeptides. In some embodiments, the one or more insertions are located within one or more genes encoding head structure polypeptides. In some embodiments, the one or more insertions are located within one or more genes encoding tail structure polypeptides. In some embodiments, the one or more insertions are located within one or more genes encoding collar structure polypeptides. In some embodiments, the one or more insertions are located within one or more genes encoding coat structure polypeptides. In some embodiments, the insertion is located within one or more genes encoding one or more base proteins. In some embodiments, the insertion is located within one or more genes encoding one or more proteins required for bacteriophage assembly. In some embodiments, the insertion is located within one or more genes encoding one or more portal proteins. In some embodiments, the insertion is located within one or more genes encoding one or more polypeptides involved in recombination. In some embodiments, the insertion is located within one or more genes encoding one or more integrases. In some embodiments, the insertion is located within one or more genes encoding one or more invertases. In some embodiments, the insertion is located within one or more genes encoding one or more transposases.In some embodiments, the insertion is located within one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the insertion is located within one or more genes encoding one or more primases. In some embodiments, the insertion is located within one or more genes encoding one or more tRNA-associated proteins. In some embodiments, the insertion is located within one or more genes encoding one or more polypeptides involved in phage insertion. In some embodiments, the insertion is located within one or more genes encoding attachment sites. In some embodiments, the insertion is located within one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the insertion is located within one or more genes encoding one or more terminase. In some embodiments, the insertion is located within one or more genes encoding one or more host genes.

[0193] In some embodiments, the insertion is located within a gene encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, or host proteins, and combinations thereof.

[0194] In some embodiments, the insertion is located within a gene encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.

[0195] In some embodiments, the insertion is located within one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within three genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within four genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within five genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within six genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within seven genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within eight genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof.In some embodiments, the insertion is located within 9 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within 10 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within 11 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within 12 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within 13 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within 10 ... or within 14 genes encoding polypeptides involved in translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within 15 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the insertion is located within at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more genes. In some embodiments, the insertion is located within one or more host proteins within the phage genome.

[0196] (inversion) In some embodiments, the inversion is in a coding region of the phage genome. In some embodiments, the inversion is inverted in a regulatory region of the phage genome. In some embodiments, the inversion comprises one or more antibiotic cassettes. Suitable antibiotic cassettes are known in the art, and non-limiting examples of such antibiotic cassettes are described herein. In some embodiments, the antibiotic is chloramphenicol. In some embodiments, the antibiotic is kanamycin. In some embodiments, the antibiotic is ampicillin. In some embodiments, the antibiotic is chloramphenicol and kanamycin. In some embodiments, the one or more inversions comprise at least about 1-500 base pairs. In some embodiments, the one or more inversions comprise at least about 500-1000 base pairs. In some embodiments, the one or more inversions comprise at least about 1000-2000 base pairs. In some embodiments, the one or more inversions comprise at least about 1000-2000 base pairs. In some embodiments, the one or more inversions comprise at least about 2000-3000 base pairs. In some embodiments, one or more inversions comprise at least about 3,000-4,000 base pairs. In some embodiments, one or more inversions comprise at least about 4,000-5,000 base pairs. In some embodiments, one or more inversions comprise at least about 5,000-6,000 base pairs. In some embodiments, one or more inversions comprise at least about 6,000-7,000 base pairs. In some embodiments, one or more inversions comprise at least about 7,000-8,000 base pairs. In some embodiments, one or more inversions comprise at least about 8,000-9,000 base pairs. In some embodiments, one or more inversions comprise at least about 9,000-10,000 base pairs. In some embodiments, one or more inversions comprise at least about 10,000-15,000 base pairs.In some embodiments, the one or more inversions are at least about 10,000-15,000 base pairs, at least about 15,000-20,000 base pairs, at least about 20,000-25,000 base pairs, at least about 25,000-30,000 base pairs, at least about 30,000-35,000 base pairs, at least about 35,000-40,000 base pairs, at least about 40,000-45,000 base pairs, at least about 45,000-50,000 base pairs, at least about 50,000-55,000 base pairs, at least about 55,000-60,000 base pairs, or at least At least about 60,000 to 65,000 base pairs, at least about 65,000 to 70,000 base pairs, at least about 70,000 to 75,000 base pairs, at least about 75,000 to 80,000 base pairs, at least about 80,000 to 85,000 base pairs, at least about 85,000 to 90,000 base pairs, at least about 90,000 to 95,000 base pairs, at least about 95,000 to 100,000 base pairs, at least about 100,000 to 110,000 base pairs, at least about 110,000 to 120,000 base pairs, at least about 120,000 to 130,000 base pairs. , at least about 130,000-140,000 base pairs, at least about 140,000-150,000 base pairs, at least about 150,000-200,000 base pairs, or at least more than about 200,000 base pairs. In one particular embodiment, 9687 base pairs are inverted. In some embodiments, the inverted nucleotides are interspersed. In some embodiments, the inverted nucleotides are contiguous.

