Engineered bacteria to reduce hyperphenylalaninemia

Abstract

To provide genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with hyperphenylalaninemia.SOLUTION: A genetically engineered bacterium comprises: a) one or more gene(s) encoding a phenylalanine ammonia lyase (PAL), wherein the gene(s) encoding a PAL is operably linked to an inducible promoter that is not associated with the PAL gene in nature; b) one or more gene(s) encoding a phenylalanine transporter, wherein the gene(s) encoding the phenylalanine transporter is operably linked to an inducible promoter that is not associated with the phenylalanine transporter gene in nature; and c) one or more gene(s) encoding a mutated fumarate and nitrate reductase (FNR) wherein the gene(s) encoding mutated FNR is operably linked to an inducible promoter that is not associated with the FNR gene in nature.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 256,05, filed November 16, 2015. No. 2, and PCT application number PCT / US2016 / filed on May 13, 2016 No. 032562, the contents of which are incorporated herein by reference in their entirety. is incorporated herein. [Background technology]

[0002] The present disclosure relates to hyperphenylalaninemia In certain aspects, the present disclosure relates to compositions and methods of treatment for reducing mammalian Genetically engineered bacteria capable of reducing hyperphenylalaninemia in animals In certain aspects, the compositions and methods disclosed herein are high in phenylalanine. The compounds may be used to treat diseases associated with phenylketonuria, such as phenylketonuria.

[0003] Phenylanine is an essential amino acid found primarily in food proteins. Typically, a small amount is utilized for protein synthesis, and the remainder is used to synthesize phenylalanine hydroxylase. in an enzymatic pathway requiring tetrahydrobiopterin and the cofactor tetrahydrobiopterin It is hydroxylated to tyrosine. Hyperphenylalaninemia is toxic and can cause brain damage. It is a group of diseases associated with excessive levels of phenylalanine that can cause Intercurrent hyperphenylalaninemia is caused by mutations in the PAH gene and / or blockade of cofactor metabolism. It is caused by a deficiency in PAH activity resulting from steroid therapy.

[0004] Phenylketonuria (PKU) is a hyperlipidemia caused by mutations in the PAH gene. A severe form of phenylalaninemia. PKU is the most common inborn error of metabolism worldwide. It is an autosomal recessive genetic disorder that occurs in approximately 1 in 3,000 births. There are over 400 different P AH gene mutations have been identified (Hoeks et al., 2009). Accumulation of alanine (Phe) can cause serious damage to the central nervous system in children and adults. If left untreated in newborns, PKU can cause irreversible brain damage. Treatment for PKU currently involves completely eliminating phenylalanine from the diet. Most natural sources of protein contain phenylalanine, an essential amino acid required for growth. This is because PKU patients need just enough phenylalanine to grow. Along with amino acid supplements, medical foods and PHE-free protein supplements are also available. This diet is difficult for patients and affects their quality of life. Give.

[0005] As mentioned above, current PKU therapy involves a significantly modified diet consisting of protein restriction. Treatment from birth generally reduces brain damage and mental retardation (H oeks et al., 2009; Sarkissian et al., 1999). However, Protein-restricted diets must be carefully monitored to ensure that essential amino acids and vitamins are not being lost. Furthermore, the use of low-protein foods is However, this presents a challenge as it is more costly than their unmodified counterparts. (Vockley et al., 2014).

[0006] Growth retardation is common in children with PKU who are kept on a low-phenylalanine diet (Dobbelaere et al., 2003). In adults, osteoporosis, maternal PKU , and new problems such as vitamin deficiencies may arise (Hoeks et al., 200 9). Excessive levels of phenylalanine in the blood that can freely cross the blood-brain barrier Nin may cause neurological disorders, behavioral disorders (e.g., irritability, fatigue), and / or physical symptoms ( For example, it may cause convulsions, skin rashes, and moldy odors. International Guidelines recommends lifelong dietary phenylalanine restriction, but this is difficult and It is widely considered to be unrealistic (Sarkissian et al., 1999) and Ongoing efforts to overcome the biggest challenge to living with KU - lifelong low pH e-dietary adherence is required” (Macleod et al., 2010).

[0007] In a subset of patients with residual PAH activity, the cofactor tetrahydrobiopterin Oral administration of THB (also known as BH4, Kuvan, or sapropterin) It can be used in conjunction with dietary restrictions to lower serum phenylalanine levels. However, cofactor therapy is costly and only suitable for mild forms of phenylketonuria. Kuvan's annual cost, for example, can be as high as $57,000 per patient. Additionally, Kuvan side effects include gastritis and severe allergic reactions ( For example, these may include wheezing, lightheadedness, nausea, and flushed skin.

[0008] The enzyme phenylalanine ammonia-lyase (PAL) converts phenylalanine to a non-toxic Unlike PAHs, they can be metabolized to trans ammonia and trans cinnamic acid. PAL does not require THB cofactor activity to metabolize phenylalanine. Oral enzyme therapy using PAL is being studied, but "PAL is available in sufficient quantities at an affordable cost." Since it was not available in humans and even animals, studies were not continued." (Sarkis A pegylated form of recombinant PAL (PEG-PAL) has also been used as an injectable However, most patients receiving PEG-PAL Any subject may suffer from injection site reactions and / or develop antibodies to the therapeutic enzyme. (Longo et al., 2014). Therefore, hyperphenylalanine Effective, reliable, and / or long-term treatment for blood-related disorders There is a significant unmet need for Summary of the Invention [Means for solving the problem]

[0009] In some embodiments, the present disclosure provides a method for the preparation of a phenylalanine metabolic enzyme (PME). In some embodiments, the present disclosure provides genetically engineered bacteria that express a nucleotide sequence encoding ... Phenylanine ammonia-lyase and / or phenylalanine hydroxylase and / or encodes and expresses L-amino acid deaminase, hyperphenylalaninemia The present invention provides a genetically engineered bacterium capable of reducing

[0010] The phenylalanine ammonia-lyase (PAL) enzyme converts phenylalanine to a non-toxic Bell's ammonia and trans-cinnamic acid Unlike PAH, PAL can metabolize phenylalanine. L-amino acid deaminase (LAAD) does not require THB cofactor activity. It catalyzes the oxidative deamination of guaranidin, producing phenylpyruvate and trace amounts of ammonia. Phenylpyruvic acid (PPA) is used in medicines, foods, and PPA is widely used in the pharmaceutical and chemical industries, and is the basis for many chiral pharmaceutical and food additives. Starting material for the synthesis of D-phenylalanine, a crude intermediate in the manufacture of Therefore, LAAD has been studied from the perspective of industrial PPA production (Hou et al. , 2015, Appl Microbiol Biotechnol. 2015 10 Mon;99(20):8391~402; “Production of phenyl pyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus m irabilis:comparison of enzymatic and who le-cell biotransformation approaches”). centre Phenylpyruvate is unable to cross the blood-brain barrier (Steele, Fed Pro c.June 1986;45(7):2060~4; “Blood-brain bar rier transport of the alpha-keto acid an This conversion contributes to the neurological phenotype of PKU. This indicates that it is useful for controlling

[0011] In some embodiments, the present disclosure provides a method for the detection of phenylalanine-metabolizing enzymes (PMEs) encoding phenylalanine metabolic enzymes (PMEs). In some embodiments, the present disclosure provides a genetically engineered bacterium that expresses a phenotype. Phenylalanine ammonia lyase (PAL) and / or phenylalanine hydroxylase Encoding L-amino acid deaminase (PAH) and / or L-amino acid deaminase (L-AAD) and providing a genetically engineered bacterium capable of expressing and reducing hyperphenylalaninemia. do.

