Bacteria for treatment of disorders

Abstract

To provide modified probiotics, pharmaceutical compositions thereof, and methods of modulating and treating disorders.SOLUTION: The present invention provides a bacterium comprising one or more genes encoding a non-native phenylalanine metabolizing enzyme (PME) and further comprising one or more phage genomes, wherein one or more phage genes in the one or more phage genomes comprise one or more mutations.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 523,225, filed Jun. 21, 2017, U.S. Provisional Application No. 62 / 552,785, filed Aug. 31, 2017, U.S. Provisional Application No. 62 / 552,829, filed Aug. 31, 2017, U.S. Provisional Application No. 62 / 614,213, filed Jan. 5, 2018, U.S. Provisional Application No. 62 / 624,299, filed Jan. 31, 2018, and U.S. Provisional Application No. 62 / 523,202, filed Jun. 21, 2017, each of which is hereby incorporated by reference in its entirety, and the entire contents of each of these are expressly incorporated by reference herein in their entireties.

Background Art

[0002] There is increasing scientific evidence suggesting that probiotic bacteria are useful for the treatment or prevention of various diseases or disorders associated with the gastrointestinal tract, including gastrointestinal disorders such as Crohn's disease and inflammatory bowel syndrome. More recently, genetically engineered bacteria have emerged as a potential new treatment modality for gastrointestinal diseases and have also opened up the field of bacterial therapy to a number of other indications, including metabolic diseases, inflammatory diseases, and cancer. One advantage of genetically engineered bacteria is that they can specifically target one or more disease mechanisms. For example, in the case of gastrointestinal disorders, the bacteria can be engineered to contain genes for, e.g., anti-inflammatory agents or agents that help heal a disrupted gastrointestinal barrier, such as those described in International Publication No. WO 2016 / 141108, e.g., genes for the expression of the short-chain fatty acid butyrate. Genetically engineered It can also be regarded as treatment modalities for various metabolic disorders, including but not limited to these. For example, as described in International Publication No. 2016090343, bacteria express one or more enzymes that metabolize phenylalanine, thereby consuming excess phenylalanine in the gastrointestinal tract and are genetically modified to treat phenylketonuria (PKU).

[0003] Bacteriophages are the most common biological entities in the world, and it has been well demonstrated that the majority of both Gram-positive and Gram- negative bacterial species contain one or more DNA bacteriophages integrated as so-called prophages in the bacterial chromosome (Cloki e et al., Phages in Nature, Bacteriophage. January - February 2011; 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, and the integrated phage DNA (i.e., prophage) is replicated in coordination with the host genome, and phage genes that damage the host are not expressed. However, bacteriophage particles can be released from cells containing intact prophages by a process called induction, during which the prophage genes necessary for lytic growth are activated, progeny phage particles are produced, and released from the cells by cell lysis (Ca sjens, Prophages and bacterial genomics: sjens, Prophages and bacterial genomics: What have we learned so far?; Mol Microbi ol. July 2003; 49(2): pp. 277 - 300) (reviewed in). In some cases, induction can occur spontaneously and randomly in a small or large fraction of bacteria carrying prophages. In other cases, specific, often undefined environmental signals can trigger simultaneous induction of specific prophages in many cells,

[0005] causing the death of bacterial cells. Not all prophages have the ability to undergo the lytic cycle. Non-functional, i.e., defective or cryptic prophages, can reach high levels of abundance in many bacteria as a result of mutation-induced 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 Nat l Acad Sci USA. August 19, 2014; 111(33): 12127

[0006] In some embodiments, the present disclosure provides a bacterium comprising one or more phage genomes, wherein one or more of the phage genomes are defective. In some embodiments, the present disclosure provides a bacterium comprising one or In some embodiments, the present disclosure provides a bacterium comprising one or more phage genomes, wherein one or more of the phage genomes are defective in that one or more phage genes are not expressed. In some embodiments, the present 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 native 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 or cryptic phages or satellite phages. In some embodiments, the bacterium encodes one or more taiiocin or a gene delivery agent. In some embodiments of the present disclosure, one or more of the phage genomes are mutated. Such mutations can include one or more deletions of part or all of the sequence of one or more phage genes. Alternatively, the mutation can include one or more insertions of one or more nucleotides into one or more phage genes. In another embodiment, the mutation can include one or more substitutions of part or all of the sequence of one or more phage genes. In another embodiment, the mutation includes one or more inversions of part or all of the sequence of one or more phage genes in the phage genome. Further, the mutation can include any combination of one or more deletions, insertions, substitutions or inversions. In certain embodiments, In some embodiments, the present 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 native 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 or cryptic phages or satellite phages.

[0007] In some embodiments of the present disclosure, one or more of the phage genomes are mutated. Such mutations can include one or more deletions of part or all of the sequence of one or more phage genes. Alternatively, the mutation can include one or more insertions of one or more nucleotides into one or more phage genes. In another embodiment, the mutation can include one or more substitutions of part or all of the sequence of one or more phage genes. In another embodiment, the mutation includes one or more inversions of part or all of the sequence of one or more phage genes in the phage genome. In another embodiment, the mutation includes one or more inversions of part or all of the sequence of one or more phage genes in the phage genome. In another embodiment, the mutation includes one or more inversions of part or all of the sequence of one or more phage genes in the phage genome. Further, the mutation can include any combination of one or more deletions, insertions, substitutions or inversions. The one or more mutations reduce or prevent the production and release of phage particles from the bacterium as compared to the same bacterium that does not have 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 the Escherichia coli strain Nissle. In some embodiments, the mutated phage genome is the Escherichia coli Nissle phage 1 genome, the Escherichia coli Nissle phage 2 genome, and / or the Escherichia coli Nissle phage 3 genome. In one embodiment, the mutated phage genome is the Escherichia coli Nissle phage 3 genome. In one embodiment, the mutation is located within one or more genes selected from the following, or comprises one or more genes selected from the following: ECOLI_N09965, 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 above in the one or more phage genomes that do not have the one or more targeted mutations In some embodiments, the bacterium is a probiotic bacterium Non-limiting examples of such probiotic bacteria include the genus Bacteroides (B acteroides), the genus Bifidobacterium , the genus Clostridium, the genus Escherichia hia), the genus Lactobacillus, and the genus Lactococcus (Lactococcus). In some embodiments, the bacterium is Escherichia coli strain Nissle. In some embodiments the mutated phage genome is the Escherichia coli Nissle phage 1 genome, Escherichia coli Nissle phage 2 genome and / or Escherichia coli Nissle phage 3 genome is. In one embodiment, the mutated phage genome is the Escherichia coli Nissle pha ge 3 genome. In one embodiment, the mutation is one or more selected from the following is located within a gene, or comprises one or more genes selected from the following: ECOLI N_09965, ECOLIN_09970, ECOLIN_09975, ECOLIN _09980, ECOLIN_09985, ECOLIN_09990, ECOLIN_ 09995, ECOLIN_10000, ECOLIN_10005, ECOLIN_1 0010, ECOLIN_10015, ECOLIN_10020, ECOLIN_10 025, ECOLIN_10030, ECOLIN_10035, ECOLIN_100 40, ECOLIN_10045, ECOLIN_10050, ECOLIN_1005 5, ECOLIN_10065, ECOLIN_10070, ECOLIN_10075 , ECOLIN_10080, ECOLIN_10085, ECOLIN_10090, ECOLIN_10095, ECOLIN_10100, ECOLIN_10105, E COLIN_10110, ECOLIN_10115, ECOLIN_10120, EC OLIN_10125, ECOLIN_10130, ECOLIN_10135, ECO LIN_10140, ECOLIN_10145, ECOLIN_10150, ECOL IN_10160, ECOLIN_10165, ECOLIN_10170, ECOLI N_10175, ECOLIN_10180, ECOLIN_10185, ECOLIN _10190, ECOLIN_10195, ECOLIN_10200, ECOLIN_ 10205, ECOLIN_10210, ECOLIN_10220, ECOLIN_1 0225, ECOLIN_10230, ECOLIN_10235, ECOLIN_10 240, ECOLIN_10245, ECOLIN_10250, ECOLIN_102 55, ECOLIN_10260, ECOLIN_10265, ECOLIN_1027 0, ECOLIN_10275, ECOLIN_10280, ECOLIN_10290 , ECOLIN_10295, ECOLIN_10300, ECOLIN_10305, ECOLIN_10310, ECOLIN_10315, ECOLIN_10320, E COLIN_10325, ECOLIN_10330, ECOLIN_10335, EC OLIN_10340 and ECOLIN_10345. In one embodiment, the mutation s, e.g., one or more deletions, are located within one or more genes selected from the following, or include one or more genes selected from the following: ECOLIN_10110, ECOL IN_10115, ECOLIN_10120, ECOLIN_10125, ECOLI N_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN _10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_ 10165, ECOLIN_10170, and ECOLIN_10175. A pharmaceutically acceptable composition comprising the bacteria disclosed herein and a pharmaceutically acceptable carrier.

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

[0009] In some embodiments, the present disclosure relates to compositions and treatment methods for reducing hyperphenylalaninemia. In some embodiments, the composition comprises genetically engineered bacteria capable of expressing phenylalanine metabolism enzyme (PME). See, for example, WO2017087580A1, the entire contents of which are incorporated herein by reference. Phenylalanine is an essential amino acid mainly found in food proteins. Typically, a small amount is utilized for protein synthesis, and the remainder is hydroxylated to tyrosine in an enzymatic pathway that requires phenylalanine hydroxylase (PAH) and the cofactor tetrahydrobiopterin. Hyperphenylalaninemia is toxic and causes brain damage. ​​​​​​​​A group of diseases associated with excessive levels of phenylalanine that may occur. Original Primary hyperphenylalaninemia is caused by a deficiency of PAH activity resulting from mutations in the PAH gene and / or disruption of cofactor metabolism by interruption.

[0010] Phenylketonuria (PKU) is a severe form of hyperphenylalaninemia caused by mutations in the PAH gene. PKU is the most common inborn error of metabolism worldwide (1 in 3,000 births) and is an autosomal recessive genetic disease that affects approximately 13,000 patients in the United States. More than 400 different P AH gene mutations have been identified (Hoeks et al., 2009). Accumulation of phenylalanine (phe) in the blood can cause severe damage to the central nervous system in children and adults and is possible. If untreated in newborns, PKU can cause irreversible brain damage and is possible. Current treatment of PKU involves completely eliminating phenylalanine from the diet. Most natural sources of protein are essential amino acids and contain phenylalanine necessary for growth This means that patients with PKU rely on medical foods and phe-free protein supplements along with amino acid supplements that supply just enough phenylalanine for growth This dietary therapy is difficult for patients and affects the quality of life and gives. Current PKU therapy requires a significantly modified diet consisting of protein restriction. Generally, treatment from birth reduces brain damage and mental retardation (Hoeks et al., 2 009; Sarkissian et al., 1999). However, a protein-restricted diet is and gives.

[0011] Current PKU therapy requires a significantly modified diet consisting of protein restriction. Generally, treatment from birth reduces brain damage and mental retardation (Hoeks et al., 2 009; Sarkissian et al., 1999). However, a protein-restricted diet is It must be closely monitored, and essential amino acids and vitamins must be supplemented in the diet. Furthermore, access to low-protein foods is a challenge because it is more costly than high-protein, unmodified foods (Vockley et al., 2014). In children with PKU, growth retardation is common with continued adherence to a low-phenylalanine diet (Dobbelaere et al., 2003). In adults, new problems such as osteoporosis, maternal PKU, and vitamin deficiencies may occur (Hoeks et al., 2009). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). 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, for example, can be as high as $57,000 per patient. (Vockley et al., 2014). In children with PKU, growth retardation is common with continued adherence to a low-phenylalanine diet (Dobbelaere et al., 2003). In adults, new problems such as osteoporosis, maternal PKU, and vitamin deficiencies may occur (Hoeks et al., 2009). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Dobbelaere et al., 2003). In adults, new problems such as osteoporosis, maternal PKU, and vitamin deficiencies may occur (Hoeks et al., 2009). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Hoeks et al., 2009). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Macleod et al., 2010). (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. (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, for example, can be as high as $57,000 per patient. However, cofactor therapy is costly and is only suitable for mild forms of phenylketonuria. The annual cost of Kuvan, for example, can be as high as $57,000 per patient. (Vockley et al., 2014). In children with PKU, growth retardation is common with continued adherence to a low-phenylalanine diet (Dobbelaere et al., 2003). In adults, new problems such as osteoporosis, maternal PKU, and vitamin deficiencies may occur (Hoeks et al., 2009). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). (Hoeks et al., 2009). Excess levels of phenylalanine in the blood that can freely cross the blood-brain barrier can also cause neurological disorders, behavioral disorders (e.g., hypersensitivity, fatigue), and / or physical symptoms (e.g., spasms, skin rashes, musty body odor). International guidelines recommend lifelong dietary phenylalanine restriction, but this is considered difficult and unrealistic (Sarkissian et al., 1999), and "continuous efforts are required to overcome lifelong compliance with the low-phe diet, the greatest challenge in living with PKU" (Macleod et al., 2010). may occur. Additionally, the side effects of Kuvan may include gastritis and severe allergic reactions (e.g., wheezing, dizziness, nausea, skin flushing).

[0013] The enzyme phenylalanine ammonia-lyase (PAL) can metabolize phenylalanine into non-toxic levels of ammonia and trans-cinnamic acid. Unlike PAH, PAL does not require THB cofactor activity to metabolize phenylalanine. Research on oral enzyme therapy using PAL has been conducted, but "since PAL is not available in sufficient quantities at affordable costs, research has not continued even in humans and animals" (Sarkisian et al., 1999). The pegylated form of recombinant PAL (PEG-PAL) is also under development as an injectable treatment modality. However, most subjects administered PEG-PAL suffered from injection site reactions and / or developed antibodies against this therapeutic enzyme (Longo et al., 2014). Thus, there remains a large unmet need for effective, reliable, and / or long-term treatments for diseases associated with hyperphenylalaninemia, including PKU. There is an unmet need for treatments that can control the patient's blood Phe levels while allowing for the consumption of more natural proteins. There is a need.

[0014] In some embodiments, the present disclosure provides genetically engineered bacteria that can encode and express phenylalanine ammonia-lyase and / or phenylalanine hydroxylase and / or L-amino acid deaminase and can reduce hyperphenylalaninemia. The phenylalanine ammonia-lyase (PAL) enzyme metabolizes phenylalanine can be metabolized to non-toxic levels of ammonia and trans-cinnamic acid acid). Unlike PAH, 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, as well as trace amounts of ammonia and hydrogen peroxide. Phenylpyruvic acid (PPA) is widely used in the pharmaceutical industry, food industry, and chemical industry. PPA is a starting material intermediate for the synthesis of D-phenylalanine, which is a raw material in the production of many chiral drugs and food additives . Therefore, LAAD has been studied from the perspective of industrial PPA production (H ou et al., 2015, Appl Microbiol Biotechnol. 2015 Oct;99(20):8391-402; "Production of phe nylpyruvic acid from L-phenylalanine usi ng an L-amino acid deaminase from Proteu s mirabilis: comparison of enzymatic and whole-cell biotransformation approaches" s mirabilis: comparison of enzymatic and whole-cell biotransformation approaches" ). Phenylpyruvic acid cannot cross the blood-brain barrier (Steele, Fed Proc. Jun 1986;45(7):2060-4; "Blood-brain barrier transport of the alpha-keto acid analogs of amino acids.", and this conversion has been shown to be useful in controlling the neurological phenotype of PKU . .

[0015] In certain aspects, the disclosure relates to genetically engineered bacteria that can reduce 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 the 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 stably maintained in vivo and / or in vitro. In certain embodiments, the genetically engineered bacteria further comprise a phenylalanine transporter gene to increase the uptake of phenylalanine. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, as well as methods of modulating and treating disorders associated with hyperphenylalaninemia. The genetically engineered bacteria can also include one or more genetic sequences related to biosafety and / or biological containment, such as a kill switch, a gene guard system, and / or auxotrophy. In some embodiments, the engineered bacteria can include an antibiotic resistance gene. The expression of these genetic sequences can 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, different promoters, or a combination thereof.

[0016] The genetically engineered bacteria can also include one or more genetic sequences related to biosafety and / or biological containment, such as a kill switch, a gene guard system, and / or auxotrophy. In some embodiments, the engineered bacteria can include an antibiotic resistance gene. The expression of these genetic sequences can 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, different promoters, or a combination thereof. The expression of these genetic sequences can 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, different promoters, or a combination thereof. It may include the use of different copies of the same promoter to regulate the gene in question, and / or the use of different promoters used in combination to regulate the expression of different genes. By using different regulatory systems or promoter systems to control gene expression, flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to temporally control gene expression) is provided, and also the ability to "fine-tune" gene expression is provided, and any or all of these regulations can help optimize gene expression and / or bacterial growth. It may include the use of different promoters that are used in combination to regulate the expression of different genes. By using different regulatory systems or promoter systems to control gene expression, flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to temporally control gene expression) is provided, and also the ability to "fine-tune" gene expression is provided, and any or all of these regulations can help optimize gene expression and / or bacterial growth. It may include the use of different promoters that are used in combination to regulate the expression of different genes. By using different regulatory systems or promoter systems to control gene expression, flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to temporally control gene expression) is provided, and also the ability to "fine-tune" gene expression is provided, and any or all of these regulations can help optimize gene expression and / or bacterial growth. It may include the use of different promoters that are used in combination to regulate the expression of different genes. By using different regulatory systems or promoter systems to control gene expression, flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to temporally control gene expression) is provided, and also the ability to "fine-tune" gene expression is provided, and any or all of these regulations can help optimize gene expression and / or bacterial growth. It may include the use of different promoters that are used in combination to regulate the expression of different genes. By using different regulatory systems or promoter systems to control gene expression, flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to temporally control gene expression) is provided, and also the ability to "fine-tune" gene expression is provided, and any or all of these regulations can help optimize gene expression and / or bacterial growth. It may include the use of different promoters that are used in combination to regulate the expression of different genes. By using different regulatory systems or promoter systems to control gene expression, flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to temporally control gene expression) is provided, and also the ability to "fine-tune" gene expression is provided, and any or all of these regulations can help optimize gene expression and / or bacterial growth.