[0197] In some embodiments, one or more inversions are located within at least about 1-500 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 500-1000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 1000-2000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 1000-2000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 2000-3000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 3000-4000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 4000-5000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 5000-6000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 6,000-7,000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 7,000-8,000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 8,000-9,000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 9,000-10,000 base pairs of the phage genome. In some embodiments, one or more inversions are located within at least about 10,000-15,000 base pairs of the phage genome.In some embodiments, the one or more inversions are within at least about 10,000-15,000 base pairs of the phage genome, within at least about 15,000-20,000 base pairs of the phage genome, within at least about 20,000-25,000 base pairs of the phage genome, within at least about 25,000-30,000 base pairs of the phage genome, within at least about 30,000-35,000 base pairs of the phage genome, within at least about 35,000-40,000 base pairs of the phage genome. within at least about 40,000 to 45,000 base pairs of the phage genome, within at least about 45,000 to 50,000 base pairs of the phage genome, within at least about 50,000 to 55,000 base pairs of the phage genome, within at least about 55,000 to 60,000 base pairs of the phage genome, within at least about 60,000 to 65,000 base pairs of the phage genome, within at least about 65,000 to 70,000 base pairs of the phage genome Within about 70,000 to 75,000 base pairs, within at least about 75,000 to 80,000 base pairs of the phage genome, within at least about 80,000 to 85,000 base pairs of the phage genome, within at least about 85,000 to 90,000 base pairs of the phage genome, within at least about 90,000 to 95,000 base pairs of the phage genome, within at least about 95,000 to 100,000 base pairs of the phage genome, within at least about 100,000 to 110,000 base pairs of the phage genome The phage genome is located within at least about 110,000-120,000 base pairs, at least about 120,000-130,000 base pairs, at least about 130,000-140,000 base pairs, at least about 140,000-150,000 base pairs, at least about 150,000-200,000 base pairs, or at least more than about 200,000 base pairs. In a particular embodiment, 9687 base pairs of the phage genome are inverted. In some embodiments, the inverted nucleotides are interspersed. In some embodiments, the inverted nucleotides are contiguous.