[0012] In some embodiments, the genetically engineered bacteria contain unnatural phenylalanine ammonium. It contains a gene encoding phenylalanine allyase (PAL), which converts phenylalanine to phenylalanine in mammals. In some embodiments, the engineered bacteria can be processed and reduced. , further comprising a gene encoding a phenylalanine transporter, e.g., PheP In some embodiments, the engineered bacteria also contain a gene encoding L-AAD. Genetically engineered bacteria may also be used for biosafety and / or biocontainment purposes. contain, for example, death switches, gene guard systems and / or auxotrophy-related In some embodiments, the engineered bacteria may contain one or more gene sequences that The expression of any of these gene sequences may be mediated by the methods disclosed herein. The promoter system can be regulated using a variety of promoter systems, such as any promoter system currently available. The promoter system may use the same promoter to regulate one or more different genes. This may involve using different promoters to regulate different genes. This may involve using copies of the gene that are different from each other and / or modulating the expression of different genes. This may include the use of different promoters in combination to achieve gene expression. Flexibility is achieved by using different regulatory or promoter systems to control expression. (e.g., the ability to differentially regulate gene expression under different environmental conditions and / or This provides the ability to "fine tune" gene expression. Any or all of these factors may affect gene expression and / or bacterial growth. It can help to optimize.

[0013] In certain embodiments, the genetically engineered bacteria are non-pathogenic and are phenylalanine toxins. In certain embodiments, phenylalanine may be introduced into the digestive tract to reduce levels of phenylalanine. Phenylanine ammonia-lyase and / or phenylalanine hydroxylase and / or L-amino acid deaminase or L-amino acid deaminase is stably produced by genetically engineered bacteria, and / or genetically engineered bacteria in vivo and / or in vitro In certain embodiments, the genetically engineered bacteria are stably maintained at 100°C. The phenylalanine transporter gene was further transduced to increase their uptake of phenylalanine. The present invention also includes pharmaceutical compositions comprising the genetically engineered bacteria, and high phenylalanine compounds. Methods for modulating and treating disorders associated with raninemia are also provided.

[0014] The present invention also provides a method for measuring and monitoring phenylalanine ammonia-lyase activity. and the use of genetically engineered bacteria expressing phenylalanine ammonia-lyase. Methods for measuring and monitoring therapeutic activity are provided. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows synthetic biology for treating phenylketonuria (PKU) and disorders characterized by hyperphenylalaninemia. [Figure 2A] FIG. 1 shows a schematic of phenylalanine hydroxylase action in phenylketonuria (PKU). [Figure 2B] FIG. 1 is a diagram showing an outline of phenylalanine hydroxylase (PAH) action. [Figure 2C] FIG. 1 is a diagram showing an outline of phenylalanine ammonia-lyase (PAL) action. [Figure 2D] FIG. 1 shows a schematic diagram of L-amino acid deaminase (LAAD; e.g., from Proteus mirabilis) action. [Figure 3] FIG. 1 shows exemplary synthetic biologies for treating phenylketonuria (PKU) and disorders characterized by hyperphenylalaninemia. [Figure 4] FIG. 1 shows exemplary synthetic biologies for treating phenylketonuria (PKU) and disorders characterized by hyperphenylalaninemia. [Figure 5] FIG. 1 shows exemplary synthetic biologies for treating phenylketonuria (PKU) and disorders characterized by hyperphenylalaninemia. [Figure 6] FIG. 1 shows the genetic organization of an example of a construct containing a PAL3-encoding gene and a Tet promoter sequence in a high-copy plasmid, such as those contained in SYN-PKU202 and SYN-PKU303. [Figure 7] FIG. 1 shows the genetic organization of an example of a construct containing a PAL3-encoding gene and an FNR promoter sequence in a low-copy plasmid, such as those contained in SYN-PKU304, SYN-PKU307, SYN-PKU305, and SYN-PKU306. [Figure 8]FIG. 1 shows the genetic organization of an example of a construct containing a PAL3-encoding gene and a Tet promoter sequence in a low-copy plasmid, such as SYN-PKU302 or SYN-PKU201. [Figure 9] FIG. 1 shows the genetic organization of an example construct, for example, contained in SYN-PKU401, which contains the cloned LAAD gene and Tet repressor gene under the control of a Tet promoter sequence. [Figure 10] FIG. 10 shows a schematic of the construction of a pheP knock-in strain, which uses recombination to insert a second copy of pheP into the Nissle lacZ gene. [Figure 11] FIG. 1 shows the genetic organization of an example of a construct containing a gene encoding PheP, a gene encoding TetR, and a Tet promoter sequence for chromosomal insertion, as contained in, for example, SYN-PKU203, SYN-PKU401, SYN-PKU402, SYN-PKU302, and SYN-PKU303. [Figure 12A] Figure 1 shows the genetic organization of an example construct containing the PAL3 gene cloned under the control of the FNR promoter sequence in a low-copy kanamycin resistance plasmid (pSC101 origin of replication). Under anaerobic and / or hypoxic conditions, PAL3 degrades phenylalanine to the non-toxic trans-cinnamic acid. [Figure 12B] FIG. 1 shows an additional copy of the endogenous E. coli high-affinity phenylalanine transporter, pheP, driven by the PfnrS promoter and inserted into the lacZ locus of the Nissle chromosome. [Figure 13A]