[0017] In some embodiments, the bacteria can express any one or more effector molecules in the presence of a disease or tissue-specific molecule or metabolite, inflammation or inflammatory response or immunosuppression, a molecule or metabolite associated with liver damage, metabolic disease, or any other metabolite that may or may not be present in the gastrointestinal tract or tumor microenvironment, under hypoxic conditions. 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 genetically engineered bacteria further include one or more of the following: (1) one or more auxotrophies known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more kill switch circuits such as any of those described herein or known in the art, (3) one or more antibiotic resistance circuits. In some embodiments, the bacteria can express any one or more effector molecules in the presence of a disease or tissue-specific molecule or metabolite, inflammation or inflammatory response or immunosuppression, a molecule or metabolite associated with liver damage, metabolic disease, or any other metabolite that may or may not be present in the gastrointestinal tract or tumor microenvironment, under hypoxic conditions. In some embodiments, the bacteria can express any one or more effector molecules in the presence of a disease or tissue-specific molecule or metabolite, inflammation or inflammatory response or immunosuppression, a molecule or metabolite associated with liver damage, metabolic disease, or any other metabolite that may or may not be present in the gastrointestinal tract or tumor microenvironment, under hypoxic conditions. In some embodiments, the bacteria can express any one or more effector molecules in the presence of a disease or tissue-specific molecule or metabolite, inflammation or inflammatory response or immunosuppression, a molecule or metabolite associated with liver damage, metabolic disease, or any other metabolite that may or may not be present in the gastrointestinal tract or tumor microenvironment, under hypoxic conditions. 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. 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. In some embodiments, the genetically engineered bacteria further include one or more of the following: (1) one or more auxotrophies known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more kill switch circuits such as any of those described herein or known in the art, (3) one or more antibiotic resistance circuits. In some embodiments, the genetically engineered bacteria further include one or more of the following: (1) one or more auxotrophies known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more kill switch circuits such as any of those described herein or known in the art, (3) one or more antibiotic resistance circuits. In some embodiments, the genetically engineered bacteria further include one or more of the following: (1) one or more auxotrophies known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more kill switch circuits such as any of those described herein or known in the art, (3) one or more antibiotic resistance circuits. In some embodiments, the genetically engineered bacteria further include one or more of the following: (1) one or more auxotrophies known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more kill switch circuits such as any of those described herein or known in the art, (3) one or more antibiotic resistance circuits. In some embodiments, the genetically engineered bacteria further include one or more of the following: (1) one or more auxotrophies known in the art and provided herein (e.g., thyA or dapB auxotrophy), (2) one or more kill switch circuits such as any of those described herein or known in the art, (3) one or more antibiotic resistance circuits. 4) any transporter described herein or known in the art for taking up biological molecules or substrates such as one or more transporters, (5) any secretion circuit described herein or otherwise known in the art, such as one or more secretion circuits, and (6) one or more combinations of such additional circuits. Compositions of bacteria are also provided, as well as methods for treating, preventing or managing one or more diseases or disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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[0023] Figure 1 shows 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 D5-phenylalanine, and SYN-PKU-2001 and euthanized 0.5 or 2 hours after administration. After euthanasia, tissue samples from different sections of the gastrointestinal tract were collected and analyzed to determine the concentrations of Phe and SYN-PKU-2001 in each section. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In one aspect, the present disclosure includes an endogenous phage, comprising one or more modifications to the phage sequence. In some embodiments, the modification changes a property of the prophage sequence. Such mutations may result in partial or complete deletion of one or more of the phage genes. one or more insertions of one or more nucleotides into one or more phage genes; One or more partial or complete replacements of one or more phage genes in the phage genome, one It contains one or more inversions of the above phage genes, or a combination thereof.

[0020] The present disclosure provides compositions comprising novel bacteria for the treatment of disorders, the bacteria being In nature, it contains one or more bacteriophages or prophages. In embodiments, the bacterium comprises one or more modifications to the genome of one or more phages. In some embodiments, one or more modifications render the phage or prophage inactive. In some embodiments, the bacteria are characterized by the expression or expression of one or more effector molecules. The cells are further genetically modified to contain one or more genes for the production of Methods for the production and use of these engineered bacteria in novel treatments for is provided.

[0021] In one embodiment, Escherichia coli Nissle serves as the starting point, parental strain, or is used as a "chassis". In one embodiment, the modified bacteriophage is a phage that is endogenous to Escherichia coli Nissle in that phage and exists in bacteria in its natural state.

[0022] In some embodiments, the genetically engineered bacterium contains one or more genes encoding one or more effectors (e.g., , PME). In some embodiments, the genetically engineered bacterium contains one or more genes encoding PAL. In some embodiments, the genetically engineered bacterium contains one or more genes encoding LAAD. In some embodiments, the genetically engineered bacterium contains one or more genes encoding PAL and one or more genes encoding LA AD. In some embodiments, the genetically engineered bacterium contains one or more genes encoding a transporter (e.g., PheP). In some embodiments, the genetically engineered bacterium contains one or more genes encoding a transporter (e.g., Phe P) and one or more genes encoding PAL. In some embodiments, the genetically engineered bacterium contains one or more genes encoding a transporter (e.g., Phe P) and one or more genes encoding LAAD. In some embodiments, the genetically engineered bacterium contains one or more genes encoding a transporter (e.g., Ph eP), one or more genes encoding LAAD, and one or more genes encoding PAL. In any of the foregoing embodiments, phe A genetically engineered bacterium for the consumption of nitrilalanine has one or more advantages over its native state. In some embodiments, the endogenous bacteriophage genome. In an embodiment, the bacteriophage is a bacteriophage genome comprising one or more genes Such mutations include deletions, insertions, substitutions and inversions. , located within one or more bacteriophage genes, or one or more bacteriophage genes Contains phage genes.

[0023] Bacteriophages are the most common biological entities in the world, and are gram-positive and gram-negative. The majority of both Mu-negative and Mu-negative bacterial species are integrated into the bacterial chromosome as so-called prophages. It has been well documented that the microbial communities of the bacteria include one or more DNA bacteriophages (Cl okie et al., Phages in Nature, Bacteriophage.201 1 Jan-Feb; 1(1):31-45). For example, two separate studies on E. coli strains The study found that 51 different functional phages were released from the 27 E. coli strains analyzed and tested. Of 107 E. coli strains tested, 83 shed at least one functional phage type. It has been shown that genomics: what have we learned so far?; Mol Microbiol. July 2003;49(2):277-300;O Sawa et al., Genotypic variations of Shiga toxin in-converting phages from enterohaemorrhage agic Escherichia coli O157:H7 isolates; J Med Microbiol (2001) 49: 565-574, and Schicklmaier et al., A comparative study on th e frequency of prophages among natural i solates of Salmonella and Escherichia co li with emphasis on generalized transduc ers. Antonie Van Leeuwenhoek (1998) 73: 49-54).

[0024] As shown in Fig. 12, almost all E. coli genomes contain intact prophages , and most of the published complete E. coli genomes contain more intact proph ages than EcN. Abbreviations: E. coli = Escherichia coli; EcN = Escherichia coli Nissle 1917; Re fseq = reference sequence.

[0025] Among Gram-positive bacteria, the genomes of Bacillus 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; N olling et al., Genome sequence and comparative analysis of the genome of Clostridium acetobutylicum. J Bacteriol (2001) 183: 4823-4838; Bolotin et al., Complete genome sequence of Lactococcus lactis subsp. lactis IL1403, analysis of the solvent-producing bacter ium Clostridium acetobutylicum. J Bacter iol (2001) 183: 4823-4838; Bolotin et al., The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. l actis IL1403. Genome Res (2001) 11: 731 -753).

[0026] DNA phages can be either lytic or temperate. Lytic phages infect bacterial cells and then program the synthesis of progeny phages, which are subsequently released from the lysed cells. In contrast, temperate DNA phages establish a stable relationship with the host bacterium. In this relationship, the integrated phage DNA (i.e., the prophage) is replicated in concert with the host genome, and phage genes that damage the host are not expressed. However, bacteriophage particles can be released from cells containing intact prophages by a process called induction, during which the prophage genes required for lytic growth are turned on, progeny phage particles are produced, and released from the cells by cell lysis (reviewed in Casjens, Prophages and bacterial genomics: what have we lea rned so far?; Mol Microbiol. July 2003;49( 2):277-300). Induction can, in some cases, be proph age-specific, or it can be caused by various stress factors such as DNA damage, nutrient deprivation, or changes in temperature or pH. When a temperate phage infects a bacterium, it inserts its DNA into the host genome, forming a prophage. The prophage can remain latent for many generations, replicating along with the host genome without causing any harm to the host. However, under certain conditions, the prophage can be induced to enter the lytic cycle, where it produces progeny phages that lyse the host cell and are released into the environment. The ability of temperate phages to switch between the lysogenic and lytic cycles allows them to adapt to changing environmental conditions and to spread within bacterial populations. This process is important in the evolution of bacteria and in the spread of antibiotic resistance genes, as temperate phages can carry antibiotic resistance genes and transfer them between bacteria during the process of induction and phage replication. induced, either prophage-specifically or by various stress factors such as DNA damage, nutrient deprivation, or changes in temperature or pH. When a temperate phage infects a bacterium, it inserts its DNA into the host genome, forming a prophage. The prophage can remain latent for many generations, replicating along with the host genome without causing any harm to the host. However, under certain conditions, the prophage can be induced to enter the lytic cycle, where it produces progeny phages that lyse the host cell and are released into the environment. The ability of temperate phages to switch between the lysogenic and lytic cycles allows them to adapt to changing environmental conditions and to spread within bacterial populations. This process is important in the evolution of bacteria and in the spread of antibiotic resistance genes, as temperate phages can carry antibiotic resistance genes and transfer them between bacteria during the process of induction and phage replication. induced, either prophage-specifically or by various stress factors such as DNA damage, nutrient deprivation, or changes in temperature or pH. When a temperate phage infects a bacterium, it inserts its DNA into the host genome, forming a prophage. The prophage can remain latent for many generations, replicating along with the host genome without causing any harm to the host. However, under certain conditions, the prophage can be induced to enter the lytic cycle, where it produces progeny phages that lyse the host cell and are released into the environment. The ability of temperate phages to switch between the lysogenic and lytic cycles allows them to adapt to changing environmental conditions and to spread within bacterial populations. This process is important in the evolution of bacteria and in the spread of antibiotic resistance genes, as temperate phages can carry antibiotic resistance genes and transfer them between bacteria during the process of induction and phage replication. released by cell lysis (Casjens, Prophages and bacterial genomics: what have we lea rned so far?; Mol Microbiol. July 2003;49( 2):277-300). Induction can, in some cases, be proph It can occur spontaneously and randomly in a small or large part of bacteria containing prophages, or specific, often undefined environmental signals can cause synchronous induction of many prophages in many cells, leading to the death of bacterial cells. In some cases, the presence of prophage sequences can also enable some bacteria to have characteristics that they do not have without phages (e.g., antibiotic resistance, the ability to exist under various environmental conditions, improved adhesion, pathogenicity, or facilitation of horizontal gene transfer) (Casjens et al., 2001). Not all prophages have the ability to undergo the lytic cycle. Non-functional prophages, i.e., defective prophages or cryptic prophages,

[0027] 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. August 19, 2014; 111(33): 12127-1213 2, and references therein). Notably, defective prophages also often contain genes encoding proteins with homologous recombination functions, as well as many genes that can provide beneficial functions to the host, including further prevention of infection or bacteriocins, and these can be useful in nutrient competition, for example, by suppressing the growth of other adjacent bacterial species.

[0028] ​​​​​​Phages can have a positive impact on gene expression and fitness in Escherichia coli in many ways. Cryptic phages, lysogenic phages, and lytic phages have been shown to provide the host with multiple benefits 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 different niches, or an increased ability to exclude competing strains. Dormant pro phages have also been shown to prevent superinfection by another phage (e.g., a lytic phage).

[0029] Several studies have shown that endogenous phages can affect the ability of bacteria to grow on specific carbon sources. In addition to lambda, active Mu, P1, and P2 prophages as well as the cryptic prophage CP4-57 increase growth under glucose-limiting conditions and other growth conditions (Edlin, G., Lin, L. & Bitner, R. Reproductive fitness of P1, P2, and M u lysogens of Escherichia coli. J. Virol . 21, 560-564 (1977); Edlin, G., Lin, L. & Kudmar, R. λ Lysogens of E. coli repr oduce more rapidly than non-lysogens. Na ture 255, 735-737 (1975); Wang, X., Kim, Y. & Wood, T. K. Control and benefits o f CP4-57 prophage excision in Escherichi a coli. E. coli biofilms. ISME J. 3, 1164-1179 (2 009). In another study, it was shown that when λ is integrated into the E. coli genome, the ability of cells to grow on poor carbon sources ceases. In this case, the metabolic limitation can confer a survival benefit on the bacteria. Slowing bacterial growth in an environment poor in glucose may help the bacteria evade detection by the immune system and increase their chances of survival.

[0030] Other survival characteristics may be affected as well. Wang et al. created a single E. coli strain lacking all nine cryptic prophages. In this study, it was shown that these prophages are beneficial for increasing growth under various conditions to withstand osmotic, oxidative and acid stress, enhancing the utilization of phosphorus and nitrogen, and influencing biofilm formation (Wang et al., Cryptic prophages help bacteria cope with adverse environm ents; DOI: 10.1038 / ncomms1146). In pathogenic bacterial proph ages, several studies have suggested that acquisition is associated with changes in the virulence of the pathogen.

[0031] Thus, one of ordinary skill in the art may expect that modifications, such as mutations or deletions of some or all of the endogenous prophages, may change the fitness of the bacteria (e.g., may have a negative impact). Furthermore, in genetically engineered bacteria in which the endogenous prophage is capable of producing this effector, some may think that the effector activity can be changed (e.g., may have a negative impact). This is because the endogenous prophage ​ is particularly applicable when present in all specimens of a particular strain subtype - this indicates that bacteria containing the prophage sequence can evolutionarily overcome the form of bacteria lacking that prophage as demonstrated.

[0032] As further described herein, prophages in Escherichia coli Nissle that can undergo lysis under specific conditions and are present in all specimens of Escherichia coli Nissle have been identified . Surprisingly, tests of bacterial fitness, residence time, and activity have shown that bacteria containing mutations or deletions in the endogenous phage are essentially the same (e.g., at least of the same magnitude) . Under similar assay conditions, there were no distinguishable differences in Phe degradation activity (in vitro or in vivo) between strains. For example, under similar assay conditions, Phe consumption is of the same magnitude between the two strains (see, for example, FIGS. 15 and 17A) . In vivo competition studies between phage-containing and phage-free strains have shown no distinguishable differences in transport or colony formation between phage-free PKU strains of Nissle

[0033] (see, for example, FIG. 19). Thus, in some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, do not change the bacterial fitness of the modified or genetically engineered bacteria. In some embodiments, engineered bacteria containing one or more phage modifications, e.g., mutations or deletions or other modifications described herein

[0034] is essentially the same as or has at least a similar bacterial fitness as the corresponding isogenic strain in the absence of phage mutation. In further embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, do not change the strain activity (e.g., effector activity or metabolic activity) of the engineered bacteria that can produce effectors compared to the corresponding isogenic strain without phage mutation. In some embodiments, non-modified bacteria or genetically engineered bacteria comprising one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have essentially the same or at least a similar bacterial strain activity (e.g., effector activity or metabolic activity) as the corresponding isogenic strain without phage mutation. Further, in some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, change (e.g., increase or decrease) the bacterial fitness of the engineered bacteria. In some embodiments, engineered bacteria comprising one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have a changed (e.g., decreased or increased) bacterial fitness compared to the corresponding isogenic strain without phage mutation. In some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, change the strain activity (e.g., effector activity or metabolic activity) of the bacteria that can produce effectors compared to the corresponding isogenic strain without phage mutation. In some embodiments, one or more phage modifications, e.g., mutations or deletions or other modifications described herein, do not change the strain activity (e.g., effector activity or metabolic activity) of the engineered bacteria that can produce effectors compared to the corresponding isogenic strain without phage mutation. is essentially the same as or has at least a similar bacterial fitness as the corresponding isogenic strain in the absence of phage mutation.