[0198] In some embodiments, inversions occur in at least about 0.1-1%, at least about 1-2%, at least about 2-3%, at least about 3-4%, at least about 4-5%, at least about 5-6%, at least about 6-7%, at least about 7-8%, at least about 8-9%, at least about 10-12%, at least about 12-14%, at least about 14-15%, at least about 15-16%, at least about 16-17%, at least about 17-18%, at least about 18-20%, at least about 19-21%, at least about 22-23%, at least about 24-25%, at least about 25-26%, at least about 26-27%, at least about 27-28%, at least about 28-30%, at least about 29-31%, at least about 30-31%, at least about 31-32%, at least In some embodiments, the inversions are located within at least about 8-9%, at least about 9-10%, at least about 10-11%, at least about 11-12%, at least about 12-13%, at least about 13-14%, at least about 14-15%, at least about 15-16%, at least about 16-17%, at least about 17-18%, at least about 18-19%, at least about 19-20%, at least about 20-21%, at least about 21-22%, at least about 22-23%, at least about 23-24%, at least about 24-25%, at least about 25-26%, at least about 26-27%, at least about 27-28%, at least about 28-29%, or at least about 29-30% of the phage genome. In some embodiments, at least about 30-40% of the phage genome is inverted. In some embodiments, the inversions are located within at least about 40-50% of the phage genome. In some embodiments, the inversion is located within at least about 50-60% of the phage genomes. In some embodiments, the inversion is located within at least about 60-70% of the phage genomes. In some embodiments, the inversion is located within at least about 70-80% of the phage genomes. In some embodiments, the inversion is located within at least about 80-90% of the phage genomes. In some embodiments, the inversion is located within at least about 90-100% of the phage genomes.

[0199] In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes contain inversions. In some embodiments, at least about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 genes contain an inversion. In some embodiments, 13 genes contain an inversion. In one embodiment, 74 genes contain an inversion.

[0200] In some embodiments, the one or more inversions are located at the beginning or 5' end of the phage genome. In some embodiments, the one or more inversions are located at the end or 3' end of the phage genome. In some embodiments, the one or more inversions are located in the middle of the phage genome. In some embodiments, the phage genes are interspersed within the bacterial genome and the inversions are located at one or more of the interspersed positions.

[0201] In some embodiments, the optimal region for inversion, i.e., the region to achieve the desired effect, can be determined by analyzing homology with other phages in other bacteria. Homologous conserved regions in phages may be suitable for inversion because they are conserved and may contain one or more essential genes. In some embodiments, a regulatory element, such as a promoter, is inverted. In some embodiments, a coding sequence is inverted. In some embodiments, one or more inverted regions contains one or more genes essential for the lytic cycle.

[0202] In some embodiments, the inversion is located within one or more genes encoding lytic genes. In some embodiments, the inversion is located within one or more genes encoding one or more proteases or lysins. In some embodiments, the inversion is located within one or more genes encoding one or more toxins. In some embodiments, the inversion is located within one or more genes encoding one or more antibiotic resistance-associated proteins. In some embodiments, the inversion is located within one or more genes encoding one or more phage translation-associated proteins. In some embodiments, the one or more inversions are located within a construct The inversions are located within one or more genes encoding structural proteins. Such structural genes include genes encoding head, tail, collar, or coat polypeptides. In some embodiments, the inversions are located within one or more genes encoding one or more platform proteins. In some embodiments, the inversions are located within one or more genes encoding one or more proteins required for bacteriophage assembly. In some embodiments, the inversions are located within one or more genes encoding one or more portal proteins. In some embodiments, the inversions are located within one or more genes encoding one or more polypeptides involved in recombination. In some embodiments, the inversions are located within one or more genes encoding one or more integrases. In some embodiments, the inversions are located within one or more genes encoding one or more invertases. In some embodiments, the inversions are located within one or more genes encoding one or more transposases. In some embodiments, the inversions are located within one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the inversions are located within one or more genes encoding one or more primases. In some embodiments, the inversions are located within one or more genes encoding one or more tRNA-associated proteins. In some embodiments, the inversion is located within one or more genes encoding one or more polypeptides involved in phage inversion. In some embodiments, the inversion is located within one or more genes encoding attachment sites. In some embodiments, the inversion is located within one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the inversion is located within one or more genes encoding one or more terminases. In some embodiments, the inversion is located within one or more genes encoding one or more host genes.

[0203] In some embodiments, the inversion is located within a gene encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, or host proteins, and combinations thereof.

[0204] In some embodiments, the inversion is located within a gene encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof.