[0014] Figure 1 shows a schematic of a non-limiting embodiment of the present disclosure. It depicts phenylalanine degradation components integrated into the E. coli Nissle chromosome. In some embodiments, an engineered, plasmid-free bacterial strain is used to prevent plasmid conjugation in vivo. In some embodiments, multiple insertions of PAL genes result in increased copy number and / or increased phenylalanine degradation activity. In some embodiments, a copy of the endogenous E. coli high-affinity phenylalanine transporter, pheP, is driven by the PfnrS promoter and inserted into the lacZ locus. [Figure 13B] 1 shows a schematic diagram of one non-limiting embodiment of the present disclosure, in which the E. coli Nissle chromosome is engineered to contain four copies of PfnrS-PAL inserted into four different insertion sites in the genome (malE / K, yicS / nepI, agaI / rsmI, and cea) and one copy of a phenylalanine transporter gene inserted into a different insertion site (lacZ). In this embodiment, the PAL gene is PAL3 obtained from P. luminescens, and the phenylalanine transporter gene is pheP from E. coli. In one embodiment, the strain is SYN-PKU511. [Figure 13C] Schematic diagram of one embodiment of the present disclosure shows an E. coli Nissle chromosome engineered to contain five copies of PAL under the control of an oxygen level-dependent promoter (e.g., PfnrS-PAL3) inserted into different chromosomal integration sites (malE / K, yicS / nepI, malP / T, agaI / rsmI, and cea), and one copy of a phenylalanine transporter gene under the control of an oxygen level-dependent promoter (e.g., PfnrS-pheP) inserted into a different chromosomal integration site (lacZ). The genome is further engineered to contain thyA auxotrophy, with the thyA gene deleted and / or replaced with an unrelated gene, as well as a kanamycin resistance gene. [Figure 14]FIG. 1 shows the genetic organization of an example of a non-limiting construct containing the gene encoding araC, the gene encoding the LAAD of Proteus mirabilis, and an arabinose-inducible promoter (ParaBAD) sequence for chromosomal insertion into the endogenous arabinose operon for chromosomal integration, as contained in, for example, SYN-PKU705. [Figure 15A] Figure 1 shows phenylalanine concentrations in samples containing bacteria expressing PAL1 from a low-copy (LC;SYN-PKU101) or high-copy (HC;SYN-PKU102) plasmid or PAL3 from a low-copy (LC;SYN-PKU201) or high-copy (HC;SYN-PKU202) plasmid induced with anhydrotetracycline (ATC) and then grown in medium supplemented with 4 mM (660,000 ng / mL) phenylalanine. Samples are removed at 0, 4, and 23 hours. Phenylanine concentrations are determined by mass spectrometry. [Figure 15B] Cinnamate levels in samples 4 and 23 hours after induction are shown. In the PAL3-expressing strain, the PAL3 gene is obtained from Photorhabdus luminescens, an enterobacterium in the same taxonomic subphylum as E. coli. [Figure 16A] Phenylanine concentrations in samples containing bacteria expressing PAL1 or PAL3 from low-copy (LC) or high-copy (HC) plasmids or additionally containing one copy of pheP driven by a chromosomally integrated Tet promoter were shown. Bacteria were induced with ATC and then grown to an OD of 2.0 in medium supplemented with 4 mM phenylalanine (660,000 ng / mL). Samples were removed at 0, 2, and 4 hours post-induction, and phenylalanine concentrations were determined by mass spectrometry. Notably, the additional copy of pheP enhanced the degradation of phenylalanine (4 mM) at 4 hours. [Figure 16B]Figure 1 shows cinnamate levels in samples 2 and 4 hours after induction. In some embodiments, cinnamate can be used as a surrogate biomarker for strain activity. Overexpression of PheP improves phenylalanine metabolism in genetically engineered bacteria. The strains analyzed in this data set are SYN-PKU101, SYN-PKU102, SYN-PKU202, SYN-PKU201, SYN-PKU401, SYN-PKU402, SYN-PKU203, SYN-PKU302, and SYN-PKU303. [Figure 17A]

[0023] Figure 1 shows a non-limiting embodiment of a PAL construct under non-inducing conditions. Figure 2 shows relatively low PAL and PheP production by preventing FNR dimerization and activating PAL and / or pheP gene expression under aerobic conditions with oxygen (O). [Figure 17B]