[0035] Further, in some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, change (e.g., increase or decrease) the bacterial fitness of the engineered bacteria. In some embodiments, engineered bacteria comprising one or more phage modifications, e.g., mutations or deletions or other modifications described herein, have a changed ( e.g., decreased or increased) bacterial fitness compared to the corresponding isogenic strain without phage mutation. In some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, change the strain activity (e.g., effector activity or metabolic activity) of the bacteria that can produce effectors compared to the corresponding isogenic strain without phage mutation. Further, in some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, do not change the strain activity (e.g., effector activity or metabolic activity) of the engineered bacteria that can produce effectors compared to the corresponding isogenic strain without phage mutation. In some embodiments, one or more modifications in the phage genome, e.g., mutations or deletions or other modifications described herein, change the strain activity (e.g., effector activity or metabolic activity) of the bacteria that can produce effectors compared to the corresponding isogenic strain without phage mutation. ​​​​​​​Alter (e.g., decrease or increase). In some embodiments, one or more ph age modifications, such as mutations or deletions or other modifications described herein, non-modified bacteria or genetically engineered bacteria have corresponding isogenic strains without phage mutations and have altered (e.g., decreased or increased) bacterial strain activity (e.g., effector activity or metabolic activity).

[0036] In some embodiments, the genetically engineered bacteria include one or more Escherichia coli Nissle bacteriophages (e.g., phage 1, phage 2, and phage 3). In some embodiments, the genetically engineered bacteria include one or more mutations in phage 3. Such mutations include deletions, insertions, substitutions, and inversions and are located within one or more phage 3 genes or include one or more phage 3 genes. In some embodiments, one or more insertions include an antibiotic cassette. In some embodiments, the mutation is a deletion. In some embodiments, the genetically engineered bacteria are located within one or more genes selected from the following or include 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, E COLIN_10010, ECOLIN_10015, ECOLIN_10020, EC OLIN_10025, ECOLIN_10030, ECOLIN_10035, ECO LIN_10040, ECOLIN_10045, ECOLIN_10050, ECOL IN_10055, ECOLIN_10065, ECOLIN_10070, ECOLI N_10075, ECOLIN_10080, ECOLIN_10085, ECOLIN _10090、ECOLIN_10095、ECOLIN_10100、ECOLIN_ 10105、ECOLIN_10110、ECOLIN_10115、ECOLIN_1 0120、ECOLIN_10125、ECOLIN_10130、ECOLIN_10 135、ECOLIN_10140、ECOLIN_10145、ECOLIN_101 50、ECOLIN_10160、ECOLIN_10165、ECOLIN_1017 0、ECOLIN_10175、ECOLIN_10180、ECOLIN_10185 、ECOLIN_10190、ECOLIN_10195、ECOLIN_10200、 ECOLIN_10205, ECOLIN_10210, ECOLIN_10220, E COLIN_10225, ECOLIN_10230, ECOLIN_10235, EC OLIN_10240、ECOLIN_10245、ECOLIN_10250、ECO LIN_10255, ECOLIN_10260, ECOLIN_10265, ECOL IN_10270, ECOLIN_10275, ECOLIN_10280, ECOLI N_10290, ECOLIN_10295, ECOLIN_10300, ECOLIN _10305、ECOLIN_10310、ECOLIN_10315、ECOLIN_ 10320、ECOLIN_10325、ECOLIN_10330、ECOLIN_1 0335、ECOLIN_10340、およびECOLIN_10345。一実施形態で The genetically engineered bacteria are ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, E COLIN_10135, ECOLIN_10140, ECOLIN_10145, EC OLIN_10150, ECOLIN_10160, ECOLIN_10165, ECO Complete deletion or deletion of one or more of LIN_10170, ECOLIN_10175, In a specific embodiment, the deletion comprises a deletion or partial deletion of ECOLIN_10110 , ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, E COLIN_10145, ECOLIN_10150, ECOLIN_10160, EC Complete deletion of OLIN_10165, ECOLIN_10170, and ECO In one embodiment, the sequence of SEQ ID NO: 130 is In one embodiment, a sequence comprising SEQ ID NO: 130 is deleted from the phage 3 genome. In one embodiment, the genetically engineered bacterium has the sequence of SEQ ID NO:28 deleted from its genome. In one embodiment, the genetically engineered bacterium comprises a modified phage genome sequence comprising the sequence The modified phage genome sequence comprises sequence number 281.

[0037] In order that this disclosure may be more readily understood, certain terms are first defined. These definitions The definition should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless otherwise defined, all technical and scientific methods used herein are within the scope of the appended claims. Terms have the same meaning as commonly understood by one of ordinary skill in the art. are described throughout the detailed description.

[0038] "Hyperphenylalaninemia" "hyperphenylalaninemic" and "hyperphenylalaninemic" "Excess phenylalanine" refers to increased or abnormally high concentrations of phenylalanine in the body. In some embodiments, high phenyl The diagnostic signal for uraninemia is at least 2 mg / dL, at least 4 mg / dL, At least 6 mg / dL, at least 8 mg / dL, at least 10 mg / dL, at least At least 12 mg / dL, at least 14 mg / dL, at least 16 mg / dL, at least at least 18 mg / dL, at least 20 mg / dL, or at least 25 mg / dL As used herein, high phenylalanine Diseases associated with phenylketonuria include, but are not limited to, phenylketonuria, classic or typical phenylephrine purpura (PPP) Phenylketonuria, atypical phenylketonuria, permanent mild hyperphenylalaninemia, non-phenyl Phenylketonuria, hyperphenylalaninemia, phenylalanine hydroxylase deficiency, Cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase These include deficiency, glaucoma, and Segawa disease. Affected individuals suffer from progressive and irreversible neurological deficits, Mental retardation, encephalopathy, epilepsy, eczema, reduced growth, microcephaly, They may suffer from tremors, limb spasms, and / or hypopigmentation (Leonard 20 Hyperphenylalaninemia may also occur secondary to other conditions, such as liver disease. be.

[0039] "Phenylalanine ammonia lyase" and "PAL" are enzymes that convert phenylalanine into Phenylalanine metabolic enzymes (P Trans-cinnamic acid is known to have low toxicity and is effective in mammals. It is converted by liver enzymes into hippuric acid, which is secreted in the urine. It can substitute for the enzyme PAH to metabolize lanin. PAL enzyme activity is related to the THB cofactor In some embodiments, PAL does not require PAL gene activity from a prokaryotic species. In an alternative embodiment, the PAL is encoded by a gene derived from a eukaryotic species. In some embodiments, PAL is encoded by, but not limited to, Chromobacter xylosoxidans dans), Pseudomonas aeruginosa nosa), Photorhabdus lumine scens), Anabaena variabilis, and Agrobacterium tumefaciens They are encoded by PAL genes from bacterial species, including P. faciens. In some embodiments, PAL is provided by the PAL gene from Anabaena variabilis. and is referred to herein as "PAL1" (Moffitt et al., 2007). In some embodiments, the PAL is a PA derived from Photorhabdus luminescence. L gene, and is referred to herein as "PAL3" (Williams, In some embodiments, PAL is produced from yeast species, such as Rhodosporum sp. From Rhodosporidium toruloides It is encoded by the PAL gene, which is involved in the transcription of IL-1 and IL-2 (Gilbert et al., 1985). In one embodiment, the PAL is derived from a plant species, such as Arabidopsis thaliana. thaliana (Wanner et al., 1999). Any suitable nucleotide and amino acid sequence of PAL, or its functional group, Fragments may be used.

[0040] "Phenylalanine hydroxylase" and "PAH" are enzymes that bind to the cofactor tetrahydrobiopeptide. The aromatic side chain of phenylalanine is used in conjunction with pterin to create tyrosine in the human body. It is used to refer to the enzymes that catalyze the hydroxylation of PAHs. The gene is located on the long (q) arm of chromosome 12 between positions 22 and 24.2. The amino acid sequences are highly conserved among mammals. The nucleic acid sequences are well known and widely available. Full-length human cDNA sequences for PAHs 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 bind to their respective keto acids, Stereospecific oxidative deamination of L-amino acids to produce ammonia and hydrogen peroxide. For example, LAAD is the enzyme that catalyzes the synthesis of phenylalanine. A number of LAAD enzymes are known in the art. Many of them belong to the genera Proteus and Providencia. dencia, and Morganella, or venom LAAD is characterized by a rapid kinetics of phenylalanine degradation (Hou et al., Appl Microbiol Technol.2015 October;99(20):8 pp. 391~402; “Production of phenylpyruvic ac id from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis:c omparison of enzymatic and whole-cell bi Most eukaryotes and Prokaryotic L-amino acid deaminases are extracellular, whereas Proteus species LAAD is intracellular. It is located in the inner membrane and faces outward toward the periplasmic space where the enzyme activity resides. As a result of localization, phenylalanine transport across the inner membrane into the cytoplasm is mediated by the Proteus LAA D-mediated phenylalanine degradation. They are easily imported into the periplasm through the outer membrane without the need for transporters and are highly efficient at utilizing substrates. Eliminate the need for a transporter, improving chances.

[0042] In some embodiments, the genetically engineered bacteria may be, but is not limited to, Proteus sp. LAAD genes from bacterial species, including those from the genera Providencia and Morganella In some embodiments, the bacterial species is Proteus mirabilis. In some embodiments, the bacterial species is Proteus bulga. In some embodiments, the genetic modification is The engineered bacterially encoded LAAD is localized to the plasma membrane and faces the periplasmic space. The catalytic activity occurs in the periplasmic space.

[0043] "Phenylalanine Metabolic Enzymes" or "PMEs" are enzymes that break down phenylalanine. The term is used to refer to any phenylalanine enzyme known in the art that is capable of Nin-metabolizing enzymes can be encoded by genetically engineered bacteria. PMEs include However, phenylalanine hydroxylase (PAH), phenylalanine ammonia ase (PAL), aminotransferase, L-amino acid deaminase (LAAD) , and phenylalanine dehydrogenase.

[0044] Phenylalanine hydroxylase, phenylalanine dehydrogenase or amino The reaction with transferase requires a cofactor, whereas LAAD and PAL require an additional In some embodiments, the cofactor is produced by a genetically engineered bacterium. The PME to be encoded requires a cofactor. In some embodiments, the cofactor is a genetic In another embodiment, the gene is provided simultaneously or sequentially with administration of the engineered bacteria. The engineered bacteria are capable of producing the cofactor. In some embodiments, the genetic engineering In some embodiments, the selected bacterium encodes a phenylalanine hydroxylase. In the genetically engineered bacteria, phenylalanine dehydrogenase is encoded. In embodiments, the genetically engineered bacteria encodes an aminotransferase. In some embodiments, the PME encoded by the engineered bacterium does not require a cofactor. Without wishing to be bound by theory, the absence of a cofactor requirement is a key factor in the enzyme-mediated catalysis of catalysis. The rate of phenylalanine decomposition depends on the availability of substrate and is controlled by the availability of cofactors. In some embodiments, the present invention is intended to include, but is not limited to, those produced by genetically engineered bacteria. In some embodiments, the PME produced by the genetically engineered bacteria is PAL. In some embodiments, the PME produced by the genetically engineered bacteria is a LAAD. encodes a combination of PMEs.

[0045] In some embodiments, the catalytic activity of the PME depends on oxygen levels. In this form, PME is catalytically active under microaerobic conditions. D catalytic activity is dependent on oxygen. In some embodiments, the LAAD is in aerobic conditions, such as microaerobic conditions. Active under hypoxic conditions. In some embodiments of the invention, the PME is administered to, for example, the colon. Functions at very low levels of oxygen or in the absence of oxygen as found. As a typical example, PAL activity does not depend on the presence of oxygen.

[0046] As used herein, an "effector" or "effector molecule" refers to a compound that An effector may refer to a molecule, such as a metabolite or a polypeptide, that exerts a function of For example, the single gene may be encoded by a single gene for secretion or display. Alternatively, the effector may require multiple genes. For example, a desired property can be obtained by a biosynthetic pathway (e.g., butyrate). A polypeptide encoded by multiple genes in a biosynthetic pathway that synthesizes a metabolic product having Similarly, catabolic enzymes, e.g., for the breakdown of toxic metabolites, may also act as effectors. Polypeptides encoded by multiple genes in a pathway can also be called effectors. These effector molecules are also referred to as "therapeutic metabolites," "therapeutic molecules" or "therapeutic Other terms used interchangeably herein as effectors include, but are not limited to, "effector polypeptides." The term "polypeptide of interest" or "polypeptides of interest" or "protein of interest" refers to a polypeptide of interest. "protein" and "protein(s) of interest."

[0047] As used herein, a "payload" refers to a genetically engineered microorganism, such as a bacterium. The present invention refers to one or more polynucleotides and / or polypeptides of interest produced by In some embodiments, the payload comprises one or more genes or operons. In some embodiments, one or more genes comprising a payload and The gene and / or operon are endogenous to the microorganism. One or more elements of the load are derived from different microorganisms and / or organisms. In embodiments, the payload is a therapeutic payload. The peptide is encoded by a gene for the biosynthesis of the molecule. Erodes are encoded by genes for the metabolism, catabolism, or breakdown of molecules. In some embodiments, the payload is encoded by a gene for the import of a molecule. In some embodiments, the payload is a gene encoding a polypeptide for export of the molecule. In some embodiments, the payload is a regulatory molecule, such as FNR. In some embodiments, the payload is a promoter or In some embodiments, payload expression is mediated by a regulatory element such as a FN repressor. In some embodiments, the payload expression is driven from an inducible promoter such as RS. The expression is driven from a constitutive promoter. In an alternative embodiment, the payload comprises a repressor element, such as a cleavage switch. The compound is produced by a synthetic or biochemical pathway, and the biosynthetic or biochemical pathway may be any In some embodiments, the gene may be endogenous to the microorganism. contains two or more payloads.

[0048] The present invention relates, inter alia, to genetically engineered bacteria, pharmaceutical compositions thereof, and high phenylalanine Methods for modulating and treating disorders associated with raninemia are included. In this study, genetically engineered bacteria express a non-natural phenylalanine ammonia lyase (PAL ), which processes and reduces phenylalanine in mammals. In some embodiments, the engineered bacteria can be In some embodiments, the engineered bacteria further comprises a gene encoding a reporter. The engineered bacteria may also contain a gene encoding a LAAD. Tee and / or biological containment, e.g., kill switches, gene guard systems and / or may comprise one or more gene sequences related to auxotrophy. These gene sequences can be regulated using various promoter systems, for example, any of the promoter systems disclosed herein, and the promoter can be the same promoter that regulates one or more different genes, or different copies of the same promoter that regulates different genes, or may include the use of different promoters used in combination to regulate the expression of different genes. The use of different regulatory systems or promoter systems for controlling gene expression provides flexibility (e.g., the ability to differentially control gene expression under different environmental conditions and / or the ability to differentially control gene expression temporally), and also provides the ability to “fine-tune” gene expression, and any or all of such regulation may help to optimize gene expression and / or bacterial growth. Genetically engineered bacteria and pharmaceutical compositions containing them can be used to metabolize phenylalanine in the body into non-toxic molecules for treating and / or preventing conditions associated with hyperphenylalaninemia including PKU. In certain embodiments, compositions containing genetically engineered bacteria can be used in the methods of the present disclosure for treating and / or preventing disorders associated with hyperphenylalaninemia.

[0049] Effector molecules also include anti-cancer molecules. “Anti-cancer molecules” refer to one or more therapeutic substances or drugs of interest produced by genetically engineered microorganisms (e.g., engineered bacteria or engineered oncolytic viruses), which can reduce and / or inhibit cell growth or replication. In some embodiments, the anti-cancer It is a therapeutic molecule useful for rating or treatment. In some embodiments, an anti-cancer molecule is a therapeutic molecule encoded by a gene. In alternative embodiments, the anti-cancer molecule is , a therapeutic molecule produced by a biochemical or biosynthetic pathway, and the biosynthetic pathway or the biochemical pathway may optionally be endogenous to the microorganism. In some embodiments, the genetically engineered microorganism can produce two or more anti-cancer molecules. Non-limiting examples of anti-cancer molecules include immune checkpoint inhibitors (e.g., CTLA-4 antibody, PD-1 antibody , PDL-1 antibody), cytotoxic agents (e.g., Cly A, FASL, TRAIL, TNF -α), immune-stimulating cytokines and costimulatory molecules (e.g., OX40, CD28, I COS, CCL21, IL-2, IL-18, IL-15, IL-12, IFN-γ, I L-21, TNF, GM-CSF), antigens and antibodies (e.g., tumor antigens, neoantigens, C txB-PSA fusion protein, CPV-OmpA fusion protein, NY-ESO-1 tumor antigen, RAF1, antibodies against immunosuppressive molecules, anti-VEGF, anti-CXR4 / CXCL12 , 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, I L-12, AFP, VEGFR2), and enzymes (e.g., E. coli CD, HSV-TK) are included. In some embodiments, the anti-cancer molecule includes RNA interference, microRNA response ​Answer or inhibition, TLR response, antisense gene regulation, target protein binding (aptamer or decoy oligo), nucleic acid molecules that mediate gene editing such as CRISPR interference are included In some embodiments, bacteria or viruses can be used as vectors for introducing DNA into mammalian cells, for example, by bacteriophage (bactofection) (Bernardes et al., 2013).

[0050] Non-limiting examples of effector molecules include "anti-inflammatory molecules" and / or "gastrointestinal barrier function enhancer molecules". Anti-inflammatory molecules and / or gastrointestinal 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-acyl phosphatidylethanolamine (NAPE), elafin (also called protease inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein, but are not limited thereto. Such molecules include compounds that inhibit pro-inflammatory molecules, for example, single-chain variable antibodies (scFv) , antisense RNA, siRNA, or shRNA that neutralizes TNF-α, IFN-γ, IL-1β, IL -6, IL-8, IL-17, and / or chemokines (e.g., CXCL-8 and CCL2). Such molecules are described herein such as those that result in IL-22 production) AHR agonists (e.g., indole acetic acid, indole-3-aldehyde, and indole) and PXR agonists (e.g., IPA) are also included. Such molecules 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). Molecules may be primarily anti-inflammatory (e.g., IL-10) or primarily gastrointestinal barrier function enhancing (e.g., GLP-2). Molecules may be both anti-inflammatory and gastrointestinal barrier function enhancing. Anti-inflammatory and / or gastrointestinal barrier function enhancer molecules may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene. Alternatively, anti-inflammatory and / or gastrointestinal barrier function enhancer molecules may be synthesized by a biosynthetic pathway that requires multiple genes (e.g., butyrate).