[0205] In some embodiments, the inversion is located within one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversion is located within two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversion is located within three genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversion is located within four genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversion is located within two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversion is directed to polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within five genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within six genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within seven genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within eight genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within nine genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within ten genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within 11 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within 12 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within 13 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof.In some embodiments, the inversions are located within 14 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within 15 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. In some embodiments, the inversions are located within at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage inversion, and combinations thereof. , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more genes. In some embodiments, the inversion is located within one or more host proteins in the phage genome.

[0206] (replacement) In some embodiments, the substitution is in a coding region of the phage genome. In some embodiments, the substitution is substituted in a regulatory region of the phage genome. In some embodiments, the substitution comprises one or more antibiotic cassettes. Suitable antibiotic cassettes are known in the art, and non-limiting examples of such antibiotic cassettes are described herein. In some embodiments, the antibiotic is chloramphenicol. In some embodiments, the antibiotic is kanamycin. In some embodiments, the antibiotic is ampicillin. In some embodiments, the antibiotic is chloramphenicol and kanamycin. In some embodiments, the one or more substitutions comprise at least about 1-500 base pairs. In some embodiments, the one or more substitutions comprise at least about 1-500 base pairs. In some embodiments, the one or more substitutions comprise at least about 500-1000 base pairs. In some embodiments, the one or more substitutions comprise at least about 1000-2000 base pairs. In some embodiments, the one or more substitutions comprise at least about 1000-2000 base pairs. In some embodiments, the one or more substitutions comprise at least about 2000-3000 base pairs. In some embodiments, the one or more substitutions comprise at least about 3000-4000 base pairs. In some embodiments, the one or more substitutions comprise at least about 4000-5000 base pairs. In some embodiments, the one or more substitutions comprise at least about 5000-6000 base pairs. In some embodiments, the one or more substitutions comprise at least about 6000-7000 base pairs. In some embodiments, the one or more substitutions comprise at least about 7000-8000 base pairs. In some embodiments, the one or more substitutions comprise at least about 8000-9000 base pairs. In some embodiments, the one or more substitutions comprise at least about 9,000-10,000 base pairs, hi some embodiments, the one or more substitutions comprise at least about 10,000-15,000 base pairs.In some embodiments, the one or more substitutions are at least about 10,000-15,000 base pairs, at least about 15,000-20,000 base pairs, at least about 20,000-25,000 base pairs, at least about 25,000-30,000 base pairs, at least about 30,000-35,000 base pairs, at least about 35,000-40,000 base pairs, at least about 40,000-45,000 base pairs, at least about 45,000-50,000 base pairs, at least about 50,000-55,000 base pairs, at least about 55,000-60,000 base pairs, at least about 60,000-65,000 base pairs, at least about 65,000-70,000 base pairs, at least about 70,000-80,000 base pairs, at least about 80,000-90,000 base pairs, at least about 90,000-100,000 base pairs, at least about 95 ... The nucleotide sequence may comprise at least about 0,000 to 75,000 base pairs, at least about 75,000 to 80,000 base pairs, at least about 80,000 to 85,000 base pairs, at least about 85,000 to 90,000 base pairs, at least about 90,000 to 95,000 base pairs, at least about 95,000 to 100,000 base pairs, at least about 100,000 to 110,000 base pairs, at least about 110,000 to 120,000 base pairs, at least about 120,000 to 130,000 base pairs, at least about 130,000 to 140,000 base pairs, at least about 140,000 to 150,000 base pairs, at least about 150,000 to 200,000 base pairs, or at least more than about 200,000 base pairs. In a specific embodiment, 9,687 base pairs are substituted. In some embodiments, the substituted nucleotides are interspersed. In some embodiments, the substituted nucleotides are contiguous.