[0023] Figure 1 depicts one non-limiting embodiment of a PAL construct under inducing conditions. Under anaerobic conditions, FNR dimerizes, resulting in upregulation of PAL and PheP production (irregular curves above "PAL" and "pheP") due to induction of PAL and PheP expression by the FNR promoter. Arrows near a rectangle or group of rectangles indicate the promoter responsible for driving transcription of such gene(s) (in the direction indicated by the arrow). Arrows above each rectangle indicate the expression product of each gene. [Figure 18] This figure shows β-galactosidase levels in samples containing bacteria harboring a low-copy plasmid expressing lacZ from an FNR promoter (Pfnr1-5) selected from the FNR promoter examples listed in Table 3. Various FNR promoters were used to generate a library of reporters induced under anaerobic / hypoxic conditions with various expression levels and dynamic ranges. These promoters contained strong ribosome binding sites. Bacterial cultures were grown under aerobic (+O2) or anaerobic (-O2) conditions. Samples were removed after 4 hours, and promoter activity, based on β-galactosidase levels, was analyzed by performing a standard β-galactosidase colorimetric assay. [Figure 19A] Figure 1 shows a schematic representation of the lacZ gene under the control of an example FNR promoter (PfnrS). LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. [Figure 19B] FnrS promoter activity as a function of β-galactosidase activity in SYN-PKU904. SYN-PKU904, a genetically engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values ​​from a standard β-galactosidase colorimetric assay are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins driving high levels of gene expression within 1 hour under anaerobic and / or hypoxic conditions. [Figure 19C] FIG. 1 shows the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen. [Figure 20A] This figure shows the phenylalanine levels produced under aerobic conditions in samples of wild-type Nissle, bacteria containing a low-copy plasmid expressing PAL3 from either the Tet or FNR promoter, or bacteria containing a single copy of pheP driven by the Tet promoter and integrated into the chromosome. Samples were incubated in medium supplemented with ATC and 4 mM phenylalanine (660,000 ng / mL). Samples were removed after 0, 2, 4, and 24 hours. Phenylalanine concentrations were determined by mass spectrometry. [Figure 20B]This figure shows the phenylalanine levels produced under anaerobic and / or hypoxic conditions in samples of wild-type Nissle, bacteria containing low-copy plasmids expressing PAL3 from the Tet or FNR promoters, or bacteria containing a single copy of pheP driven by the Tet promoter and integrated into the chromosome. Samples were incubated in medium supplemented with ATC and 4 mM phenylalanine (660,000 ng / mL). Samples were removed after 0, 2, 4, and 24 hours. Phenylalanine concentrations were determined by mass spectrometry. These data suggest that the FNR-responsive fnrS promoter is as effective at activating PAL3 expression as a tetracycline-inducible promoter under anaerobic conditions. [Figure 21] Phenylanine concentrations in cultures of synthetic probiotic strains with or without an additional chromosomal copy of pheP inserted. After 1.5 hours of growth, cultures were placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. Four hours after induction, bacteria were resuspended in assay buffer containing 4 mM phenylalanine. Aliquots were removed from the cell assay every 30 minutes for 3 hours for phenylalanine quantification by mass spectrometry. The phenylalanine degradation rates of strains containing an additional pheP copy (SYN-PKU304 and SYN-PKU305; left) were higher than those of strains without an additional pheP copy (SYN-PKU308 and SYN-PKU307; right). [Figure 22] FIG. 1 shows trans-cinnamate concentrations (PAL activity) of strains containing single PAL3 insertions at various chromosomal locations. [Figure 23] FIG. 1 shows trans-cinnamate concentrations (PAL activity) of strains containing multiple PAL3 insertions at various chromosomal locations. [Figure 24]This figure shows phenylalanine concentration over time in cultures of the synthetic probiotic strain SYN-PKU511. After 2.5 hours of growth, the cultures were placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. After 3.5 hours of induction in medium containing phenylalanine, whole-cell extracts were prepared every 30 minutes for 3 hours, and phenylalanine was quantified by mass spectrometry. SYN-PKU511 contains five copies of the gene encoding an anaerobically (FNR)-regulated phenylalanine ammonia-lyase (PAL) integrated at five chromosomal locations and a gene encoding an anaerobically regulated high-affinity Phe transporter (pheP) integrated at the lacZ locus. [Figure 25A] This figure shows phenylalanine concentrations in cultures of SYN-PKU401, a synthetic probiotic strain containing a high-copy pUC57-plasmid carrying a LAAD driven by a Tet-inducible promoter. Cells were grown in shake flasks at 37°C and induced with TCA for 2 hours at early log phase. Cells were centrifuged and resuspended in assay buffer containing phenylalanine. Cells were measured at various cell concentrations and oxygen levels. Cells were incubated aerobically in 14 ml culture tubes with shaking at 250 rpm (1 ml), in microaerophilic conditions (1 ml) without shaking in 1.7 ml conical tubes, or anaerobically in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. Aliquots were removed from the cell assay every 30 minutes for 2 hours for phenylalanine quantification by mass spectrometry. Phenylalanine concentrations under aerobic conditions using two cell densities are shown. Figures A and B were repeated under the same experimental conditions. The activity under aerobic conditions is approximately 50 μmol / hr / 1e9 cells. [Figure 25B]This figure shows phenylalanine concentrations in cultures of SYN-PKU401, a synthetic probiotic strain containing a high-copy pUC57-plasmid carrying a LAAD driven by a Tet-inducible promoter. Cells were grown in shake flasks at 37°C and induced with TCA for 2 hours at early log phase. Cells were centrifuged and resuspended in assay buffer containing phenylalanine. Cells were measured at various cell concentrations and oxygen levels. Cells were incubated aerobically (1 ml) in 14 ml culture tubes with shaking at 250 rpm, in microaerophilic conditions (1 ml) in 1.7 ml conical tubes without shaking, or anaerobically in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. Aliquots were removed from the cell assay every 30 minutes for 2 hours for phenylalanine quantification by mass spectrometry. Figures A and B were repeated under the same experimental conditions. Phenylanine concentrations are shown for cells grown aerobically, microaerobically, or anaerobically. [Figure 26A] Phenylanine concentrations before and after feeding in an in vivo mouse model of PKU. At the start of the study, homozygous BTBR-Pahenu2 mice were administered water supplemented with 100 micrograms / mL of ATC and 5% sucrose. Mice were fasted overnight (10 hours) by removing chow, and blood samples were collected the following morning by mandibular bleeding to determine baseline phenylalanine levels. Mice were re-fed chow and, one hour later, were gavaged with 100 microliters (5 x 109 CFU) of bacteria (SYN-PKU302 or control Nissle) and then fed for an additional two hours. Serum phenylalanine concentrations were determined two hours after gavage. [Figure 26B] Figure 1 shows the percent (%) change in blood phenylalanine concentration before and after feeding as a group mean for females or males (p<0.01). [Figure 27A]Figure 1 shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean body weight), mice were orally gavaged with 200 μL of HO (n=30), SYN-PKU901 (n=33), or SYN-PKU303 (n=34). Blood phenylalanine concentrations 2 hours after phenylalanine injection are shown. These data demonstrate that oral administration of the genetically engineered probiotic strain SYN-PKU303 significantly reduces blood phenylalanine levels in mice compared to mice treated with mock (HO) or the parental strain (SYN-PKU901) (*, p<0.05; ***, p<0.001; ****, p<0.00001). SYN-PKU303 can disrupt intestinal recirculation of phenylalanine. [Figure 27B] Figure 1 shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean body weight), mice were orally gavaged with 200 μL of HO (n=30), SYN-PKU901 (n=33), or SYN-PKU303 (n=34). Blood phenylalanine concentrations 4 hours after phenylalanine injection are shown. These data demonstrate that oral administration of the genetically engineered probiotic strain SYN-PKU303 significantly reduces blood phenylalanine levels in mice compared to mice treated with mock (HO) or the parental strain (SYN-PKU901) (*, p<0.05; ***, p<0.001; ****, p<0.00001). SYN-PKU303 can disrupt intestinal recirculation of phenylalanine. [Figure 28]Figure 28 shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean body weight of group), mice were gavaged with 200 μL of HO (n=30), SYN-PKU901 (n=33), SYN-PKU303 (n=34), or SYN-PKU304 (n=34). Blood phenylalanine concentrations after phenylalanine injection indicate that SYN-PKU304 (a low-copy plasmid containing fnrS-PAL) is at least as effective as SYN-PKU303 (a high-copy plasmid containing Tet-PAL) in reducing circulating Phe levels in the intestinal recirculation model. [Figure 29A] Blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram average group body weight), mice were gavaged with HO, SYN-PKU901, SYN-PKU303, or SYN-PKU304. Blood phenylalanine concentrations 2 hours after phenylalanine injection are shown. These data demonstrate that oral administration of the genetically engineered probiotic strains SYN-PKU303 and SYN-PKU304 significantly reduced blood phenylalanine levels in mice compared to mice treated with mock (HO) or the parental strain (SYN-PKU901) (*, p<0.05; **, p<0.01; ***, p<0.001; ** **, p<0.0001). [Figure 29B]Figure 1 shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean body weight), mice were orally gavaged with HO, SYN-PKU901, SYN-PKU303, or SYN-PKU304. Blood phenylalanine concentrations 4 hours after phenylalanine injection are shown. These data demonstrate that oral administration of the genetically engineered probiotic strains SYN-PKU303 and SYN-PKU304 significantly reduced blood phenylalanine levels in mice compared to mice treated with mock (HO) or the parental strain (SYN-PKU901) (*, p<0.05; **, p<0.01; ***, p<0.001; ** **, p<0.0001). [Figure 29C]

[0041] Figure 29B shows blood phenylalanine concentrations relative to baseline following subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean group body weight), mice were gavaged with HO, SYN-PKU901, SYN-PKU303, or SYN-PKU304. A scatter plot of the data shown in Figure 29A is shown. [Figure 29D]