[0051] Effector molecules also include metabolic effector molecules. "Metabolic effector molecules" and / or "satiety effector molecules" include n-acyl-phosphatidylethanolamine (NAPE), n- ​Acyl-ethanolamine (NAE), ghrelin receptor antagonist, peptide YY3 -36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK 22, CCK8, bombesin family molecules, bombesin, gastrin-releasing peptide (G RP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin , pancreatic polypeptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonist, nicotinamide adenine dinucleotide (NAD), nicotinamide mon onucleotide (NMN), nucleotide riboside (NR), nicotinamide, and ni cotinic acid (NA) are included, but not limited to these. Such molecules also include compounds that inhibit molecules that promote metabolic diseases (e.g., dipeptidyl peptidase-4 (DPP4 ) or single-chain variable antibodies (scFv) that inhibit the ghrelin receptor, antisense RNA, siRNA, or shRNA). Metabolic and / or satiety effector molecules can be encoded by a single gene, for example, glucagon-like peptide 1 is encoded by the GLP-1 gene. In some embodiments, a genetically engineered bacterium containing a gene sequence for the production or catabolism of tryptophan and / or one of its metabolites further includes a gene sequence for the expression of one or more metabolic effector molecules and / or satiety effector molecules. Another non-limiting example of an effector molecule is the co-owned international patent application PCT / U filed concurrently.

[0052] ​S2016 / 34200 (filing date: May 25, 2016), PCT / US2017 / 01 3072 (filing date: January 11, 2017), PCT / US2017 / 016603 (filing date: February 3, 2017), PCT / US2017 / 016609 (filing date: February 4, 2016), PCT / US2017 / 017563 (filing date: 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: 2016 June 10), PCT / US2016 / 069052 (filing date: December 28, 2016 ), PCT / US2016 / 32562 (filing date: May 13, 2016), PCT / US 2016 / 062369 (filing date: November 16, 2016) and PCT / US2017 / 013072, the contents of which are hereby incorporated by reference in their entirety into this specification.

[0053] In certain embodiments, a new or improved effector (e.g., PME) can be identified according to methods known in the art or described herein and encoded by a genetically engineered bacterium. In some embodiments, the enzyme encoded by the genetically engineered bacterium is a wild-type enzyme isolated from a virus, prokaryote, or eukaryote. In some embodiments, the enzyme sequence has been further modified or mutated to increase one or more specific properties of the enzyme, such as stability or catalytic activity.

[0054] ​​​​​​The term "phenylalanine metabolite" refers to a metabolite generated as a result of the breakdown of phenylalanine. The metabolite is produced directly from phenylalanine by an enzyme that uses phenylalanine as a substrate, or indirectly by different enzymes downstream in the metabolic pathway that act on phenylalanine metabolite substrates. In some embodiments, the phenylalanine metabolite is produced by genetically engineered bacteria encoding 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 are phenylpyruvic acid and phenyl-lactic acid. Other examples include phenylacetate, phenylethylamine, and phenylacetylglutamine.

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

[0057] "Phenylalanine transporter" is used to refer to a membrane transport protein capable of transporting phenylalanine into bacterial cells (see, for example, Pi et al., 1991 for reference). 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 derived from bacterial species including, but not limited to, Acinetobacter calcoaceticus, Salmonella enterica, and Escherichia coli. Other phenylalanine transporters include those encoded by the aroP gene, which transports three aromatic amino acids including phenylalanine with high affinity and, together with PheP, the Aageneral amino acid permease that accounts for the largest part of phenylalanine uptake. Furthermore, low levels of phenylalanine transport activity are traced to the activity of the LIV-I / LS system, which consists of two periplasmic binding proteins, the LIV binding protein (LIV-I system) and the LS binding protein (LS system), and the membrane components LivHMGF, a branched-chain amino acid transporter. In some embodiments, the phenylalanine transporter is encoded by the aroP gene derived from bacterial species. In some embodiments, the phenylalanine transporter is the LIV binding protein and the LS binding protein. ​​​​​​​​​​​It is encoded by the LivHMGF gene derived from a bacterial species. In some embodiments the genetically engineered bacterium comprises more than one phenylalanine transporter selected from the pheP, aroP, and LIV-I / LS systems .

[0058] The terms "phenylalanine" and "Phe" are used to refer to the amino acid having the formula C 6 H 5 CH 2 CH(NH 2 )COOH . Phenylalanine is a precursor of tyrosine, dopamine , norepinephrine, and epinephrine. L-phenylalanine is an essential amino acid and is the form of phenylalanine mainly found in food proteins. The stereoisomer D-phenylalanine is found in lesser 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" means a mechanism (e.g., a protein, a protein, or a protein complex) for importing (import) molecules such as amino acids, peptides (dipeptides , tripeptides, polypeptides, etc.), toxins, metabolites, substrates, and other biomolecules from the extracellular environment into the microorganism .

[0060] "Operably linked" means bound to a regulatory region sequence so as to enable the expression of a nucleic acid sequence, e.g., a nucleic acid sequence acting cis, e.g., a gene encoding PAL . ​​It refers to. The regulatory region can direct the transcription of the target gene and can be a nucleic acid including a promoter sequence, an enhancer sequence, a response element, a protein recognition site, an inducible element, a promoter control element, a protein binding sequence, 5' and 3' untranslated regions, a transcription start site, a termination sequence, a polyadenylation sequence, and an intron.

[0061] An "inducible promoter" refers to a regulatory region operably linked to one or more genes, and the expression of the gene increases in the presence of an inducer of the regulatory region. An "inducible promoter" refers to a regulatory region operably linked to one or more genes, and the expression of the gene increases in the presence of an inducer of the regulatory region. 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), and in the presence of an inducer of the regulatory region, the effector molecule is expressed. An "indirectly inducible promoter" refers to a regulatory system including two or more regulatory regions, for example, a first regulatory region operably linked to a gene encoding a second regulatory region (e.g., a transcriptional regulatory factor 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 suppressed, thereby activating or suppressing the expression of the effector molecule. Both directly inducible promoters and indirectly inducible promoters are included in "inducible promoters". In the presence of an inducer of the first regulatory region, the second regulatory region can be activated or suppressed, thereby activating or suppressing the expression of the effector molecule. Both directly inducible promoters and indirectly inducible promoters are included in "inducible promoters". promoters".

[0063] "Exogenous environmental conditions" or "environmental conditions" refer to those to which the promoters described herein are directly or an indirectly induced setting or situation. The phrase refers to environmental conditions that are external to the engineered microorganism but endogenous or natural to the host target environment. Thus, "exogenous" and "endogenous" can be used interchangeably to refer to environmental conditions that are endogenous to the mammalian body but external or exogenous to 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 upper gastrointestinal tract of the mammalian host. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of the mammalian host. In some embodiments, the exogenous environmental conditions are specific to the small intestine of the mammalian host. In some embodiments, the exogenous environmental conditions are low acid, microaerophilic, or anaerobic conditions such as those in the mammalian gastrointestinal environment. In some embodiments, the exogenous environmental conditions refer to the presence of molecules or metabolites specific to the mammalian gastrointestinal tract in a healthy or diseased state, such as propionate. In some embodiments, the exogenous environmental conditions are specific to the tumor microenvironment. In some embodiments, the exogenous environmental conditions are molecules or metabolites specific to the tumor microenvironment. In some embodiments, the exogenous environmental conditions are tissue-specific 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 include a pH-dependent promoter. In some embodiments, the genetically engineered microorganisms of the present disclosure include an oxygen-level-dependent promoter. In some aspects, the bacteria have evolved transcription factors that can detect oxygen levels. Different signaling pathways respond to different oxygen levels . In some embodiments, the exogenous environmental conditions are a low pH environment. In some embodiments, the genetically engineered microorganisms of the present disclosure include a pH-dependent promoter. In some embodiments, the genetically engineered microorganisms of the present disclosure include an oxygen-level-dependent promoter. In some aspects, the bacteria have evolved transcription factors that can detect oxygen levels. Different signaling pathways respond to different oxygen levels It can be induced by a bell and occurs with different dynamics.

[0064] As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. It can be induced by a bell and occurs with different dynamics. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production. As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to the external settings or situations or environmental conditions of the engineered microorganisms and are related to the in vitro culture conditions of the microorganisms. "Exogenous environmental conditions" may also refer to the conditions during the growth, production, and manufacture of organisms. Such conditions include, but are not limited to, aerobic culture conditions, anaerobic culture conditions, low nitrogen culture conditions, and other conditions under set oxygen concentrations. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganisms are grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does 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., PME such as PAL or LAAD), the induction rate of the transporter (e.g., PheP), and the overall viability and metabolic activity of the strain during strain production.

[0065] "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 the binding and / or activation of the corresponding transcription factor activates downstream gene expression. "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 the binding and / or activation of the corresponding transcription factor activates downstream gene expression. "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 the 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. is included. Corresponding FNR-responsive promoters, ANR-responsive promoters, and D NR-responsive promoters are known in the art (see, for example, Castiglion e 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) is highly expressed under conditions of low or no environmental oxygen, from the Escherichia coli Nissle fumarate and nitrate reductase gene S (fnrS) (Durand and Storz, 2 010; Boysen et al., 2010). The PfnrS promoter is activated under anaerobic and / or hypoxic conditions by the global transcriptional regulator FNR, which is found naturally in Nissle. Under anaerobic and / or hypoxic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with the iron-sulfur clusters in the FNR dimer, converting them to an inactive form. Thus, the PfnrS inducible promoter is employed to modulate the expression of proteins or RNAs. PfnrS is used interchangeably in this application as FNRS, fnrS, FNR, P-FNRS promoter, and other such related names that denote the promoter PfnrS.

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

Table 1

[0069] As used herein, an “adjustable regulatory region” is under direct or indirect control of a transcription factor and refers to a nucleic acid sequence capable of activating, suppressing, derepressing, or controlling gene expression in response to the level of an inducer. In some embodiments, the adjustable regulatory region includes a promoter sequence. The inducer can be an RNS or other inducer described herein, and the adjustable regulatory region can be an RNS-responsive regulatory region or other responsive regulatory region described herein. The adjustable regulatory region can be operably linked to a gene sequence or gene cassette for the production of one or more payloads, e.g., a butyrogenic or other gene cassette or gene sequence. For example, in certain embodiments, the adjustable regulatory region is an RNS-derepressible regulatory region, and in the presence of RNS, the RNS-sensing transcription factor no longer binds to and / or suppresses the regulatory region, thereby enabling the expression of the operably linked gene or gene cassette. In this case, the adjustable regulatory region derepresses the expression of the gene or gene cassette in response to the RNS level. Each gene or gene cassette can be operably linked to an adjustable regulatory region that is directly or indirectly controlled by at least one

[0070] transcription factor capable of sensing an RNS. 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 Yes. In some embodiments, the exogenous environmental conditions are biological molecules involved in the inflammatory response, for example, molecules present in inflammatory disorders of the gastrointestinal tract. In some embodiments, the exogenous environmental conditions or signals are naturally present or not naturally present in the environment in which the recombinant bacterial cells are present. In some embodiments, the exogenous environmental conditions or signals are, for example, by the generation or removal of a biological state and / or the administration or removal of a biological molecule artificially generated.

[0071] In some embodiments, the exogenous environmental conditions and / or signals stimulate the activity of an inducible promoter . In some embodiments, the exogenous environmental conditions and / or signals that help activate the 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 invention, for example, tetracycline, arabinose, or any biological molecule that helps activate the inducible promoter. In some embodiments, the exogenous environmental conditions and / or signals are added to the culture medium containing the recombinant bacterial cells of the invention. In some embodiments, the exogenous environmental conditions that help activate the inducible promoter are naturally present in the mammalian gastrointestinal tract (e.g., hypoxia or anaerobic conditions, or biological molecules involved in the inflammatory response). In some embodiments, the loss of exposure to the exogenous environmental conditions (e.g., in vivo) results in the absence of the exogenous environmental conditions for the promoter to be induced (e.g., aerobic environment outside the gastrointestinal tract), thus inhibiting the activity of the inducible promoter. As used herein, "non-natural" or "unnatural" means not naturally present in a particular environment or not occurring naturally. In some embodiments, the loss of exposure to exogenous environmental conditions (e.g., in vivo) inhibits the activity of the inducible promoter because the exogenous environmental conditions are not present for the promoter to be induced (e.g., aerobic environment outside the gastrointestinal tract). As used herein, "non-natural" A nucleic acid sequence typically refers to a nucleic acid sequence that does not normally exist in bacteria, such as an additional copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or sub-strain of bacteria, or a sequence that has been modified and / or mutated compared to an unmodified sequence from the same subtype of bacteria. In some embodiments, the non-natural nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-natural nucleic acid sequence can be one or more genes in a regulatory region, promoter, gene, and / or gene cassette. In some embodiments, "non-natural" refers to two or more nucleic acid sequences that are not found in nature in the same relationship to each other. The non-natural nucleic acid sequence can be present on a plasmid or chromosome. Further, multiple copies of any regulatory region, promoter, gene, and / or gene cassette can be present in the bacteria, and one or more copies of the regulatory region, promoter, gene, and / or gene cassette can be mutated or otherwise modified as described herein. In some embodiments, the 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 a gene cassette that perform multiple different functions. In some embodiments, the genetically engineered bacteria of the invention contain a gene encoding an effector molecule (e.g., PME), and the gene is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule or a promoter operably linked to a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule is operably linked to a promoter that is directly or indirectly inducible and not naturally associated with the gene, such as the FNR promoter operably linked to a gene encoding an effector molecule, or a second effector molecule operatively linked to a ParaBAD promoter that may be linked.

[0072] A "constitutive promoter" is a promoter that can promote continuous transcription of a coding sequence or gene under its control and / or to which it is operatively linked. Refers to. Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, constitutive E. coli σ promoter (e.g., osmY pro moter (International Genetically Engineere S d Machine (iGEM) standard biological parts registry name BBa_J459 92; BBa_J45993)), constitutive E. coli σ promoter (e.g., htpG heat shock promoter (BBa_J45504)), constitutive E. coli σ 32 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015) ), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), G 70 lnRS promoter (BBa_K088007), lacZ promoter (BBa_K 119000; BBa_K119001); M13K07 gene I promoter (BBa _M13101); M13K07 gene II promoter (BBa_M13102), M 13K07 gene III promoter (BBa_M13103), M13K07 gene I V promoter (BBa_M13104), M13K07 gene V promoter (BBa _M13105), M13K07 gene VI promoter (BBa_M13106), M 13K07 gene I V promoter (BBa_M13104), M13K07 gene V promoter (BBa _M13105), M13K07 gene VI promoter (BBa_M13106), M 13K07 gene VIII promoter (BBa_M13108), M13110 (BB a_M13110)), a constitutive Bacillus subtilis (Bacillus subti lis) σ A promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), P liaG (BBa_K823000) 、P lepA (BBa_K823002), P veg (BBa_K823003)), the con stitutive Bacillus subtilis σ B promoter (e.g., promoter ctc (BBa_ K143010), promoter gsiB (BBa_K143011)), Salmonella ( Salmonella) promoter (e.g., Pspv2 from Salmonella (BBa_ K112706), Pspv from Salmonella (BBa_K112707)), bacteri ophage T7 promoter (e.g., T7 promoter (BBa_I712074; B Ba_I719005; BBa_J34814; BBa_J64997; BBa_K11 3010; BBa_K113011; BBa_K113012; BBa_R0085; B Ba_R0180; BBa_R0181; BBa_R0182; BBa_R0183; B Ba_Z0251; BBa_Z0252; BBa_Z0253)), bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and their functional fragments are included.