[0207] In some embodiments, the one or more substitutions are located within at least about 1-500 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 500-1000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 1000-2000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 1000-2000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 2000-3000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 3000-4000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 4000-5000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 5000-6000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 6,000-7,000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 7,000-8,000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 8,000-9,000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 9,000-10,000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 10,000-15,000 base pairs of the phage genome. In some embodiments, the one or more substitutions are located within at least about 10,000-15,000 base pairs of the phage genome, at least about 15,000-20,000 base pairs of the phage genome, at least about 2,000 base pairs of the phage genome. within at least about 0 to 25,000 base pairs of the phage genome, within at least about 25,000 to 30,000 base pairs of the phage genome, within at least about 30,000 to 35,000 base pairs of the phage genome, within at least about 35,000 to 40,000 base pairs of the phage genome, within at least about 40,000 to 45,000 base pairs of the phage genome, within at least about 45,000 to 50,000 base pairs of the phage genome, within at least about 50,000 to 55,000 base pairs of the phage genome, within at least about 55,000 to 60,000 base pairs of the phage genome, within at least about 60,000 to 65,000 base pairs of the phage genome, within at least about 65,000 to 70,000 base pairs of the phage genome, within at least about 70,000 to 75,000 base pairs of the phage genome, within at least about 75,000 to 80,000 base pairs of the phage genome, The phage genome is located within at least about 80,000 to 85,000 base pairs, at least about 85,000 to 90,000 base pairs, the phage genome, at least about 90,000 to 95,000 base pairs of the phage genome, at least about 95,000 to 100,000 base pairs of the phage genome, at least about 100,000 to 110,000 base pairs of the phage genome, at least about 110,000 to 120,000 base pairs of the phage genome, at least about 120,000 to 130,000 base pairs of the phage genome, at least about 130,000 to 140,000 base pairs of the phage genome, at least about 140,000 to 150,000 base pairs of the phage genome, at least about 150,000 to 200,000 base pairs of the phage genome, or within more than at least about 200,000 base pairs of the phage genome. In one particular embodiment, 9687 base pairs of the phage genome are replaced. In some embodiments, the replaced nucleotides are interspersed. In some embodiments, the replaced nucleotides are contiguous.

[0208] In some embodiments, the substitutions are at least about 0.1-1%, at least about 1-2%, at least about 2-3%, at least about 3-4%, at least about 4-5%, at least about 5-6%, at least about 6-7%, at least about 7-8%, at least about 8-9%, at least about 9-10%, at least about 10-11%, at least about 11-12%, at least about 12-13%, at least about 13-14%, at least about 14-15% of the phage genome. , at least about 15-16%, at least about 16-17%, at least about 17-18%, at least about 18-19%, at least about 19-20%, at least about 20-21%, at least about 21-22%, at least about 22-23%, at least about 23-24%, at least about 24-25%, at least about 25-26%, at least about 26-27%, at least about 27-28%, at least about 28-29%, or at least about 29-30% of the phage genome. In some embodiments, at least about 30-40% of the phage genome is substituted. In some embodiments, substitutions are located within at least about 40-50% of the phage genome. In some embodiments, substitutions are located within at least about 50-60% of the phage genome. In some embodiments, substitutions are located within at least about 60-70% of the phage genome. In some embodiments, substitutions are located within at least about 70-80% of the phage genome. In some embodiments, the substitutions are located within at least about 80-90% of the phage genome, hi some embodiments, the substitutions are located within at least about 90-100% of the phage genome.

[0209] In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes contain substitutions. In some embodiments, at least about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 1 In some embodiments, 15, 116, 117, 118, 119, or 120 genes contain substitutions. In some embodiments, 13 genes contain substitutions. In one embodiment, 74 genes contain substitutions.

[0210] In some embodiments, the one or more substitutions are located at the beginning or 5' end of the phage genome. In some embodiments, the one or more substitutions are located at the end or 3' end of the phage genome. In some embodiments, the one or more substitutions are located in the middle of the phage genome. In some embodiments, the phage genes are dispersed within the bacterial genome and the substitutions are located at one or more of the dispersed positions.

[0211] In some embodiments, the optimal region for replacement, i.e., the region for achieving the desired effect, can be determined by analyzing homology with other phages in other bacteria. Homologous conserved regions in phages are conserved and may contain one or more essential genes, and therefore may be suitable for replacement. In some embodiments, a regulatory element, such as a promoter, is replaced. In some embodiments, a coding sequence is replaced. In some embodiments, one or more replacement regions include one or more genes essential for the lytic cycle.