[0046] Figure 29B shows blood phenylalanine concentrations relative to baseline following subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean group body weight), mice were gavaged with HO, SYN-PKU901, SYN-PKU303, or SYN-PKU304. A scatter plot of the data shown in Figure 29B is shown. [Figure 30A]Figure 1 shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean body weight per group), mice were gavaged with 200 μL of HO (n=12), 200 μL of SYN-PKU901 (n=12), or 100, 200, or 400 μL of SYN-PKU304 (n=12 per treatment group). A dose-dependent decrease in blood phenylalanine levels is shown in SYN-PKU304-treated mice compared with mock-treated (HO) or parental strain (SYN-PKU901)-treated mice (*30% decrease; p<0.05). This experiment represents one of eight studies of this same design, each demonstrating that SYN-PKU304 can disrupt intestinal recirculation of phenylalanine. [Figure 30B] Figure 1 shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Thirty and 90 minutes after injection of phenylalanine (0.1 mg / gram mean body weight per group), mice were gavaged with 200 μL of HO (n=12), 200 μL of SYN-PKU901 (n=12), or 100, 200, or 400 μL of SYN-PKU304 (n=12 per treatment group). A dose-dependent decrease in blood phenylalanine levels is shown in SYN-PKU304-treated mice compared with mock-treated (HO) or parental strain (SYN-PKU901)-treated mice (*30% decrease; p<0.05). This experiment represents one of eight studies of this same design, each demonstrating that SYN-PKU304 can disrupt intestinal recirculation of phenylalanine. [Figure 31A] Figure 1 shows a schematic of PKU-specific phenylalanine metabolites. The conversion of phenylalanine to phenylpyruvate and phenyllactic acid in the absence of functional PAH is shown. These metabolites can be detected by mass spectrometry as described in Examples 24-26 or by other means. [Figure 31B]Figure 1 shows a schematic of PAL-specific phenylalanine metabolites. The conversion of phenylalanine by PAL3 to trans-cinnamic acid is outlined, which is further metabolized to hippuric acid by hepatic enzymes. These metabolites can be detected by mass spectrometry or other means, as described in Examples 24-26. [Figure 32A] Blood phenylalanine concentrations relative to baseline and phenylalanine concentrations. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL of HO (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at 800 μL (2.9e10 cfu / mouse). Blood phenylalanine concentrations relative to baseline were shown. The total metabolic activity of SYN-PKU304 was calculated to be 81.2 μmol / h, with a total reduction of ΔPhe relative to SYN-PKU901 of 45% (P<0.05). [Figure 32B] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL (n=12) of HO, SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at 2.9e10 cfu / mouse. Blood phenylalanine concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 32C] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL (n=12) of HO, SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at 2.9e10 cfu / mouse. Blood phenylpyruvate concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 32D]Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL (n=12) of HO, SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at 2.9e10 cfu / mouse. Blood phenyllactic acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 32E] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL (n=12) of HO, SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at 2.9e10 cfu / mouse. Blood t-cinnamic acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 32F] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL (n=12) of HO, SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at 2.9e10 cfu / mouse. Blood hippuric acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 33A]Blood phenylalanine concentrations relative to baseline and phenylalanine concentrations. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total HO (n = 9), SYN-PKU801 (n = 12), or SYN-PKU517 (n = 12) at 800 μL (3.6 e10 cfu / mouse). Blood phenylalanine concentrations relative to baseline were shown. The total metabolic activity of SYN-PKU517 was calculated to be 39.6 μmol / h, with a 17% overall reduction in ΔPhe relative to SYN-PKU801 (P < 0.05). [Figure 33B] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total of HO (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) (3.6e10 cfu / mouse). Blood phenylalanine concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 33C] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total HO (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) (3.6e10 cfu / mouse). Blood phenylpyruvate concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 33D]Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total HO (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) (3.6e10 cfu / mouse). Blood phenyllactic acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 33E] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total HO (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) (3.6e10 cfu / mouse). t-Cinnamic acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 33F] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total HO (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) (3.6e10 cfu / mouse). Blood hippuric acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 34A]Blood phenylalanine concentrations relative to baseline and phenylalanine concentrations. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL of HO (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at 800 μL (3.6e10 cfu / mouse). Blood phenylalanine concentrations relative to baseline were shown. The total metabolic activity of SYN-PKU705 was calculated to be 133.2 μmol / h, with a 30% overall reduction in ΔPhe relative to SYN-PKU901 (P<0.05). [Figure 34B] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total of HO (n=12), SYN-PKU901 (n=12), or 8 μL (3.6e10 cfu / mouse) of SYN-PKU705 (n=12). Blood phenylalanine concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 34C] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with 800 μL total of HO (n=12), SYN-PKU901 (n=12), or 8 μL (3.6e10 cfu / mouse) of SYN-PKU705 (n=12). Blood phenylpyruvate concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 34D]Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL of HO (n=12), SYN-PKU901 (n=12), or 8 μL (3.6e10 cfu / mouse) of SYN-PKU705 (n=12). Blood phenyllactic acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 34E] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL of HO (n=12), SYN-PKU901 (n=12), or 8 μL (3.6e10 cfu / mouse) of SYN-PKU705 (n=12). Blood t-cinnamic acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 34F] Absolute levels of phenylalanine and PKU- and PAL-specific metabolites after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Thirty and 90 minutes after phenylalanine injection, mice were gavaged with a total of 800 μL of HO (n=12), SYN-PKU901 (n=12), or 8 μL (3.6e10 cfu / mouse) of SYN-PKU705 (n=12). Blood hippuric acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 35]This figure shows phenylalanine and two toxic analogs, p-fluoro-DL-phenylalanine and o-fluoro-DL-phenylalanine, useful in an untargeted approach to select for PAL enzymes with increased activity. p-Fluoro-DL-phenylalanine and o-Fluoro-DL-phenylalanine are incorporated into cellular proteins in place of phenylalanine, causing cell death. These compounds are readily incorporated by PheP and, as shown below, can serve as substrates for PAL, allowing them to be used in genetic selection and screening to identify strains with improved Phe-consuming activity. Mutations that allow more efficient PAL metabolism can prevent the incorporation of phenylalanine analogs into cellular proteins, thus allowing growth at high concentrations of the analogs. [Figure 36] 1 is a bar graph showing the rate of trans-cinnamic acid production when phenylalanine or various phenylalanine-containing peptides are used as substrates. The results indicate that the PKU strain was able to rapidly degrade Phe, even in the form of dipeptides and tripeptides, and that the engineered bacteria could be administered with food, since dietary proteins would be degraded into dipeptides and tripeptides and available as bacterial substrates. [Figure 37] This is a bar graph showing the effect of various copy numbers of pheP, PAL, and further addition of LAAD on the rate of phenylalanine degradation in vitro. The results demonstrate that increasing the copy number of PAL increases the rate of phenylalanine degradation. Addition of the high-affinity transporter pheP abolishes transport limitation, resulting in higher PAL activity. Transporter copy number does not increase the rate (PAL, not transport (pheP), is limiting). In the presence of oxygen, LAAD can degrade Phe at an extremely high rate. [Figure 38A]Bar graphs depicting measurements from characterization of the phenylalanine intestinal recirculation model. PKU mice were maintained on Phe-free chow and subcutaneously injected with phenylalanine (0.1 mg / kg body weight) at T=0. Blood samples were taken at the indicated time points to determine serum phenylalanine (Phe) kinetics after injection. Whisker plots depicting the distribution of total