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

[0074] In some embodiments, the genetically engineered bacteria are active in the digestive tract (e.g., expressing one or more payloads (e.g., PME)). In some embodiments, the genetically engineered bacteria are active in the large intestine (e.g., expressing one or more payloads ). In some embodiments, the genetically engineered bacteria are active in the small intestine (e.g expressing one or more payloads). In some embodiments, the genetically engineered bacteria are active in the small intestine and large intestine. Without wishing to be bound by theory, phenylalanine degradation may be very effective in the small intestine because amino acid absorption, e.g., phenylalanine absorption, occurs in the small intestine . Prevention or reduction of phenylalanine uptake into the blood can avoid an increase in Phe levels and the resulting toxicity of Phe . Furthermore, extensive intestinal recycling of amino acids between the intestine and the body may enable 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 o f enterorecirculation of amino acids and in PKU" (Chang et al., "A new theory of enterorecirculation of amino acids and its implications in PKU" (Chang et al., "A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino ac ids using oral enzyme-artificial cells, as in removing phenylalanine in phenylke tonuria; Artif Cells Blood Substit Immob il Biotechnol. 1995; 23(1): 1-21). Blood-derived phenylalanine circulates in the small intestine (see, e.g., FIG. 15), and can be removed by active bacteria at that site. In some embodiments, the genetically engineered bacteria pass through the small intestine. In some embodiments, the genetically engineered bacteria have an increased residence time in the digestive tract. In some embodiments, the genetically engineered bacteria colonize the small intestine or the large intestine. In some embodiments, the genetically engineered bacteria colonize the colon. In some embodiments, the genetically engineered bacteria have an increased residence time in the digestive tract. In some embodiments, the genetically engineered bacteria do not colonize the digestive tract. As used herein, the term "hypoxic" means a level, amount, or concentration of oxygen that is lower than the level, amount, or concentration of oxygen (O

[0075] ) present in the atmosphere (e.g., < 21% O 2 ; < 160 torr O ). Thus, the term "hypoxic condition(s)" or "hypoxic environment" refers to a condition or environment that includes an oxygen level lower than that present in the atmosphere. In some embodiments, the term "hypoxic" refers to the mammalian digestive tract, e.g., the lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon 2 ; < 160 torr O 2 ). Thus, the term "hypoxic condition(s)" or "hypoxic environment" refers to a condition or environment that includes an oxygen level lower than that present in the atmosphere. In some embodiments, the term "hypoxic" refers to the mammalian digestive tract, e.g., the lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon , oxygen (O 2 It refers to the level, amount, or concentration of In some embodiments, the term "hypoxia" refers to an oxygen level of 0 to 60 mmHg. 2 (0 to 60 torr O 2 ) (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, 3 7, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mm Hg O 2 (a) any or all fractional increases thereof; (e.g., 0.2mmHg, 0.5mmHg O 2 , 0.75 mm Hg O 2 , 1.25 mm Hg O 2 , 2.175 mm Hg O 2 , 3.45 mm Hg O 2 , 3.7 5 mm Hg O 2 , 4.5 mm Hg O 2 , 6.8 mm Hg O 2 , 11.35mmHg O 2 , 46.3 mm Hg O 2 , 58.75mmHg, etc., but these examples are The decimals listed here are for illustrative purposes only and are in no way meant to be limiting. In some embodiments, "hypoxia" refers to a blood pressure of less than about 60 mmHg. O 2 (For example, 0 to about 60 mmHg O 2 The term "hypoxia" also refers to levels, amounts, or concentrations of O between 0 and 60 mmHg (including 0 mmHg and 60 mmHg), e.g., 0 - 5 mmHg O 2 levels, amounts, or concentrations in the range of, e.g., 0 - 5 mmHg O 2 , <1.5 mmHg O 2 , 6 - 10 mmH g, <8 mmHg, 47 - 60 mmHg, etc., can be referred to, but these exemplary ranges are provided here for illustrative purposes only and are in no way meant to be limiting. For example, see Albenberg et al., Gastroenterology, Vol. 147 (5):1055 - 1063 (2014); Bergofsky et al., J Clin. Invest., Vol. 41 (11):1971 - 1980 (1962); Crompt on et al., J Exp. Biol., Vol. 43:473 - 478 (1965); He et al. , PNAS (USA), Vol. 96:4586 - 4591 (1999); McKeown , Br. J. Radiol., Vol. 87:20130676 (2014) (doi: 10.1259 / brj.20130676). In each of the foregoing references, the oxygen levels found in the gastrointestinal tracts of various mammalian species have been discussed, and each of these references is hereby incorporated by reference in its entirety herein. In some embodiments, the term "hypoxia" refers to the level, amount, or concentration of oxygen (O ) found in organs or tissues other than the mammalian gastrointestinal tract, e.g., the urogenital tract, tumor tissue, etc., where the oxygen is present at a reduced level, e.g., a hypoxic or anoxic level. In some embodiments, "hypoxia" refers to a partially aerobic, semi - aerobic , microaerobic, nano - aerobic 2 environment, etc. where the oxygen is present at a reduced level, e.g., a hypoxic or anoxic level In some embodiments, "hypoxia" refers to a partially aerobic, semi - aerobic (se mi aerobic), microaerobic, nano - aerobic aerobic), microoxic, hypoxic, anoxic, and / or the level, amount or concentration of oxygen (O 2 ) present under 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 (O ) refers to the amount of dissolved oxygen (``DO''), which is the level of free, non-compound oxygen (O ) present in a liquid, typically expressed as milligrams per liter (mg / L), parts per million (ppm 2 ; 1 mg / L = 1 ppm) or micromoles (umole) (1 μmol O = 0 2 .022391 mg / L O ) and is reported in Fondriest Environmental, Inc., “Dissolved Oxygen”, the Basics of Environmental Measurements, November 19, 2013, www.fondriest.com / environmental-measurements / parameters / water-quality / dissolved-oxygen / >. In some embodiments, the term “hypoxic” refers to a level, amount, or concentration of oxygen (O ) of about 6.0 mg / L DO or less, such as 6.0 mg / L, 5.0 mg / L, 4.0 mg / L, 3.0 mg 2 / L, 2.0 mg / L, 1.0 mg / L, or 0 mg / L, and fractions thereof, such as 3.25 mg / L, 2.5 mg / L, 1.75 mg / L, 1.5 mg / L, 1.2 2 5 mg / L, 0.9 mg / L, 0.8 mg / L, 0.7 mg / L, 0.6 mg / L, 0. 5 mg / L, etc. In some embodiments, the term “hypoxic” refers to a level, amount, or concentration of oxygen (O ) of about 6.0 mg / L DO or less, 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, and fractions thereof, such as 3.25 mg / L, 2.5 mg / L, 1.75 mg / L, 1.5 mg / L, 1.2 2 5 mg / L, 0.9 mg / L, 0.8 mg / L, 0.7 mg / L, 0.6 mg / L, 0. 5 mg / L, etc. In some embodiments, the term “hypoxic” refers to a level, amount, or concentration of oxygen (O ) of about 6.0 mg / L DO or less, 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, and fractions thereof, such as 5 mg / L, 0.4 mg / L, 0.3 mg / L, 0.2 mg / L and 0.1 mg / L are DO. These exemplary decimals are listed here for illustrative purposes and are in no way intended to be limiting. The oxygen level in a liquid or solution can be reported as a percentage of air saturation or a percentage of oxygen saturation (the ratio of the dissolved oxygen (O 2 ) concentration in the solution to the maximum amount of dissolved oxygen in a solution at a constant temperature, pressure, and salt concentration under a stable equilibrium). A solution that contains no oxygen producers or consumers and is well oxygenated (e.g., a mixed and / or stirred solution) is 100% air saturated. In some embodiments, the term "hypoxic" refers to 40% or less air saturation, 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%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, and any and all fractional increases 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%, 0.5%, 0.44%, 0.3%, 0.25%, 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 40% (including 0% and 40%), e.g., 0 - 5%, 0.05 - 0.1%, 0.1 - 1%, 1 - 5%, 5 - 10%, 10 - 15%, 15 - 20%, 20 - 25%, 25 - 30%, 30 - 35%, 35 - 40%, etc.). 0.1 to 0.2%, 0.1 to 0.5%, 0.5 to 2.0%, 0 to 10%, 5 to 10%, 1 0 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, etc.) are included. The exemplary decimals and ranges listed here are for illustrative purposes only and are in no way meant to be limiting In some embodiments, the term "hypoxic" means an oxygen saturation of 9% or less such as, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0% O 2 saturation and all and any fractional increases thereof (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%, 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% (including 0% and 9%) (e.g , 0 to 5%, 0.05 to 0.1%, 0.1 to 0.2%, 0.1 to 0.5%, 0.5 to 2. 0%, 0 to 8%, 5 to 7%, 0.3 to 4.2% O 2 etc.) are included. The exemplary decimals and ranges listed here are for illustrative purposes only and are in no way meant to be limiting .

[0076] [Table A] Intestinal oxygen pressure

Table 2

[0077] In some embodiments, the promoter described herein is directly or indirectly affected by the conditions in a culture vessel (e.g., a flask or fermenter or other suitable culture vessel ) Induced, and in the culture vessel, the strain is grown or maintained prior to in vivo administration. In non-limiting examples of such conditions provided during culturing of the strain prior to in vivo administration include low oxygen, anaerobic, microaerophilic, 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, the conditions within the culture vessel are set to 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" means a genetic sequence, e.g., a nucleic acid sequence. A gene, gene sequence, or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. A gene, gene sequence, or genetic sequence is meant to include a sequence encoding a protein or polypeptide, and also means to include genetic sequences that do not encode a protein or polypeptide, e.g., regulatory sequences, leader sequences, signal sequences, or other non-protein-coding sequences.

[0079] "Microorganism" refers to a microscopic, typically single-celled, ultramicroscopic ​​​​of (submicroscopic), or ultramicroscopic pic) sized organisms or microorganisms. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, certain algae, and protozoa. In some embodiments, the microorganism is engineered to produce one or more therapeutic molecules or proteins for one or more purposes (a "engineered microorganism"). In certain embodiments, the microorganism is engineered to take up and catabolize specific metabolites or other compounds from its environment, such as the gastrointestinal tract. In certain embodiments, the microorganism is engineered to synthesize and release specific beneficial metabolites or other compounds (synthetic or naturally occurring) into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered

[0080] "Non-pathogenic bacteria" refers to bacteria that are unable to cause disease or adverse reactions in a host. In some embodiments, the non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, the non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, the non-pathogenic bacteria are commensal bacteria that are present in the normal microbiota of the gastrointestinal tract. Examples of non-pathogenic bacteria include, but are not limited to, the genera Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium Enterococcus, Escherichia, Lactobacillus, Lactococcus ​Actococcus), Saccharomyces, and Strepto coccus, for example, Bacillus coagulans (Bacillus coagulans), Bacillus subtilis, Bacteroides fragilis (Bacteroides fragilis), Bacteroides sub tilis (Bacteroides subtilis), Bacteroides thetaiota omicron (Bacteroides thetaiotaomicron), Bifido bacterium bifidum (Bifidobacterium bifidum), Bifido bacterium infantis (Bifidobacterium infan tis), Bifidobacterium lactis (Bifidobacterium la ctis), Bifidobacterium longum (Bifidobacterium lo ngum), Clostridium butyricum (Clostridium butyric um), Enterococcus faecium , Escherichia coli, Lactobacillus acidophilus (L actobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus casei (L actobacillus casei), Lactobacillus johnsonii (Lactob acillus johnsonii), Lactobacillus paracasei (Lactoba cillus paracasei), Lactobacillus plantarum (Lactoba cillus plantarum), Lactobacillus reuteri (Lactobaci L. reuteri), Lactobacillus rhamnosus s rhamnosus), Lactococcus lactis lactis), and Saccharomyces boulardii are included (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,797 ; U.S. Patent No. 5,589,168; U.S. Patent No. 7,731,976). Natural pathogenic bacteria may be genetically engineered to reduce or eliminate pathogenicity.

[0081] "Probiotics" is used to refer to live, non - pathogenic microorganisms, such as bacteria, that can confer health benefits on a host organism containing an appropriate amount of the microorganisms. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and / or subtypes of non - pathogenic bacteria are currently recognized as probiotics. Examples of probiotic bacteria include, but are not limited to, the genus Bifidobacteria, the genus Escherichia, the genus Lactobacillus, and the genus Saccharomyces, such as Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia 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. 7,731,976). 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797; U.S. ​​​​​(Patent No. 6,835,376). Probiotics are variants or mutations of bacteria. strains (Arthur et al., 2012; Cuevas-Ramos et al., 201 0 years; Olier et al., 2012; Nougayrede et al., 2006). Non-pathogenic Bacteria can 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 used to enhance or improve probiotic properties. It may also be genetically engineered to be

[0082] As used herein, a "stably maintained" or "stable" bacterium is a non- Bacterial host cells carrying native genetic material, e.g. genes encoding effector molecules. The term is used to refer to a method for producing a gene that contains non-native genetic material and in which the non-native genetic material is retained, expressed, and / or propagated. As described above, it is either integrated into the host genome or expressed as a self-replicating extrachromosomal plasmid. Stable bacteria can be grown in vitro, e.g., in culture medium, and / or in A stable bacterium can survive and / or grow in vivo, e.g., in the gastrointestinal tract. For example, a stable bacterium can be The plasmid or chromosome carrying the PAL gene is stably maintained in the host cell. Even genetically modified bacteria containing genes encoding vector molecules (e.g., PAL) Preferably, the effector is thereby expressed in a host cell, and the host cell is and / or capable of surviving and / or proliferating in vivo. The copy number affects the stability of expression of non-native genetic material, e.g., the PAL gene. In some embodiments, the copy number is determined by the presence of non-native genetic material, such as a PAL gene or It affects the expression level of the PAH gene.

[0083] As used herein, the terms "modulate" and "treat" and their etymological equivalents refer to the amelioration of a disease, disorder, and / or condition, or at least one distinguishable symptom thereof. In another embodiment, "modulate" and "treat" refer to the amelioration of at least one measurable physical parameter, which may not necessarily be distinguishable 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 distinguishable symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to retarding or reversing the progression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition may include alleviating or eliminating the relevant symptoms without necessarily including the eradication of the underlying disease. For example, phenylketonuria is caused by a congenital genetic mutation for which there is no known cure. Phenylketonuria may also be secondary to other conditions, such as liver disease. Treating phenylketonuria may include alleviating or eliminating excessive phenylalanine and / or related symptoms and does not necessarily include the eradication of the underlying disease. As used herein, the terms "prevent" and their etymological equivalents refer to delaying the onset or preventing a given disease, disorder, and / or condition or at least one distinguishable symptom thereof from occurring. 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 distinguishable symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to retarding or reversing the progression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition may include alleviating or eliminating the relevant symptoms without necessarily including the eradication of the underlying disease. For example, phenylketonuria is caused by a congenital genetic mutation for which there is no known cure. Phenylketonuria may also be secondary to other conditions, such as liver disease. Treating phenylketonuria may include alleviating or eliminating excessive phenylalanine and / or related symptoms and does not necessarily include the eradication of the underlying disease. As used herein, the terms "prevent" and their etymological equivalents refer to delaying the onset or preventing a given disease, disorder, and / or condition or at least one distinguishable symptom thereof from occurring. 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 distinguishable symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, "modulate" and "treat" refer to retarding or reversing the progression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition may include alleviating or eliminating the relevant symptoms without necessarily including the eradication of the underlying disease. For example, phenylketonuria is caused by a congenital genetic mutation for which there is no known cure. Phenylketonuria may also be secondary to other conditions, such as liver disease. Treating phenylketonuria may include alleviating or eliminating excessive phenylalanine and / or related symptoms and does not necessarily include the eradication of the underlying disease. As used herein, the terms "prevent" and their etymological equivalents refer to delaying the onset or preventing a given disease, disorder, and / or condition or Reducing the risk of suffering from such diseases, disorders, and / or conditions related symptoms refers to.

[0084] Those in need of treatment may include individuals already having a specific medical disease and those at risk of having the disease or ultimately being susceptible to the disease. The need for treatment is evaluated, 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 of acceptance of treatment of a subject having the disease.

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

[0086] The terms "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 the organism and does not inactivate the biological activity and properties of the administered bacterial compound. Adjuvants are included in these terms.

[0087] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the 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 glycol, and surfactants such as polysorbate 20.