[0212] In some embodiments, the substitution is located within one or more genes encoding lytic genes. In some embodiments, the substitution is located within one or more genes encoding one or more proteases or lysins. In some embodiments, the substitution is located within one or more genes encoding one or more toxins. In some embodiments, the substitution is located within one or more genes encoding one or more antibiotic resistance-associated proteins. In some embodiments, the substitution is located within one or more genes encoding one or more phage translation-associated proteins. In some embodiments, the substitution is located within one or more genes encoding structural proteins. Such structural genes include genes encoding head, tail, collar, or coat polypeptides. In some embodiments, the substitution is located within one or more genes encoding one or more platform proteins. In some embodiments, the substitution is located within one or more genes encoding one or more proteins required for bacteriophage assembly. In some embodiments, the substitution is located within one or more genes encoding one or more portal proteins. In some embodiments, the substitution is located within one or more genes encoding one or more polypeptides involved in recombination. In some embodiments, the substitution is located within one or more genes encoding one or more integrases. In some embodiments, the substitution is located in one or more genes encoding one or more invertases. In some embodiments, the substitution is located in one or more genes encoding one or more transposases. In some embodiments, the substitution is located in one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the substitution is located in one or more genes encoding one or more primases. In some embodiments, the substitution is located in one or more genes encoding one or more tRNA-associated proteins.In some embodiments, the substitution is located in one or more genes encoding one or more polypeptides involved in phage substitution. In some embodiments, the substitution is located in one or more genes encoding an attachment site. In some embodiments, the substitution is located in one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the substitution is located in one or more genes encoding one or more terminases. In some embodiments, the substitution is located in one or more genes encoding one or more host genes.

[0213] In some embodiments, the substitutions are mediated by a variety of mechanisms, including cell lysis, phage structure, phage assembly, Located within genes encoding one or more polypeptides involved in one or more of phage packaging recombination, replication or translation, phage substitution, or host proteins, and combinations thereof.

[0214] In some embodiments, the substitutions are located within genes encoding one or more polypeptides involved in one or more of cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage substitution, and combinations thereof.

[0215] In some embodiments, the substitutions are located within one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within three genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within four genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within two genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within five genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within six genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within seven genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within eight genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof.In some embodiments, the substitutions are located within 9 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within 10 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within 11 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within 12 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within 13 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions encode polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage displacement, and combinations thereof. In some embodiments, the substitutions are located within 14 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage replacement, and combinations thereof. In some embodiments, the substitutions are located within at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 2 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more genes. In some embodiments, the substitutions are located within one or more host proteins within the phage genome.

[0216] (Phage in E. coli Nissle) In some embodiments described herein, the genetically engineered bacterium is the engineered Escherichia coli strain Nissle 1917 (E. coli Nissle). As described in more detail in the Examples herein, routine testing procedures identified bacteriophage production from E. coli Nissle 1917 (E. coli Nissle; E. coli Nissle) and related engineered derivatives. To identify the source of the bacteriophage, a collaborative bioinformatics evaluation of the genomes of E. coli Nissle and engineered derivatives was conducted, analyzing the strain genome sequences for evidence of prophage, evaluating any identified prophage elements for the potential for functional phage production, comparing any functional phage elements to other known phages identified from bacterial genome sequences, and assessing the frequency with which prophage elements are found in other sequenced E. coli genomes. Evaluation tools included phage prediction software (PHAST and PHASTER), SPAdes genome assembler software, software for mapping low-divergence sequences against larger reference genomes (BWA-MEM), genome sequence alignment software (MUMmer), and the National Center for Biotechnology Information (NCBI) non-redundant database. Evaluation results indicate that the analyzed E. coli Nissle and engineered derivatives contained three candidate prophage elements (Figure 1), with two of the three (phage 2 and phage 3) containing most of the genetic features characteristic of intact phage genomes (Figures 2, 3, and 4). Two other possible phage elements (Figures 5A and 5B) were also identified. Notably, the engineered strains did not contain any additional phage elements not identified in the parent E. coli Nissle, indicating that the plaque-forming units produced by these strains originate from one of these endogenous phages. Further analysis, described herein, identified phage 3 as the plaque-forming phage (phage 3).Interestingly, phage 3 is unique to E. coli Nissle among a collection of nearly 6000 sequenced E. coli genomes, although related sequences limited to short regions of homology to other putative prophage elements are found in a small number of genomes. As described in more detail in the Examples, phage 3 was found to be inducible and cause bacterial lysis upon induction, whereas none of the other phages were inducible.