phenylalanine (Phe) levels in mouse blood. [Figure 38B] This is a bar graph showing measurements from the characterization of the phenylalanine intestinal recirculation model. PKU mice were maintained on a Phe-free chow diet and subcutaneously injected with phenylalanine (0.1 mg / kg body weight) at T=0. Blood samples were taken at the indicated time points to determine serum phenylalanine (Phe) kinetics after injection. This is a whisker plot showing the distribution of changes in phenylalanine (Phe) levels from T0 in mouse blood. [Figure 38C] This bar graph shows measurements from the characterization of the phenylalanine intestinal recirculation model. PKU mice were maintained on a Phe-free chow diet and injected subcutaneously with phenylalanine (0.1 mg / kg body weight) at T=0. Blood was collected at the indicated time points to determine serum phenylalanine (Phe) kinetics after injection. Phe levels are shown to steadily increase over at least 6 hours. [Figure 39] This graph shows the total labeled phenylalanine concentration measured during subcutaneous 13C-Phe challenge to determine the extent of recirculation in a PKU(enu2) mouse model. At TO, all mice were maintained on a Phe-free diet with low-Phe water until injection with 0.1 mg / kg 13C-Phe. Blood and intestinal effluent samples were collected at 0, 20 min, and 2 h, and phenylalanine concentrations were determined by LC-MS. The occurrence of intestinal recirculation of labeled 13C-Phe was confirmed. Furthermore, unlabeled Phe concentrations were also determined, revealing high levels of pre-existing (unlabeled) phenylalanine in the small intestine (data not shown). [Figure 40A]Figure 1 shows graphs depicting amino acid content in various compartments in wild-type and enu2- / - mice. Measurements of amino acid content in blood are shown. As expected, phenylalanine (Phe) levels were higher in the blood of enu2- / - mice. No other significant differences were observed between wild-type and enu2- / - mice. [Figure 40B] Figure 1 shows graphs depicting amino acid content in various compartments in wild-type and enu2- / - mice. Measurements of amino acid content in the small intestine are shown. As expected, phenylalanine (Phe) levels were elevated in the blood of enu2- / - mice. No other significant differences were observed between wild-type and enu2- / - mice. [Figure 40C] Figure 1 shows graphs depicting amino acid content in various compartments in wild-type and enu2- / - mice. Measurements of amino acid content in the large intestine are shown. As expected, phenylalanine (Phe) levels were elevated in the blood of enu2- / - mice. No other significant differences were observed between wild-type and enu2- / - mice. [Figure 41A] This graph shows absolute phenylalanine concentrations following gavage of a strain containing low-copy PfnrS-PAL, chromosomal PfnrS-pheP (SYN-PKU305) in a fast-feed model of PKU. In this model, Enu2 mice were maintained on normal chow until the time of dosing. Food was removed at TO. Mice were dosed hourly for 3 hours (4 doses total: 0, 1, 2, and 3 hours), and blood was collected at 4 hours to determine serum Phe concentrations. Probiotic (SYN-PKU305)-treated mice show a more rapid decline in serum Phe. [Figure 41B]This graph shows the change in phenylalanine relative to baseline following gavage of a strain containing low-copy PfnrS-PAL, chromosomal PfnrS-pheP (SYN-PKU305) in a fast-feed model of PKU. In this model, Enu2 mice were maintained on normal chow until the time of dosing. Food was removed at TO. Mice were dosed hourly for 3 hours (4 doses total: 0, 1, 2, and 3 hours), and blood was collected at 4 hours to determine serum Phe concentrations. Mice treated with the probiotic (SYN-PKU305) show a more rapid decline in serum Phe. [Figure 42A] This graph shows the effect of administering SYN-PKU305 (containing low-copy pSC101-PfnrS-PAL3 and chromosome lacZ::PfnrS-pheP) in drinking water with gelatin as a carrier after Phe challenge (5e9 cells / ml). Enu2 mice were maintained on a normal chow diet throughout the experiment. [Figure 42B] This graph shows the effect of administering SYN-PKU305 (containing low-copy pSC101-PfnrS-PAL3 and chromosome lacZ::PfnrS-pheP) in drinking water with gelatin as a carrier after Phe challenge (5e9 cells / ml). Enu2 mice were maintained on a normal chow diet throughout the experiment. [Figure 43A] This table shows that in vitro and in vivo activity correlate, but maximum in vivo activity is reached at approximately 50% reduction in deltaPhe. [Figure 43B] 1 is a line graph showing that in vitro and in vivo activity correlate, but maximum in vivo activity is reached at approximately 50% reduction in deltaPhe. [Figure 44A]1 is a bar graph showing trans-cinnamic acid (TCA) levels in blood following subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were gavaged with a total of 750 μL of HO (n = 12), SYN-PKU901 (streptomycin-resistant Nissle; n = 12; 3 × 250 μL, totaling 1 × 10 e11 cfu / mouse for three gavage doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions of PAL (2XfnrS-PAL(malEK, malPT)), and two chromosomal insertions of pheP (2XfnrS-pheP(lacZ, HA1 / 2)), and one chromosomal insertion of LAAD (Para::LAAD), and dapA auxotrophy, and chloramphenicol resistance; n = 12; 3 × 250 μL, totaling 1 × 10 e11 cfu / mouse for three gavage doses). Serum TCA concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 44B] 1 is a bar graph showing hippuric acid levels in blood following subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were gavaged with a total of 750 μL of HO (n = 12), SYN-PKU901 (streptomycin-resistant Nissle; n = 12; 3 × 250 μL, totaling 1 × 10 e11 cfu / mouse for three gavage doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions of PAL (2XfnrS-PAL(malEK, malPT)), and two chromosomal insertions of pheP (2XfnrS-pheP(lacZ, HA1 / 2)), and one chromosomal insertion of LAAD (Para::LAAD), and dapA auxotrophy, and chloramphenicol resistance; n = 12; 3 × 250 μL, totaling 1 × 10 e11 cfu / mouse for three gavage doses). Serum hippuric acid concentrations are shown at 0 and 4 hours after phenylalanine injection. [Figure 44C]1 is a bar graph showing urinary trans-cinnamic acid (TCA) concentrations following subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were gavaged with a total of 750 μL of HO (n = 12), SYN-PKU901 (streptomycin-resistant Nissle; n = 12; 3 × 250 μL, totaling 1 × 10e11 cfu / mouse for three gavage doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions of PAL (2XfnrS-PAL(malEK, malPT)), and two chromosomal insertions of pheP (2XfnrS-pheP(lacZ, HA1 / 2)), and one chromosome of LAAD (Para::LAAD), and dapA auxotrophy, and chloramphenicol resistance; n = 12; 3 × 250 μL, totaling 1 × 10e11 cfu / mouse for three gavage doses). Urinary TCA concentrations 4 hours after phenylalanine injection are shown. [Figure 44D]1 is a bar graph showing urinary hippuric acid concentrations following subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were gavaged with a total of 750 μL of HO (n = 12), SYN-PKU901 (streptomycin-resistant Nissle; n = 12; 3 × 250 μL, totaling 1 × 10e11 cfu / mouse for three gavage doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions of PAL (2XfnrS-PAL(malEK, malPT)), and two chromosomal insertions of pheP (2XfnrS-pheP(lacZ, HA1 / 2)), and one chromosome of LAAD (Para::LAAD), and dapA auxotrophy, and chloramphenicol resistance; n = 12; 3 × 250 μL, totaling 1 × 10e11 cfu / mouse for three gavage doses). Urinary hippuric acid concentrations 4 hours after phenylalanine injection are shown. Low levels of TCA were present in both urine and serum. Even lower levels of hippuric acid were detected in serum. The highest levels of hippuric acid were detected in urine. This indicates that most of the TCA produced by the bacteria is converted to hippuric acid in the liver and excreted in the urine. Similar levels of metabolites were measured in urine when other effective PKU strains were administered. [Figure 45A] Figure 1 shows the amount of TCA recovered 4 hours after oral gavage of TCA (0.0125, 0.025, 0.05, or 0.1 mg / g TCA). PKU mice were orally gavaged with 0.0125, 0.025, 0.05, or 0.1 mg / g TCA (three mice per cage, two cages per group). Four hours after gavage, urine, feces, and blood were collected and analyzed for TCA and its major degradation product, hippuric acid. No significant amounts of TCA or hippuric acid were detected in the blood or feces (data not shown). Observations revealed nearly complete recovery of TCA in the form of hippuric acid. These data indicate that 1 mole of hippuric acid found in urine is equivalent to 1 mole of Phe converted to TCA in the small intestine of PKU mice upon challenge with the PKU strain. [Figure 45B]FIG. 1 shows the amount of hippuric acid recovered 4 hours after oral gavage of TCA (0.0125, 0.025, 0.05, or 0.1 mg / g TCA). [Figure 46] Figure 1 shows the kinetics of TCA conversion to hippuric acid in PKU mice (enu2- / -) over 6 hours after Phe challenge. Animals were placed in metabolic cages (3 mice per cage, 2 cages per group), and urine samples were collected 1, 2, 3, 4, 5, and 6 hours after TCA administration. TCA is converted to hippuric acid and excreted in the urine by 4 hours. [Figure 47A]