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

[0089] As used herein, the term "antibody" or "antibodies" means to encompass all variations of antibodies and fragments thereof having one or more specific binding specificities. Thus, the term "antibody" or "antibodies" includes full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (ScFv, camelids), Fab, Fab', multimeric versions of these fragments (e.g., F(ab')2), single-domain antibodies (sdAB, VH fragments), heavy-chain antibodies (HCAb), nanobodies, diabodies, and minibodies. An antibody can have two or more binding specificities, e.g., bispecificity. The term "antibody" also means to include so-called antibody mimetics. Antibody mimetics can specifically bind to an antigen but can be single amino acid chain molecules that do not have an antibody-related structure and can be small molecules (e.g., 3 - 30 kDa). Antibody mimetics include affibody (Z domain of protein A) molecules, affilin (γ-B crystallin), ubiquitin, affimer (cystatin), affitin (Sac7d) (from Sulfolobus acidocaldarius), alphabody (triple helix coiled coil), anticalin (Antic H H ​​​​​​​​​​​​​Alin (lipocalin), Avimer (domains of various membrane receptors), DARPin (ankyrin repeat motif), Fynomer (SH3 domain of Fyn) Kunitz domain peptide (Kunitz domain of various protease inhibitors), ecallantide (Kalbitor), and monobody are included, but not limited thereto. In certain embodiments, the term "antibody" or "antibodies" means to refer to single-chain antibodies (plural optional), single-domain antibodies (plural optional), and camel antibodies (plural optional). The usefulness of antibodies in the treatment of cancer and additional anti-cancer antibodies can be found, for example, in Scott et al., "Antibody Therapy for Cancer, Nature Reviews Cancer, April 2012, Volume 12", which is incorporated herein by reference in its entirety. "Single-chain antibody" or "single-chain antibodies (plural optional)" typically refers to a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and optionally a linker or peptide containing a disulfide bond or the like. Single-chain antibodies lack the constant Fc region found in conventional antibodies. In some embodiments, the single-chain antibody is a naturally occurring single-chain antibody, such as a camel antibody. In some embodiments, the single-chain antibody is a synthetic, engineered, or modified single-chain antibody. In some embodiments, the single-chain antibody can retain substantially the same antigen specificity as the original immunoglobulin despite the addition of a linker and the removal of the constant region. In some

[0090] aspects, the single-chain antibody can be a "scFv antibody", which is described, for example, in U.S. Patent No. 4,946,778, the content of which is incorporated herein by reference in its entirety. ​​​​​​​​As described, optionally linked with a short linker peptide of 10 to about 25 amino acids refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (without the constant region). The Fv fragment is the smallest fragment that retains the binding site of the antibody, and its binding site can, in some embodiments, 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 V L sequences of the scFv can be connected via the N-terminus of VH connected to the C-terminus of VL, or via the C-terminus of VH connected to the N-terminus of VL. The ScFv fragment is an independent folding entity that can be fused, without distinction, 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 linker can be glycine-rich (providing flexibility) and serine or also threonine-rich (increasing solubility). Short linkers can prevent the association of the two domains and result in multimers (diabodies, tribodies, etc.). Long linkers can result in proteolysis or weak domain association (described by Voelkel et al., 2011 ). Linkers of 15 to 20 amino acids or 18 to 20 amino acids in length are most frequently used. Further non-limiting examples of linkers, including other flexible linkers, are described by Chi et al., 2013 (Adv Drug Deliv Rev. October 15, 2013 ; 65(10):1357-1369, Fusion Protein Linker s: Property, Design and Functionality), the content of which is hereby incorporated by reference in its entirety. Flexible linkers can cause proteolysis or weak domain association (described by Voelkel et al., 2011 ). Linkers of 15 to 20 amino acids or 18 to 20 amino acids in length are most frequently used. Further non-limiting examples of linkers, including other flexible linkers, are described by Chi et al., 2013 (Adv Drug Deliv Rev. October 15, 2013 ; 65(10):1357-1369, Fusion Protein Linker s: Property, Design and Functionality), the content of which is hereby incorporated by reference in its entirety. Flexible linkers are described, and the content thereof is hereby incorporated by reference in its entirety herein. Flexible linkers It also contains small or polar amino acids such as glycine and serine, but 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, KESGSSEQ LAQFRSLD and EGKSSGSESKST, (Gly)8, and Gly- and Ser-rich flexible linkers such as GSAGSAAGSGEF. As used herein, "single-chain antibody" includes single-domain antibodies including camel 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 having a camelid scaffold derived from camels, llamas, or alpacas. Camelids produce functional antibodies lacking a light chain. 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 the six CDRs in classical antigen-binding molecules (Fab) or single-chain variable fragments (scFv). Camelid antibodies can achieve binding affinities comparable to those of conventional antibodies. Antibodies based on the camelid scaffold can be produced using methods well known in the art. Cartilaginous fish also have heavy-chain antibodies (Ig ​​​​​​​​​​​​​​​​There is a NAR (nanoantibody, "novel antigen receptor of immunoglobulin"), and from this, a single-domain antibody called a VNAR fragment can be obtained. Alternatively, the dimeric variable domains derived from IgG from humans or mice can be split into monomers. Nanobodies are single-chain antibodies derived from the light chain. The term "single-chain antibody" also refers to antibody mimetics. In some embodiments, the antibodies expressed by the engineered microorganisms are bispecific. In certain embodiments, the bispecific antibody molecule comprises an scFv or a fragment thereof, having binding specificity for a first epitope, and the scFv or a fragment thereof has binding specificity for a second epitope. Antigen-binding fragments or antibody moieties include bispecific scFv (diabodies), bispecific scFv antibodies in which the antibody molecule recognizes two different epitopes, single-binding domains (dAbs), and minibodies.

[0091] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural environment. A specific level of purification is not required. Recombinant-produced polypeptides and proteins expressed in host cells including, but not limited to, bacterial cells or mammalian cells can be separated, fractionated, or partially or The monomeric single-chain diabody (scDb) can be readily assembled in bacteria and mammalian cells and exhibits improved stability under physiological conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001) 14(10):815 - 823 (describing optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies)).

[0092] fully purified by any suitable technique. ​​​​​​​​​​​For the purposes of the present invention, is considered to be isolated, in the same way as a substantially purified natural or recombinant polypeptide. A recombinant peptide, polypeptide or protein is produced by recombinant DNA technology, i.e., a peptide, polypeptide or protein produced from a cell, microorganism or mammal transformed with an exogenous recombinant DNA expression construct encoding the polypeptide. Most bacterial cultures express proteins or peptides that are usually not glycosylated. Fragments, derivatives of the aforementioned polypeptides, analogs or variants, and any combinations thereof are also included as polypeptides. The terms "fragment", "variant", "derivative" and "analog" include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide, and any polypeptide that retains 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 bioactive fragments 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 include conservative or non-conservative amino acid substitutions, deletions or additions. As used herein, the term "polypeptide" includes "polypeptide" (singular) and "polypeptides" (plural), and refers to a molecule consisting of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). "Polypeptide" also includes molecules having a branched structure, as well as multimers, dimers, trimers, etc. formed by non-covalent associations of polypeptides. "Derivative" refers to a polypeptide that has been modified by chemical modification of one or more amino acid residues. Modifications include, but are not limited to, acetylation, acylation, phosphorylation, glycosylation, sulfation, methylation, hydroxylation, amidation, alkylation, carboxylation,

[0093] As used herein, the term "polypeptide" includes "polypeptide" (singular) and "polypeptides" (plural), and refers to a molecule consisting of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). "Polypeptide" also includes molecules having a branched structure, as well as multimers, dimers, trimers, etc. formed by non-covalent associations of polypeptides. The term refers to any chain(s) of two or more amino acids and does not necessarily refer to a product of a specific length. Thus, "peptide", "dipeptide", "tripeptide", " oligopeptide", "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids ( s) is included within the definition of "polypeptide", and the term "polypeptide" may be used in place of any of these terms 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" also, without limitation, refers to the products of post-expression modification of polypeptides, including glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be of natural biological origin or produced by recombinant techniques. In other embodiments, the polypeptide is produced by a genetically engineered bacterium or virus of the present invention. The polypeptides of the present invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more amino acids, 100 or more, 200 or more, 500 or more, 1000 or more, or 2,000 or more amino acids. A polypeptide may have a defined three-dimensional structure but does not necessarily have such a structure. A polypeptide having a defined three-dimensional structure is referred to as folded, and a polypeptide that does not possess a defined three-dimensional structure but has a number of different ... ... ... ... ... ... ... ... ... ... A polypeptide that can adopt the conformation is said to be unfolded. The term "pe ptide" or "polypeptide" may refer to an amino acid sequence corresponding to a protein or a part of a protein, or may refer to an amino acid sequence corresponding to a non-protein sequence, such as a regulatory peptide sequence, a leader peptide sequence, a signal peptide sequence, a linker peptide sequence, and sequences selected from other peptide sequences.

[0094] Polypeptides also include fusion proteins. As used herein, the term "var iant" 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 result from well-known in vitro recombinant techniques. A fusion pro tein may have a structural function similar (but not necessarily to the same degree) to the individual original proteins that are components of the fusion protein, and / or a regulatory function similar (but not necessarily to the same degree), and / or a biochemical function similar (but not necessarily to the same degree) and / or immunological activity (but not necessarily to the same degree). "Derivative" includes, but is not limited to, peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. The "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide with the sequence of the second peptide. An amino acid of one peptide is the same or similar if it is identical or When the amino acid substitution is a conservative substitution, it is similar to the corresponding amino acid of the second peptide. The conser vative substitutions include those described in Dayhoff, M.O., ed., The Atlas of Pro tein Sequence and Structure 5, National B iomedical Research Foundation, Washington , D.C. (1978), and Argos, EMBO J. 8 (1989), 77 9-785. For example, amino acids belonging to one of the following groups represent conservative changes or conservative substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Me t, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu. As used herein, the term "sufficiently similar" means that the first and second

[0095] amino acid sequences have a sufficient or minimal number of identical or equivalent amino acid residues such that they have a common structural domain and / or a common functional activity, when compared to the second amino acid sequence. For example, 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 common structural domain ​​ An amino acid sequence containing is defined herein as being sufficiently similar. Preferably a variant is sufficiently similar to the amino acid sequence of the peptide of the present invention. Such a va riant generally retains the functional activity of the peptide of the present invention. A variant is one or more amino acid deletions, additions, and / or substitutions, and peptides having different amino acid sequences from natural and wild-type pep tides, respectively. They may be naturally occurring variants and artificially designed variants.

[0096] As used herein, the terms "linker", "linker peptide" or "pep tidyl linker" or "linker" refer to a synthetic or unnatural or natural amino acid sequence that connects or couples two polypeptide sequences, for example, couples two polypeptide domains. As used herein, the term "synthetic" refers to an amino acid sequence that does not occur naturally. Exemplary linkers are described herein . Further exemplary linkers are provided in U.S. Patent Application Publication No. 20140079701 , the content of which is incorporated herein by reference in its entirety.

[0097] As used herein, the term "codon-optimized sequence" refers to a sequence that has been modified from an existing coding sequence or, for example, designed to improve the translation of a transcribed RNA molecule transcribed from a coding sequence in a host cell or organism, or to improve the transcription of a coding sequence. Codon optimization includes, but is not limited to, selecting codons for a coding sequence to match the codon preferences of the expression host organism. This includes selecting codons for the coding sequence to A process is included. The term "codon-optimized" refers to modifying codons in the gene or coding region of a nucleic acid molecule to reflect the typical codon usage frequency of a host organism without changing the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or two or more, or a significant 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 that has been modified from an existing coding sequence or is designed to improve translation in an expression host cell or organism of a transcribed RNA molecule transcribed from a coding sequence, or to improve transcription of the coding sequence. In some embodiments, improving transcription and / or translation includes increasing the level of transcription and / or translation. In some embodiments, improving transcription and / or translation includes decreasing the level of transcription and / or translation. In some embodiments, codon optimization is used to finely tune the expression level from a target construct. Codon optimization includes, but is not limited to, a process that includes selecting codons of a coding sequence to match the codon preference of an expression host organism. Many organisms exhibit a bias or preference for using specific codons to encode the insertion of specific amino acids into a growing polypeptide chain. Codon preference or codon bias, the differences in codon usage frequency among organisms, is permitted by the degeneracy of the genetic code and is more fully documented among many organisms. Codon bias often correlates with the translation efficiency of messenger RNA (mRNA), and the translation efficiency of that messenger RNA (mRNA) is, without changing the polypeptide encoded by the nucleic acid molecule, to reflect the typical codon usage frequency of the host organism. This refers to modifying codons in the gene or coding region of the nucleic acid molecule. Such optimization includes replacing at least one, or two or more, or a significant 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 that has been modified from an existing coding sequence or, for example, is designed to improve translation in an expression host cell or organism of a transcribed RNA molecule transcribed from a coding sequence, or to improve transcription of the coding sequence. In some embodiments, improving transcription and / or translation includes increasing the level of transcription and / or translation. In some embodiments, improving transcription and / or translation includes decreasing the level of transcription and / or translation. In some embodiments, codon optimization is used to finely tune the expression level from a target construct. Codon optimization includes, but is not limited to, a process that includes selecting codons of a coding sequence to match the codon preference of an expression host organism. Many organisms exhibit a bias or preference for using specific codons to encode the insertion of specific amino acids into a growing polypeptide chain. Codon preference or codon bias, the differences in codon usage frequency among organisms, is permitted by the degeneracy of the genetic code and is more fully documented among many organisms. Codon bias often correlates with the translation efficiency of messenger RNA (mRNA), and the translation efficiency of that messenger RNA (mRNA) is, The preference or bias of codons, the differences in codon usage frequency among organisms, is permitted by the degeneracy of the genetic code and is more fully documented among many organisms. Codon bias often correlates with the translation efficiency of messenger RNA (m RNA), and the translation efficiency of that messenger RNA (mRNA) is, Rather, it is thought to depend on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The preference for selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be adjusted for optimal gene expression in a given organism based on codon optimization.

[0098] As used herein, the terms "secretion system" or "secreted protein" refer to natural or non-natural secretion mechanisms that can secrete or export a protein of interest or a therapeutic protein from a microorganism, such as the 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 comprises an "export 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 can remove this tag before secreting the protein of interest from the engineered bacteria. For example, in type V auto-secretion-mediated secretion, the N-terminal peptide export tag is the native S ​ Removed during the transfer of the "passenger" peptide from the cytoplasm to the periplasmic compartment by the ec system. Furthermore, when the auto-secretor translocates across the outer membrane, the C-terminal secretion tag is removed by autocatalytic or protease-catalyzed cleavage, releasing the protein of interest into the extracellular environment.

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

[0100] Effectors also include immune checkpoint inhibitors. An "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 the activation or function of T cells 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 co-stimulatory or inhibitory interactions of T cell responses and regulate and maintain self-tolerance and physiological immune responses. For example, systemic immunotherapy using CTLA-4 inhibitors can alter immune regulation, induce immune dysfunction, and result in opportunistic autoimmune disorders (see, e.g., Kong et al., 2014).

[0101] As used herein, a genetically engineered microorganism, e.g., a bacterium or phage or molecule that has been "engineered to harm" 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 as compared to a control (e.g., an untreated control under the same conditions or an unmodified microorganism of the same subtype).

[0102] As used herein, a bacterium or phage molecule that has been "engineered to activate" or "stimulate" a genetically engineered microorganism, e.g., 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 as 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 a virus that infects bacteria and replicates within the bacteria. As used herein, "phage" or bacteriophage generically refers to prophage, lysogenic phage, dormant phage, temperate phage, intact phage, defective phage, cryptic phage, and satellite phage, phage tail bacteriocins, teriosins, as well as gene delivery agents.

[0104] As used herein, the term "prophage" means the genomic material of a bacteriophage that is integrated into a replicon of a host cell and replicates with the host. Proph When the phage is specifically activated, it can produce phages. In some cases, the prophage cannot produce phages or has not produced phages (i.e., defective prophage or cryptic prophage). In some cases, the prophage also means satellite phage. The terms "prophage" and "endo- genic phage" are used interchangeably herein.

[0105] As used herein, the terms "temperate phage" or "temperate bacterio- phage" or "prophage" are used interchangeably to refer to phages that are present within the DNA of a bacterial host and replicate 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 as 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 in its natural state with respect to another defined element. 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 native or natural state functions as a precursor strain for an engineered strain. As a result, the same one or more endogenous phages ​​​​​​​ or a prophage may also be present in the genetically engineered bacterium if, for example, the progenitor or parental strain contains such an endogenous phage or prophage. Thus, the genetically engineered bacterium contains a prophage in its natural state (wherein the phage is a defined element in its natural state).

[0107] The term "endogenous phage" or "endogenous prophage" also refers to phages present in the natural state of the bacterium (and its parental strain).

[0108] As used herein, the terms "phage knockout" or "inactivated phage" refer to a phage that can no longer produce and / or package phage particles, or is modified to produce fewer phage particles than the wild-type phage sequence. In some embodiments, an inactivated phage or phage knockout refers to the inactivation of a temperate phage in the 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, and inversions at one or more positions within the phage genome, e.g., 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 the same. For example, isogenic mutants typically have one that has one or more known genes or proteins Refers to two strains that are identical except for containing defined mutations in quality. Thus a phage-free or phageless strain has the corresponding isogenic type strain containing a prophage, and the prophage can be induced to release phage particles from the bacterial cell. Can.

[0110] As used herein, the adjectives "phage-free", "phage free" and "phageless" are used interchangeably to characterize bacteria or strains containing one or more prophages (one or more of which are modified). The modification can result in the loss of the ability of the prophage to be induced or the ability to release phage particles. Alternatively, the modification can result in lower efficiency or lower frequency of induction, or lower efficiency or lower frequency of phage release, compared to the isogenic strain without the modification. The ability to induce and release phage can be measured using a plaque assay as described herein. Compared to the isogenic strain without the modification, it can result in lower efficiency or lower frequency of induction, or lower efficiency or lower frequency of phage release. The ability to induce and release phage can be measured using a plaque assay as described herein. The ability to induce and release phage can be measured using a plaque assay as described herein. The ability to induce and release phage can 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. In which the phage genes necessary for lysis are not expressed. Refers to bacteria 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 a part of the life cycle of a lysogenic prophage in which lytic phage genes are activated, phage particles are produced, and lysis occurs. In which lytic phage genes are activated, phage particles are produced, and lysis occurs. Refers to a 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. Direct, i.e., the induced prophage starts the production and release of phage particles. . Induction is often stimulated by damage to the bacterial DNA and may or may not involve excision of the prophage from the bacterial chromosome.

[0114] In some embodiments, the genetically engineered bacteria are useful for the treatment, prevention, management, reduction of severity, improvement, or cure of 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 oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathies, bullous pemphigoid, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackievirus myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler 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 syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain - Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch - Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4 - related sclerosing disorders, immunomodulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert - Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Ménière's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren ulcer, Muscular Harbermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry - Romberg syndrome, neuralgic amyotrophy (Parsonnage - Turner syndrome) P pediatric A autoimmune N europsychiat ric D disorders A associated with S treptococ cus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry - Romberg syndrome, neuralgic amyotrophy (Parsonnage - Turner syndrome) , pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia , POEMS syndrome, polyarteritis nodosa, type I, type II, and type III polyglandular autoimmune syndrome, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, erythroid leukemia, Raynaud's phenomenon, reactive arthritis, reflex sympathetic neurodystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, episcleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis , vesiculobullous dermatosis, vitiligo, and Wegener's granulomatosis are included, but are not limited thereto . In some embodiments, the disorder is graft-versus-host disease.