[0217] Prophages are very common among E. coli strains, and E. coli Nissle contains a relatively small number of prophage sequences compared to the average number found in the set of well-characterized, sequenced E. coli genomes. Therefore, the presence of prophages in the engineered strains is part of the natural state of this species, and the prophage characteristics of the analyzed engineered strains were consistent with those of the progenitor strain, E. coli Nissle.

[0218] Table D lists the genes contained within the genome of phage 3. Table E shows the sequence of phage 3. Table F shows the sequences of genes contained in E. coli Nissle phage 3. Table G shows the sequences of polypeptides encoded by the genome of E. coli Nissle phage 3.

[0219] [Table D] Phage 3 Genome [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0220] [Table E] Phage 3 genome sequence [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11] [Table 6-12]

[0221] [Table F] Gene sequences contained in E. coli Nissle phage 3 [Table 7-1] [Table 7-2]

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

[0222] [Table G] Polypeptide sequences encoded by the genome of E. coli Nissle phage 3 [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9]

[0223] In any of these embodiments, the bacteria described herein comprise one or more modifications or mutations in the E. coli Nissle phage 3 genome. In some embodiments, the modification alters the properties or behavior of phage 3. In some embodiments, the modification or mutation prevents phage 3 from entering or completing the lytic process. In some embodiments, the modification or mutation reduces the ability of phage 3 to enter the lytic process. In some embodiments, the modification or mutation prevents E. coli Nissle phage 3 from infecting other bacteria of the same or different type.

[0224] In some embodiments, the modification or mutation alters (e.g., increases or decreases) the fitness of the bacterial host. In some embodiments, the modification or mutation does not substantially affect bacterial fitness, such that the bacterial fitness is substantially the same as the fitness of an isogenic strain without the modification or mutation. In some embodiments, the modification or mutation alters (e.g., increases or decreases) a desired effector function, e.g., of the genetically engineered bacterium. In some embodiments, the modification or mutation improves a desired effector function, e.g., of the genetically engineered bacterium. In some embodiments, the modification or mutation does not substantially affect the effector function, such that the effector function is substantially the same as the effector function of an isogenic strain without the modification or mutation. In some embodiments, the effector circuit is first engineered into the genome, and then the phage is engineered. In some embodiments, a new chassis with the modified phage3 is generated prior to engineering the effector function(s).