[0023] Figure 1 is a schematic diagram of a non-limiting embodiment of a genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the genetic organization of an exemplary bacterial chromosome containing phenylalanine degradation components integrated into the E. coli Nissle chromosome is shown. SYN-PKU707 contains three chromosomal insertions of PAL3 (3XfnrS-PAL(malP / T, yicS / nepI, malE / K)) and two copies of pheP (2XfnrS-pheP(lacZ, agaI / rsmI)). SYN-PKU707 also contains one copy of a mutant FNR transcription factor, FNRS24Y (Para::FNRS24Y). [Figure 47B]

[0023] Figure 1 is a schematic diagram of a non-limiting embodiment of a genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the genetic organization of an exemplary bacterial chromosome (e.g., as found in strain SYN-PKU708) is shown. The bacterial chromosome contains three chromosomal insertions of PAL3 (3XfnrS-PAL(malP / T, yicS / nepI, malE / K)) and two copies of pheP (2XfnrS-pheP(lacZ, agaI / rsmI)). The bacterial chromosome contains one copy of a mutant FNR transcription factor, i.e., FNRS24Y (Para::FNRS24Y), and one copy of LAAD inserted at the same insertion site (arabinose operon), transcribed in a bicistronic message from the endogenous arabinose promoter. The bacterial chromosome further contains a dapA auxotroph (deltaDapA). [Figure 47C]

[0023] Figure 1 is a schematic diagram of a non-limiting embodiment of a genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the gene organization of an exemplary bacterial chromosome (e.g., as found in SYN-PKU709) is shown. The bacterial chromosome contains three insertions of PAL3 (3XfnrS-PAL(malP / T, yicS / nepI, malE / K)) and two copies of pheP (2XfnrS-pheP(lacZ, agaI / rsmI)). The bacterial chromosome further contains one copy of LAAD, which is inserted into the arabinose operon and whose expression is driven by the native Para promoter (Para::LAAD). The genome is further modified to delete the dapA gene and contain a dapA auxotrophy. [Figure 47D]

[0023] Figure 1 is a schematic diagram of a non-limiting embodiment of a genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the gene organization of an exemplary bacterial chromosome (e.g., as found in SYN-PKU710) is shown. The bacterial chromosome contains three insertions of PAL3 (3XfnrS-PAL(malP / T, yicS / nepI, malE / K)) and two copies of pheP (2XfnrS-pheP(lacZ, agaI / rsmI)). The bacterial chromosome also contains one copy of LAAD, which is inserted into the arabinose operon and expression is driven by the native Para promoter (Para::LAAD). The bacterial chromosome also contains two copies of IPTG-inducible PAL3 (2XLacIPAL, exo / cea and rhtC / rhtB), a dapA auxotrophy, and all antibiotic resistance is cured. [Figure 47E]

[0023] Figure 1 is a schematic diagram of a non-limiting embodiment of a genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the gene organization of an exemplary bacterial chromosome (e.g., as found in SYN-PKU712) is shown. SYN-PKU712 corresponds to SYN-PKU707, which is essentially auxotrophic for dapA. The bacterial chromosome contains three copies of fnrSPAL3 and two copies of fnrSpheP integrated into the chromosome, along with knock-in Para-FNRS24Y and deltadapA (DAP auxotrophy). SYN-PKU712 corresponds to SYN-PKU707, which is essentially auxotrophic for dapA. [Figure 47F]

[0023] Figure 1 is a schematic diagram of a non-limiting embodiment of a genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the gene organization of an exemplary bacterial chromosome (e.g., as found in SYN-PKU711) is shown. SYN-PKU711 corresponds to SYN-PKU708, which is essentially deficient in dapA auxotrophy. The bacterial chromosome contains three copies of -fnrSPAL3 and two copies of fnrSpheP integrated into the chromosome, and a knocked-in Para-FNRS24Y-LAAD. [Figure 48A]

[0033] Figure 1 shows the genetic organization of exemplary constructs. A construct containing FNRS24Y driven by an arabinose-inducible promoter and araC in the reverse orientation is shown. SYN-PKU707 and SYN-PKU712 are examples of strains containing such constructs (integrated into the arabinose operon). An exemplary sequence includes SEQ ID NO: 64. In some embodiments, FNRS24Y expression (from the construct shown in Figure 48A or Figure 48B) is induced as part of the oxygen bypass switch shown in Figure 50. In some embodiments, the construct shown is used in combination with hypoxia-inducible PAL3, pheP, and / or LAAD constructs. In some embodiments, the construct is located on a plasmid, e.g., a low-copy or high-copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In a non-limiting example, the construct shown is knocked into the E. coli arabinose operon and driven by the endogenous arabinose promoter. [Figure 48B]