[0115] In some embodiments, the disease is a metabolic disease. As used herein, "metabolic disease" includes type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedl syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Orr Bright 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 Loss; Src homology 2B1 (SH2B1) deficiency; prohormone convertase 1 / 3 deficiency; me lanocortin-4 receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary mal formation, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; fragile X syndrome ; Borjeson-Forsmann-Lehmann syndrome; Alström syndrome ; Cohen syndrome; and ulnar-mammary syndrome, including but not limited to these.

[0116] In some embodiments, the disorder is cancer. "Cancer" or "cancerous" is used to refer to a physiological state characterized by unregulated cell proliferation. In some embodiments, the cancer refers to a tumor. "Tumor" is used to refer to the growth or proliferation of any neoplastic cells, or any preneoplastic or cancerous cells or tissues. A tumor can be either malignant or benign. Types of cancer include adrenal cancer, adrenocortical cancer, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytoma, brain stem glioma, craniopharyngioma, ependymoma), bronchial tumor, central nervous system tumor, breast cancer, Castleman 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, heart cancer, Kaposi 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., Epstein-Barr virus-related lymphoma, Burkitt lymphoma, cutaneous T- cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma), malignant mesothelioma, multiple myeloma, myelodysplasia ; myelodysplastic syndrome ; myelodysplastic syndromes Syndromes, nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovary 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, thymic cancer, thyroid cancer, rare pediatric cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor are included but are not limited thereto. Side effects of cancer treatment include 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, thirst, dysphagia, edema, fatigue, hair loss (alopecia), infections, infertility, lymph edema, stomatitis, nausea, pain, peripheral neuropathy, dental caries, urinary tract infections, and / or problems with memory and concentration may be included but are not limited thereto (National Cancer Institute, USA). In some embodiments, the disorder is a hyperammonemia 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 disease, phenylketonuria.

[0118] Exemplary circuits for the treatment, prevention, reduction of severity, management, improvement, and cure of one or more of the above disorders are described in co-owned international patent applications PCT / US2016 / 34200 (filing date May 25, 2016), PCT / US2017 / 013072 (filing date January 11, 2017), PCT / US2017 / 016603 (filing date February 3, 2017), PCT / US2017 / 016609 (filing date: February 4, 2016), PCT / US2017 / 017563 (filing date: February 10, 2017), PCT / US2017 / 017552( filing date: February 10, 2017), PCT / US2016 / 044922 (filing date 2016 July 29), PCT / US2016 / 049781 (filing date: August 31, 2016) , PCT / US2016 / 37098 (filing date: June 10, 2016), PCT / US2 016 / 069052 (filing date: December 28, 2016), PCT / US2016 / 32 562 (filing date: May 13, 2016), PCT / US2016 / 062369 (filing date November 16, 2016), and as described in PCT / US2017 / 013072 which are hereby incorporated by reference in their entirety.

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

[0120] The phrase "and / or" when used between elements in a listing is intended to mean either (1) only a single listed element is present, or (2) more than one of the listed elements 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 phrase "and / or" can be used interchangeably with "at least one" or "one or more" of the elements in the listing.

[0121] (bacteria) In some embodiments, the bacteria disclosed herein are capable of expressing an endogenous phage genome. In some embodiments, the bacterium comprises one or more mutations or modifications that enhance its natural In some embodiments, the phage includes a bacteriophage in its natural or native 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 a defective prophage. In some embodiments, the one or more mutations cause the phage to enter the lytic cycle. In some embodiments, the one or more mutations prevent the lytic cycle. influence the ability of a given population to undergo a lytic phase, e.g. In some embodiments, the one or more mutations reduce the frequency or number of bacteria in the In some embodiments, one or more of the phages are Natural mutations can alter (e.g., increase or decrease) the fitness of bacteria. In some embodiments, one or more mutations alter effector function (e.g., increase or decrease 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 are produced or In any of these embodiments, the bacteria is Alternatively, in any of these embodiments, the bacteria may be in its natural state. It can be further genetically engineered to contain a gene sequence encoding one or more effector molecules. It can be.

[0122] In some embodiments, bacteria containing one or more mutant phages can be used as a bacterial chassis, to which a gene circuit is added or modified. In some embodiments, the bacteria are non - pathogenic bacteria. In some embodiments, the bacteria

[0123] are symbiotic bacteria. In some embodiments, the bacteria are probiotic bacteria. In some embodiments, the bacteria are natural pathogenic bacteria that are 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, the genera Bacillus, Bacteroides, Bifidobacterium, Brevibacterium, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, for example, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei Kasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus lactis, and Saccharomyces boulardii are included therein. In certain embodiments, the bacterium is 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 la ctis, selected from the group consisting of.

[0124] In some embodiments, the bacterium is one of the most characterized probiotics evolved Gram-negative bacteria of the Enterobacteriaceae family, Escherichia coli strain Nissle 1917 (E. coli Nissle) (Ukena et al., 2007). The strain is characterized by its complete harmlessness (S chultz, 2008), has GRAS (Generally Recognized As Safe eneral g ly ecognized r s a afe)) status (Reister s et al., 2014, underlined by the author). By genome sequencing, it was confirmed that Escherichia coli Niss le lacks important virulence factors (e.g., Escherichia coli α-hemolysin, P-pilus adhesion factor) (Schultz, 2008). Furthermore, Escherichia coli Nissle does not possess pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is non-invasive, and is not uropathic (Schultz, 2008). It has been shown not to be of original nature (Sonnenborn et al., 2009). As early as in 19 In 17, Escherichia coli Nissle was packaged into a medicinal capsule called Mutaflor for therapeutic use. It is generally recognized that the therapeutic effect and safety of Escherichia coli Nissle have been proven in a persuasive manner (Ukena et al., 2007 ). ). )

[0125] In some embodiments, the bacteria of the present disclosure are tumor-targeting bacteria. The tumor-targeting bacteria are described in the international patent application PCT / US2017 / 013072 filed on January 11, 2017, published as WO 2017 / 123675, the content of which is incorporated herein by reference in its entirety ). ).

[0126] One of ordinary skill in the art will understand 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 introduced into each other. For example, the PAL gene derived 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 and that can be used in accordance with the present disclosure contains one or more mutations or modifications to one or more endogenous prophage genomes. In some embodiments, the modification to the endogenous prophage genome includes one or more deletions, insertions, substitutions, or inversions within the prophage genome, or combinations thereof. In some embodiments, the modification is to the prophage genome ). ). ). ). One or more deletions within the phage. 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 insertions include a genetic sequence encoding the antibiotic cassette described herein. In some embodiments, one or more genes within the phage genome are replaced with alternative genetic sequences. In some embodiments, the replacement includes a genetic sequence encoding the 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. The unmodified Escherichia coli Nissle and the genetically engineered bacteria of the present invention can be destroyed, for example, by defense factors in the digestive 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, the residence time is calculated for a human subject. The in vivo residence time can be calculated for the genetically engineered bacteria of the present invention (see, for example, FIG. 68 of WO2017087580, the entire content of which is incorporated herein by reference). In some embodiments, the genetically engineered bacteria include a gene encoding PAL, and the PAL gene is operably linked to a promoter that is directly or indirectly inducible.

[0128]

[0129] ​​​​​​​​​​​​​​​In some embodiments, the bacterium comprises a non-natural 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 PAH, and the PAH gene is operably linked to a directly or indirectly inducible promoter. In some embodiments, the bacterium comprises a non-natural 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 LAAD, and the LAAD gene is operably linked to a directly or indirectly inducible promoter. In some embodiments, the bacterium comprises a non-natural 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 is phenyl comprises an additional copy of a native gene encoding an alanine transporter, and phenylala nine transporter gene is operably linked to a promoter that is directly or indirectly inducible. In alternative embodiments, the bacterium comprises a gene encoding a non-native phenylalanine transporter, and the phenylalanine transporter gene is operably linked to a promoter that is directly or indirectly inducible. 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 the expression of PheP and PAL and / or PAH and / or LAAD. In some embodiments, the promoter that controls the expression of PheP is different from the promoter that controls the expression of PAL and / or PAH and / or LAAD. In some embodiments, the promoter that controls the expression of PheP is one or more of the promoters disclosed herein. In some embodiments, the promoter operably linked to PAL, PAH, LAAD, and / or pheP is directly or indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the mammalian gastrointestinal tract. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the mammalian small intestine. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the mammalian large intestine. In some embodiments, the promoter that controls the expression of PheP is different from the promoter that controls the expression of PAL and / or PAH and / or LAAD. In some embodiments, the promoter that controls the expression of PheP is one or more of the promoters disclosed herein. In some embodiments, the promoter that controls the expression of PheP is different from the promoter that controls the expression of PAL and / or PAH and / or LAAD. In some embodiments, the promoter that controls the expression of PheP is one or more of the promoters disclosed herein. In some embodiments, the promoter that controls the expression of PheP is different from the promoter that controls the expression of PAL and / or PAH and / or LAAD. In some embodiments, the promoter that controls the expression of PheP is one or more of the promoters disclosed herein. In some embodiments, the promoter that controls the expression of PheP is one or more of the promoters disclosed herein.

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

[0134] In some embodiments, the promoter is directly or indirectly induced prior to in vivo administration. Such conditions provided during the cultivation of the strain prior to in vivo administration include, but are not limited to, hypoxic, anaerobic, microaerobic or aerobic conditions, other defined oxygen levels (such as those exemplified below), the presence of arabinose, IPTG, rhamnose or the presence of other chemical and / or nutritional inducers described herein or known in the art. In some embodiments, the conditions in the culture vessel are set to specific oxygen levels, 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% -

[0135] (bacteriophage) In some embodiments, the bacteria of the present invention contain one or more lysogenic phages, dormant phages, temperate phages, intact phages, defective phages, cryptic phages or satellite phages, or bacteriocins / phage tails or gene delivery agents in their natural state. In some embodiments, the prophage or bacteriophage is present in all isolates of the particular bacteria of interest. In some embodiments, the bacteria are probiotic It is a genetically engineered derivative of a parental strain containing one or more cterio phages. Therefore, such bacteria of the present invention may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types. In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). The bacteriophage genome size ranges 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., Pasteurel In any of the embodiments described herein, the bacteria may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types. In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). It is a genetically engineered derivative of a parental strain containing one or more cterio phages. Therefore, such bacteria of the present invention may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types. In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). The bacteriophage genome size ranges 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., Pasteurel In any of the embodiments described herein, the bacteria may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types.

[0136] In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). The bacteriophage genome size ranges 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., Pasteurel In any of the embodiments described herein, the bacteria may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types. In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). It is a genetically engineered derivative of a parental strain containing one or more cterio phages. Therefore, such bacteria of the present invention may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types. In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). The bacteriophage genome size ranges 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., Pasteurel

[0137] The bacteriophage genome size ranges 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., Pasteurel In some embodiments, the modification or mutation alters the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation alters the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not alter the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not alter the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it). In any of the embodiments described herein, the bacteria may be in their natural state or may be further genetically modified to contain a circuit for the expression or production of one or more effector molecules. In any of the embodiments described herein, the bacteria contain one or more modifications or mutations 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 types. from the la phage F108), to the genome of Bacillus megaterium phage G of about about 500 kbp (Hatfull and Hendrix ; Bacteriophages and their Genomes, Curr Opin Virol. October 1, 2011; 1(4): 298 - 303, and the references therein). The phage genome can encode from less than 10 genes to hundreds of genes. There are also some examples of phages integrated into bacterial plasmids, but temperate phages or prophages are usually integrated into the chromosome of the bacterial host (Waldor MK, Friedman DI, Adhya S eds Phage s Their Role in Bacterial Pathogenesis a nd Biotechnology. Washington, DC: ASM Press; 2005 , pp. 37 - 54, Little, Lysogeny, Prophage Ind uction, and Lysogenic Conversion). In some cases the phage is always at the same position within the bacterial host chromosome, and this position is specific to each phage, i.e., different phages are at different positions. Other phages are more permissive in that they can be integrated at a number of different locations.

[0138] Accordingly, the bacteria of the present disclosure contain one or more phage genomes of variable length. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 1 bp to 10 kb. In one embodiment, the bacteria contain a bacteriophage genome with a length of at least about 1 bp to 1 It contains a bacteriophage genome in the range of 0 kb. In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 10 kb to 20 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 20 kb to 30 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 30 kb to 40 kb. In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 30 kb to 40 kb. In one embodiment , the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 30 kb to 40 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 40 kb to 50 kb. In one embodiment , the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 40 kb to 50 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 50 kb to 60 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 60 kb to 70 kb. In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 70 kb to 80 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 80 kb to 90 kb. In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 90 kb to 100 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 100 kb to 1 20 kb. In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 120 kb to 140 kb . In one embodiment, the genetically engineered bacteria contains a bacteriophage genome with a length in the range of at least about 140 kb to 1 It includes a bacteriophage genome within a range of 60 kb. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 160 kb to 180 kb In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 180 kb to 2 00 kb. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 200 kb to 180 kb In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 160 kb to 2 50 kb. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 250 kb to 300 kb In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 300 kb to 3 50 kb. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 350 kb to 400 kb In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 400 kb to 5 00 kb. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length in the range of at least about 500 kb to 1000 kb phage genome. In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length exceeding 1000 kb In one embodiment, the genetically engineered bacteria contain a bacteriophage genome with a length exceeding 1000 kb.

[0139] In some embodiments, the bacteria of the present invention contain one or more phage genomes containing one or more genes encoding one or more polypeptides. In one embodiment, the genetic engineering In some embodiments, the bacteria of the present invention contain one or more phage genomes containing one or more genes encoding one or more polypeptides. In one embodiment, the genetic engineering The engineered bacteria contain a bacteriophage genome containing at least about 1 to 5 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 5 to 10 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 10 to 15 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 15 to 20 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 20 to 25 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 25 to 30 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 30 to 35 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 35 to 40 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 40 to 45 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 45 to 50 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 50 to 55 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 55 to 60 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 60 to 65 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 65 to 70 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 70 to 75 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 75 to 80 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 80 to 85 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 85 to 90 genes. In one embodiment, the engineered bacteria contain a bacteriophage genome containing at least about 90 to 95 genes. comprises a bacteriophage genome containing the son. In one embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 70 - 75 genes. One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 75 - 80 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least also about 80 - 85 genes. In one embodiment , the genetically engineered bacteria comprises a bacteriophage genome containing at least about 85 - 90 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 90 - 9 5 genes. In one embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 95 - 100 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 100 - 115 genes. In one embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 115 - 120 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 120 - 125 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 125 - 130 genes . In one embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 130 - 135 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 135 - 140 genes One embodiment, the genetically engineered bacteria comprises a bacteriophage genome containing at least about 135 - 140 genes. One In an embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 140-145 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 145-150 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 150-160 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 160-170 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 170-180 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 180-190 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 190-200 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing at least about 200-300 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing more than about 300 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing more than about 300 genes. In one embodiment, the genetically engineered bacterium contains a bacteriophage genome containing more than about 300 genes. In some embodiments, the phage is always or almost always at the same location or position within the chromosome of a particular species of bacterial host. In some embodiments, the phage is found integrated at different positions within the host chromosome of a particular species. In some embodiments, the phage is on a plasmid.

[0140] The presence of the prophage sequence is also in bacteria that are not present in isogenic strains without the phage. In some embodiments, the phage is always or almost always at the same location or position within the chromosome of a particular species of bacterial host. In some embodiments, the phage is found integrated at different positions within the host chromosome of a particular species. In some embodiments, the phage is on a plasmid.

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

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

[0143] In some embodiments, the prophage can be a defective prophage or a cryptic prophage. A defective prophage can no longer undergo the lytic cycle. A cryptic prophage may not be able to undergo the lytic cycle or may never have undergone the lytic cycle. Functional studies of the entire prophage repertoire in bacterial genomes suggest that most prophages are defective at some level: excision, virion formation, lysis, or infectivity (Bobay et al., 2014). Defective prophages or cryptic prophages result from decay by mutation and / or loss of one or more genes essential for the lytic cycle over thousands of bacterial replication cycles. cycles. ​​​​​​​​can reach high levels of abundance in bacteria (Bobay et al., Pervasive dome stication of defective prophages by bact eria, Proc Natl Acad Sci USA). Notably, defective prophages also encode genes for proteins with homologous recombination functions, additional mechanisms for preventing infection, or bacteriocins (which may be useful in competition for nutrients, for example, by inhibiting the growth of other neighboring bacterial species) that can confer adaptive or advantageous functions to the host. For example, some defective proph ages have been characterized in Escherichia coli K-12 (e.g., Rac, e14, DLP12, and QIN) and also Bacillus subtilis (e.g., 186 and SKIN) (Casjens, 2001, and references therein). Each of these phages carries several functional genes. For example, Rac encodes the RecE homologous recombination sys tem.

[0144] Thus, in some embodiments, the bacteria include one or more defective or cryptic pro phages. In some embodiments, the prophage genes confer a homologous recombination function. In some embodiments, the prophage genes confer a function for preventing further infection. In some embodiments, the prophage genes confer bacteriocin. In some embodiments, the phage genes increase carbon utilization, improve resistance to osmotic, oxidative, and acid stress to increase growth under various conditions (), enhance phosphorus and nitrogen utilization, or affect biofilm formation. This promotes growth under adverse conditions.

[0145] In some embodiments, the bacterium contains one or more satellite phage genomes. The satellite phage is a functional phage in other respects without its own structural protein genes, and is configured with a genome for encapsulation by the structural proteins of another specific phage (Six and Klug, Bacteriophage P4: a satellite virus depending on a helper such as prophage P2, Virology, Volume 51, Number 2, February 1 973, pages 327 - 344). Thus, in some embodiments, the bacterium contains a phage genome without its own structural genes.