[0225] In some embodiments, the bacteria are at least about 1-2, at least about 2-3, at least about 3-4, at least about 4-5, at least about 5-6, at least about 6-7, at least about 7-8, at least about 8-9, at least about 9-10, at least about 10-11, at least about 11-12, at least about 12-13, at least about 13-14, at least about 14-15, at least about 15-16, at least about 16-17, at least about 17-18, at least about 18-19, at least about 19-20, at least about 20-21, at least about 21-22, at least about 22 ~23, at least about 23-24, at least about 24-25, at least about 25-26, at least about 26-27, at least about 27-28, at least about 28-29, at least about 29-30, at least about 30-31, at least about 31-32, at least about 32-33, at least about 33-34, at least about 34-35, at least about 35-36, at least about 36-37, at least about 37-38, at least about 38-39, at least about 39-40, at least about 40-41, at least about 41-42, at least about 42-43, at least about 43-44, at least Also about 44-45, at least about 45-46, at least about 46-47, at least about 47-48, at least about 48-49, at least about 49-50, at least about 50-51, at least about 51-52, at least about 52-53, at least about 53-54, at least about 54-55, at least about 55-56, at least about 56-57, at least about 57-58, at least about 58-59, at least about 59-60, at least about 60-61, at least about 61-62, at least about 62-63, at least about 63-64, at least about 64-65, at least about 65-66, at least about 66-67, at least about 67-68, at least about 68-69, at least about 69-70, at least about 70-71, at least about 71-72, at least about 72-73, and at least about 73-74, at least about 74-75, at least about 75-76, at least about 76-77, at least about 77-78, at least about 78-79, at least about 79-80, at least about 80-81, at least about 81-82, at least about 82-83, at least about 83-84, at least about 84-85, at least about 85-86, at least about 86-87, at least about 87-88, at least about 88-89, at least about 89-90, at least about 90-91, at least about 91-92, at least about 92-93, at least about 93-94, at least about 94-95, at least about 95-96, at least about 96-97, at least about 97-98, at least about 98-99, at least about 99-100, or at least about 100 or more modifications or mutations.

[0226] In some embodiments, the modification or mutation reduces entry or completion of the phage 3 lytic process by at least about 1-2 fold, at least about 2-3 fold, at least about 3-4 fold, at least about 4-5 fold, at least about 5-10 fold, at least about 10-100 fold, or at least about 100-1000 fold. In some embodiments, the modification or mutation reduces entry or completion of the phage 3 lytic process completely.

[0227] In some embodiments, the modification or mutation reduces entry or completion of the phage 3 lytic process by at least about 1% to 10%, at least about 10% to 20%, at least about 20% to 30%, at least about 30% to 40%, at least about 40% to 50%, at least about 50% to 60%, at least about 60% to 70%, at least about 70% to 80%, at least about 80% to 90%, or at least about 90% to 100%.

[0228] In some embodiments, the modification or mutation prevents the E. coli Nissle phage 3 genome from infecting other bacteria of the same or different types by at least about 1-2 fold, at least about 2-3 fold, at le...

Claims

1. A bacterium comprising the Escherichia coli Nissle (ECOLIN) phage 3 genome, the phage comprises one or more mutations in one or more phage genes associated with lytic growth, horizontal gene transfer, cell lysis, phage structure, phage assembly, phage packaging, recombination, replication, translation, phage insertion, and combinations thereof; The mutation comprises a complete or partial deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175.

2. 2. The bacterium of claim 1 , wherein the one or more mutations reduce or prevent release of phage particles from the bacterium compared to the same bacterium not having the one or more targeted mutations in the phage genome.

3. 3. The bacterium of claim 1 or 2, which is a probiotic bacterium selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Escherichia coli strain Nissle, Lactobacillus, and Lactococcus.

4. 4. The bacterium of claim 3 which is Escherichia coli strain Nissle.

5. 5. The bacterium of claim 1, wherein the mutation comprises a complete deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, and ECOLIN_10170, and a partial deletion of ECOLIN_10175.

6. The bacterium according to any one of claims 2 to 5, wherein the deletion comprises SEQ ID NO:

130.

7. The bacterium according to any one of claims 2 to 5, wherein the deletion consists of SEQ ID NO:

130.

8. The bacterium according to any one of claims 1 to 7, comprising one or more additional genetic modifications.

9. The bacterium of claim 8 , wherein the one or more additional genetic modifications comprise one or more mutations in one or more endogenous genes.

10. The bacterium of claim 8 , wherein the one or more additional genetic modifications comprise the addition of one or more non-native genes.

11. The bacterium of any one of claims 1 to 10, further comprising antibiotic resistance.

12. A pharma- ceutically acceptable composition comprising the bacterium according to any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

13. The composition of claim 12 formulated for oral administration.

Images