[0146] In some embodiments, the bacterium contains one or more tellurions. Many bacteria, both gram - positive and gram - negative, produce various particles similar to phage tails that are functional without phage heads (referred to as tellurions), many of which have been shown to have bacteriocin properties (reviewed in Ghequire and Mot, The Tailoci n Tale: Peeling off Phage; Trends in Mic robiology, October 2015, Volume 23, Number 10). Phage - tail - like bacteriocins are classified into two different families: contractile phage - tail - like (R - type) and non - contractile but flexible ones (F - type). Thus, in some embodiments, the bacterium contains one or more tellurions that confer bacteriocins or other beneficial properties.

[0147] In some embodiments, the bacterium comprises one or more gene transfer agents. The gene transfer agent (G TA) is a phage-like element encoded by some bacterial genomes. The GTA is similar to a phage but lacks the characteristic abilities that define a typical phage and packages random fragments of host cell DN A and horizontally transfers them to other bacteria of the same species (La ng et al., Gene transfer agents: phage-like ele ments of genetic exchange, Nat Rev Micro biol. June 11, 2012;10(7):472-482). Thus, DNA can replace the resident cognate chromosomal region by homologous recombination. However, since most of the particles do not have the genes encoding GTA, these particles cannot replicate as a virus.

[0148] In some embodiments, the bacterium comprises one or more filamentous virions. The filamentous virion is incorporated as a dsDNA prophage (Marvin DA et al., Structur e and assembly of filamentous bacterioph ages, Prog Biophys Mol Biol. April 2014;114(2 ):80-122).

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

[0150] In some embodiments, the modifications or mutations substantially do not affect the effector function, and the effector function is substantially the same as that of the isogenic strain without the modification or mutation. Table H provides a non-limiting example of probiotic bacteria and a list of the number of potential bacteriophages contained in the bacterial genome determined by Phaster scoring. Table I provides a list of Clostridium strains and potential phage genomes. Phaster is a web server for bioinformatics identification of phage sequences in organisms (http: / / phaster.ca ​ / )。Phaster scoring is described in detail in phaster.ca and Zhou et al. (“PH AST: A Fast Phage Search tool”, Nucl. Acids Res. (2011) 39 (suppl 2): W347 - W352) and Arn dt et al. (Arndt et al. (2016) PHASTER: a better, faste r 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, dubious, or incomplete) in a given bacterial genomic sequence. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In method 2, if the number of specific phage organisms identified by Phaster in the given bacterial genomic sequence is more than 50% of the total number of CDSs in that region, that phage organism is considered a major potential phage for that region; the percentage of the total number of that phage organism in this table in the total number of proteins in that region is calculated and then multiplied by 1 00; the percentage of the length of that phage organism in the length of that region is calculated and then multiplied by 50 (considering the encapsulation ability of the phage head). In method 3, specific phage - related keywords (“capsid”, “head”, “integrase” In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. In the first method, if the number of specific phage organisms identified by Phaster in that region is 100% or more of the total number of CDSs in that region, a total score of 150 is marked for that region. If less than 100%, methods 2 and 3 are used. ", "base", "tail", "fiber", "coat", "transposase", "portal" where any of (e.g., "terminase", "protease" or "lysine") is present case, increase the score by 10 for each keyword found. If the size of the region is more than 30Kb the score is increased by 10. If at least 40 proteins are present in the region, the score is increased by 10. If all of the phage-related proteins and virtual proteins exceed 70% of the total number of proteins in the region, the score is increased by 10 Compare the total score of Method 2 with the total score of Method 3, and select the larger one as the total score of the region. If the total score of the region is less than 70, it is marked as incomplete, if it is between 7 0 and 90, it is marked as suspicious, and if it exceeds 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 within an existing prophage or bacteriophage genome. These modifications change the properties or behavior of the prophage. In some embodiments, the modification or mutation prevents the prophage from entering or completing the lytic process For example. In some embodiments, the modification or mutation prevents the phage from infecting other bacteria of the same or different types.

[0154] In some embodiments, the modification or mutation changes the fitness of the bacterial host (e.g., decreases or increases it). In some embodiments, the modification or mutation changes, for example, the desired effector function of the genetically engineered bacteria (e.g., decreases or increases it). In some embodiments, the modification or mutation does not change the fitness of the bacterial host (e.g., does not decrease or increase it). In some embodiments, the modification or mutation does not change, for example, the desired effector function of the genetically engineered bacteria (e.g., does not decrease or increase it).

[0155] In some embodiments, the modification or mutation improves phenylalanine consumption. In some embodiments, phenylalanine consumption remains at a level similar to that observed in isogenic strains containing unmodified phages. In some embodiments, the modification or mutation has substantially no effect on bacterial fitness, and the bacterial fitness is substantially the same as that of an isogenic strain without the modification or

[0156] In some embodiments, the bacteria have 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 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 19, at least about 19 to 20, at least about 2 0 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 3 0, at least about 30 to 31, at least about 31 to 32, at least about 32 to 33 pieces, 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 39, at least about 39 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 46 to 47, at least about 47 to 48, at least about 48 to 49, at least about 49 to 50, at least about 50 to 51, at least about 51 to 52, at least about 52 to 53, at least 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 6 0 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 7 0, 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 an effector function, e.g., phenylalanine consumption. In some embodiments, an effector function, e.g., phenylalanine consumption, remains the same as that observed in isogenic strains containing unmodified phage. In some embodiments, the modification or mutation has substantially no effect on bacterial fitness, and the 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 is in an isogenic strain without phage modification Compared with [comparison object], the entry or completion of the prophage lysis process is reduced 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 100 - 100 0 fold. In some embodiments, the modification or mutation completely prevents the entry or completion of the prophage lysis process.

[0159] In some embodiments, the modification or mutation reduces the entry or completion of the prophage lysis process by at least about 1% - 1 0%, 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 6 0% - 70%, at least about 70% - 80%, at least about 80% - 90%, or at least about 90% - 100% compared to the isogenic strain without phage modification.

[0160] In some embodiments, the modification or mutation prevents the phage, whether of the same or different types, from infecting other bacteria 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 the isogenic strain without phage modification. In some embodiments, the modification or mutation prevents the phage, whether of the same or different types, from infecting other bacteria. ​ Completely prevent infection by other bacteria. In some embodiments, the modification or mutation is at least about 1% - 10%, at least about 10% - 20%, at least about 20% - 3 0%, 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 8 0% - 90%, or at least about 90% - 100% to prevent the phage from infecting other bacteria of the same or different types.

[0161] In some embodiments, the modification or mutation is at least about 1 - 2 times, at least about 2 - 3 times, at least about 3 - 4 times, at least about 4 - 5 times, at least about 5 - 10 times, at least about 10 - 100 times, or at least about 100 - 1000 times compared to the isogenic strain without phage modification, to change the fitness of the bacterial host (e.g., decrease or increase). In some embodiments, the modification or mutation is, for example, at least about 1% - 10%, at least about 10% - 20% compared to the isogenic strain without phage modification, at least about 20% - 30%, at least about 30% - 40%, at least about 40% - 5 0%, 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% to change the fitness of the bacterial host (e.g., decrease or increase). In some embodiments, the modification or mutation is, for example, at least about 1 - 2 times, at least about 2 - 3 times, at least about 3 - 4 times to change the desired effector function of the genetically engineered bacteria, for example, at least about 1 - 2 times, at least about 2 - 3 times, at least about 3

[0162] In some embodiments, the modification or mutation is, for example, to change the desired effector function of the genetically engineered bacteria by at least about 1 - 2 times, at least about 2 - 3 times, at least about 3 - 4 times, at least about 4 - 5 times, at least about 5 - 10 times, at least about 10 - 100 times, or at least about 100 - 1000 times. ​​~4-fold, at least about 4- to 5-fold, at least about 5- to 10-fold, at least about 10- to 100-fold or changed (e.g., decreased or increased) by at least about 100- to 1000-fold In some embodiments, the modification or mutation is, for example, the desired effector function of the genetically engineered bacterium, compared to the isogenic strain without phage modification, 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 also about 60% to 70%, at least about 70% to 80%, at least about 80% to 90%, or at least about 90% to 100% changed (e.g., decreased or increased).

[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 can 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 within 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 the reversal of two or more nucleotides within a polynucleotide sequence In some embodiments, the mutation comprises one or more inversions within the phage genome sequence. As used herein Refers to the case where a segment containing a nucleotide is reversed end-to-end within a polynucleotide sequence. In some embodiments, the inversion can be governed by a specific flippase. Exemplary circuits including multiple levels of control are illustrated herein, the entire content of which is incorporated by reference herein and is also described in co-owned and pending PCT application PCT / US2016 / 03 9434.

[0164] In some embodiments, the modification in the phage genome is a combination of two or more of insertions, deletions, substitutions, or inversions within one or more phage genome genes.

[0165] In any of the embodiments described herein, the modification can result in one or more frameshift mutations in one or more genes within the phage genome. As used herein, a frameshift mutation (also referred to as a framing error or reading frame shift) 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 3. The earlier in the sequence the deletion or insertion occurs, the more the protein changes. In any of the embodiments described herein, the modification can result in one or more missense mutations in one or more genes within the phage genome. As used herein, a missense mutation refers to the 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 non-functional. In any of the embodiments described herein, the modification is a phage This may result in one or more nonsense mutations in one or more genes in the genome. As used herein, a nonsense mutation is any of the 20 mutations specified by the genetic code. The sense codon corresponding to one of the amino acids is changed to a chain termination codon, thereby This refers to a mutation in which the target polypeptide is truncated.

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

[0167] (mutation) In some embodiments, the one or more mutations are at least about 1-500 base pairs long. In some embodiments, the one or more mutations are at least about 5 In some embodiments, the phage genome comprises one or more of: The mutants contain at least about 1000-2000 base pairs of the phage genome. In an embodiment, the one or more mutations are at least about 1000-2000 base pairs in a fragment of the It contains a digenome. In some embodiments, one or more mutations contain a phage genome of at least about 200 to 3000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 3000 to 4000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 4000 to 5000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 5000 to 6000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 6000 to 7000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 7000 to 8000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 8000 to 9000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 9000 to 10000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 10000 to 15000 base pairs. In some embodiments, one or more mutations contain a phage genome of at least about 1000 0 to 15000 base pairs, at least about 15000 to 20000 base pairs of phage genome, at least about 20000 to 25000 base pairs of phage genome, at least about 25000 to 30000 base pairs of phage genome, at least about 30000 to 35 000 base pairs of phage genome, at least about 35000 to 40000 base pairs of phage genome, at least about 45000 to 50000 base pairs of phage genome, at least about 5 0000 to 55000 base pairs of phage genome, at least about 55000 to 60000 base pairs of phage genome, at least about 60000 to 65000 base pairs of phage genome, at least about 65000 to 70000 base pairs of phage genome, at least about 70000 to 75000 base pairs of phage genome, at least about 75000 to 80000 base pairs of phage genome, at least about 80000 to 85000 base pairs of phage genome, at least about 85000 to 90000 base pairs of phage genome, at least about 90000 to 95000 base pairs of phage genome, at least about 95000 to 100000 base pairs of phage genome. a phage genome of 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, at least about 70,000 to 75,000 base pairs of phage genome, at least about 75,000 to 80,000 base pairs of ph age genome, at least about 85,000 to 90,000 base pairs of phage genome, at least about 90,000 to 95,000 base pairs of phage genome, at least about 95,000 to 100 000 base pairs of phage genome, at least about 100,000 to 110,000 base pairs of pha ge genome, at least about 110,000 to 120,000 base pairs of phage genome, at least about 120,000 to 130,000 base pairs of phage genome, at least about 130,000 to 140,000 base pairs of phage genome, at least about 140,000 to 150,000 base pairs of phage genome, at least about 150,000 to 200,000 base pairs of phage genome , or includes a phage genome of more than at least about 200,000 base pairs. In a specific embodiment , the phage genome of 9687 base pairs is mutated. In some embodiments, the mutated nucleotides are scattered. In some embodiments, the mutated nucleotides are continuous. In some embodiments, at least about 0.1 to 1% of the phage genome , 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 1 4 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 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 2 4%, 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% is mutated. In some embodiments, at least about 30 to 4 0% of the phage genome is mutated. In some embodiments, at least about 40 to 50% of the phage genome is mutated. In some embodiments, at least about 50 to 60% of the phage genome is mutated. In some embodiments, at least about 60 to 70% of the phage genome is mutated. In some embodiments, at least about 70 to 80% of the phage genome is mutated. In some embodiments, the phage genome has at least about 80 to 90% mutated. In some embodiments, the phage genome has at least about 90 to 100% 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, 3 9, 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, 7 9, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 10 4, 105, 106, 107, 108, 109, 110, 111, 112, 113, 11 4, 115, 116, 117, 118, 119, or 120 genes are mutated . In some embodiments, 13 genes are completely or partially mutated . In one embodiment, 74 genes are completely or partially mutated.

[0169] In some embodiments, 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% - 1 2%, 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 1 7% - 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% - 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 also 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 also 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 also about 80% - 81%, at least about 81% - 82%, at least about 82% - 83%, at least 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% is completely or partially mutated.

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

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

[0172] In some embodiments, the mutation is located within or encompasses one or more genes encoding lytic genes. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more proteases or lysins. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more toxins. In some embodiments the mutation is located within or encompasses one or more genes encoding one or more antibiotic resistance-related proteins. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more phage translation-related proteins. In some embodiments, 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, one or more mutations are located within or encompass one or more genes encoding head structure polypeptides. In some embodiments one or more mutations are located within or encompass one or more genes encoding tail structure polypeptides. In some embodiments, one or more mutations are located within or encompass one or more genes encoding collar structure polypeptides. In some embodiments, one or more mutations are It is located within or encompasses one or more genes. In some embodiments, one or more mutations are located within or encompass one or more genes encoding the polypeptide of the coat structure . In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more basal proteins. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more proteins required for bacteriophage construction. In some embodiments, the mutation is located within or encompasses 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 genes encoding one or more transposases. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more primases. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more transposases . In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in replication or translation. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more primases. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more primases . In some embodiments, the mutation is one or more is located within or encompasses one or more genes encoding tRNA-related proteins. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in phage insertion. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in phage insertion. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in phage insertion. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in attachment sites. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in attachment sites. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more polypeptides involved in packaging. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more terminases. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more host genes. In some embodiments, the mutation is located within or encompasses one or more genes encoding one or more host genes.

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

[0175] In some embodiments, the mutation is located within or encompasses one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. , phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutation is located within or encompasses two genes 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 mutation is located within or encompasses one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. , phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutation is located within or encompasses two genes 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 mutation is located within or encompasses one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. , phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutation is located within or encompasses three genes 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 mutation is located within or encompasses one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. , phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutation is located within or encompasses four genes 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 mutation is located within or encompasses one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. , phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutation is located within or encompasses two genes 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 mutation is located within or encompasses one gene encoding a polypeptide involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. , phage packaging recombination, replication or translation, phage insertion, and combinations thereof. In some embodiments, the mutation is located within or encompasses five genes 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 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 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 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 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 eleven genes encoding polypeptides involved in cell lysis, phage structure, phage Located within or encompassing them. In some embodiments, the mutation is Involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or Translation, phage insertion, and polypeptides involved in combinations thereof, encoding Located within or encompassing 12 genes. In some embodiments, The mutation is located within or encompasses 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 mutation is located within or encompasses 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 mutation is located within or encompasses 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 mutation is located within or encompasses at least about 16, 17, 18, Involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. Located within or encompassing polypeptides involved in combinations thereof. In some embodiments, the mutation is located within or encompasses 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 mutation is located within or encompasses 15 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. Involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. Located within or encompassing 15 genes encoding polypeptides involved in combinations thereof. In some embodiments, the mutation is located within or encompasses 15 genes encoding polypeptides involved in cell lysis, phage structure, phage assembly, phage packaging recombination, replication or translation, phage insertion, and combinations thereof. Involve...

Claims

1. A bacterium comprising an Escherichia coli Nissle (ECOLIN) phage 3 genome for use in treating hyperphenylalaninemia or a disease associated with hyperphenylalaninemia in a subject, comprising: 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. A bacterium for use as described in claim 1, wherein the one or more mutations reduce or prevent the release of phage particles from the bacterium compared to the same bacterium not having the one or more targeted mutations in the one or more phage genomes.

3. The bacterium for use according to claim 1, wherein the bacterium is a probiotic bacterium selected from the group consisting of Bacteroides, Bifidobacterium, Clostridium, Escherichia, Escherichia coli strain Nissle, Lactobacillus, and Lactococcus.

4. The bacterium for use according to claim 3, wherein the bacterium is Escherichia coli strain Nissle.

5. The bacterium for use according to 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. A bacterium for use as described in claim 5, wherein the deletion includes sequence number 130.

7. A bacterium for use as described in claim 5, wherein the deletion consists of sequence number 130.

8. A bacterium for use as described in claim 1, comprising one or more additional genetic modifications.

9. A bacterium for use as described in claim 8, wherein the one or more additional genetic modifications include one or more mutations in one or more endogenous genes.

10. A bacterium for use as described in claim 8, wherein the one or more additional genetic modifications include the addition of one or more non-native genes.

11. A bacterium for use as described in claim 1, wherein the bacterium further comprises antibiotic resistance.

12. The bacterium for use as described in claim 1, wherein the bacterium is present in a composition formulated for oral administration.

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