[0033] Figure 1 shows the genetic organization of exemplary constructs. A construct is shown in which FNRS24Y and LAAD are expressed in a bicistronic message from an arabinose-inducible promoter. AraC is transcribed in the opposite direction. SYN-PKU708 is an example of a strain containing such a construct (integrated into the arabinose operon). Exemplary sequences include SEQ ID NO: 73. In some embodiments, FNRS24Y expression (from the construct shown in Figure 48A or Figure 48B) is induced as part of the oxygen bypass switch shown in Figure 50. In some embodiments, the constructs shown are used in combination with hypoxia-inducible PAL3, pheP, and / or LAAD constructs. In some embodiments, the constructs are located on a plasmid, e.g., a low-copy or high-copy plasmid. In some embodiments, the constructs are located on a plasmid component of a biosafety system. In some embodiments, the constructs are integrated into the bacterial chromosome at one or more locations. In a non-limiting example, the constructs shown are knocked into the E. coli arabinose operon and driven by the endogenous arabinose promoter. [Figure 49]The genetic organization of an exemplary construct containing an inverted LacI and an IPTG-inducible promoter driving expression of PAL3 is shown. SYN-PKU710 is a non-limiting example of a strain containing such a construct. In SYN-PKU710, lacPAL is inserted into the exo / cea locus. Exemplary sequences include SEQ ID NO: 74. In some embodiments, the construct is useful for pre-induction and pre-loading of therapeutic strains prior to in vivo administration under aerobic conditions and in the presence of an inducer (e.g., IPTG). In some embodiments, the construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible PAL3 constructs (e.g., constructs induced by hypoxia, arabinose, or IPTG). In some embodiments, the construct is used in combination with a hypoxia-inducible construct active in an in vivo environment. In some embodiments, the construct is located on a plasmid, e.g., a low-copy or high-copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which can be either provided on a plasmid or integrated into the bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inducible promoter, e.g., hypoxia, arabinose, or IPTG. In some embodiments, the construct is used in combination with a LAAD expression construct. In some embodiments, the construct PAL3 sequence is the original sequence from Photorhabdus chemiluminescens. In some embodiments, the PAL3 sequence is codon-optimized for expression in E. coli. In some embodiments, the construct is located on a plasmid, e.g., a low-copy or high-copy plasmid. In some embodiments, the construct is used in a biosafety system, such as the systems shown in Figures 61A, 61B, 61C, and 61D.In some embodiments, the construct is integrated into the genome at one or more of the locations described herein. [Figure 50]

[0023] Figure 1 illustrates an "oxygen bypass switch" useful for aerobic pre-induction of strains containing one or more PMEs and / or Phe transporters under the control of a hypoxic FNR promoter in an in vitro culture vessel (e.g., a flask, fermentor, or other vessel used during cell growth, cell expansion, fermentation, harvest, purification, formulation, and / or manufacturing). In some embodiments, it may be desirable to pre-load the strain with an active PME, e.g., PAL3 and / or LAAD, prior to administration of the strain. This may be accomplished by pre-inducing expression of the enzymes when growing the strain (e.g., in a flask, fermentor, or other suitable vessel) and preparing it for in vivo administration. In some embodiments, the strain is induced under anaerobic and / or hypoxic conditions, e.g., to induce FNR promoter activity and drive expression of one or more PMEs and / or Phe transporters. In some embodiments, it may be desirable to prepare, pre-load, and pre-induce the strain under aerobic or microaerobic conditions. This allows for more efficient growth and, in some cases, reduces the accumulation of toxic metabolites. FNRS24Y is a mutant form of FNR that is more resistant to oxygen inactivation and is therefore able to activate the FNR promoter under aerobic conditions (see, e.g., Jervis AJ, Proc Natl Acad Sci USA 2009 Mar. 24, vol. 106(12):4659-64, "The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion," the contents of which are incorporated herein by reference in their entirety).See Proc Natl Acad Sci USA, March 24, 2009, Vol. 106(12):4659-64, "The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion," the contents of which are incorporated herein by reference in their entirety. In the oxygen bypass system described above, FNRS24Y is induced by the addition of arabinose and drives the expression of PAL3 and pheP by binding to and activating the FNR promoter under aerobic conditions. Therefore, this system utilizes the strong FNR promoter to obtain high-level expression of PAL3 and PheP, allowing the strain to be efficiently grown, produced, or manufactured under aerobic conditions while being effectively pre-induced and pre-loaded. The system does not interfere with or compromise in vivo activation, as the mutant FNRS24Y is no longer expressed in the absence of arabinose, and wild-type FNR binds to the FNR promoter to drive expression of PAL3 and PheP in vivo. The system can also be used to drive LAAD expression from the FNR promoter during strain production (not shown). In other embodiments described herein, LAAD expression can also be induced aerobically, for example, with arabinose. Thus, in some embodiments, LAAD and FNRS24Y can be induced simultaneously. In some embodiments, FNRS24Y and LAAD are transcribed in a bicistronic message, and their expression is driven by the arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression from the endogenous Para promoter. In some embodiments, FNRS24Y-LAAD is knocked into the arabinose operon, allowing expression from the endogenous Para promoter.In some embodiments, a LacI promoter and IPTG induction are used in the above system (instead of Para and arabinose induction). In some embodiments, a rhamnose-inducible promoter is used in the above system. In some embodiments, a temperature-sensitive promoter is used to drive expression of FNRS24Y. [Figure 51] This is a bar graph showing the in vitro PAL activity of SYN-PKU707 (3XPfnrS-PAL3; 2XPfnrSpheP; Para-fnrS24Y) as measured by the rate of trans-cinnamic acid (TCA) production. Cells were induced aerobically or anaerobically in the presence or absence of arabinose. Cultures were grown in 10 ml, 20 ml, or 30 ml flasks. Arabinose-inducible expression of fnrS24Y results in high levels of activity under aerobic conditions in 10 ml, 20 ml, or 30 ml flasks. Furthermore, activation in the absence of arabinose is maintained under anaerobic conditions. These results indicate that the strain can be efficiently pre-induced under aerobic conditions prior to in vivo administration. These results also indicate that anaerobic activation without arabinose, e.g., "in vivo" activation, may be conserved in this strain. [Figure 52] This bar graph shows blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. At 1, 2, and 3 hours after phenylalanine injection, mice were gavaged three times with a total of 750 μL of HO (n=9), SYN-PKU901 (n=9), or 800 μL of SYN-PKU707 (n=9) (1×10e11 cfu / mouse). Blood and urine were collected 4 hours after injection. Blood phenylalanine concentrations relative to baseline are shown; the total metabolic activity of SYN-PKU707 was calculated as 269 μmol / h, and the total reduction in ΔPhe was 49% (P<0.05) versus SYN-PKU901 (P<0.05). [Figure 53]This bar graph shows the absolute hippuric acid levels recovered from urine after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. At 1, 2, and 3 hours after phenylalanine injection, mice were gavaged three times with a total of 750 μL of HO (n=9), SYN-PKU901 (n=9), or 800 μL of SYN-PKU707 (n=9) (1x10e11 cfu / mouse). Blood and urine were collected 4 hours after injection. Urinary hippuric acid concentrations 4 hours after phenylalanine injection are shown. These results indicate that approximately 15-20% of the injected phenylalanine is converted to hippuric acid. Phenylalanine is converted to TCA in the small intestine, and TCA is converted to hippuric acid in the liver. [Figure 54A] Figure 1 shows the recovery of absolute urinary unlabeled hippuric acid levels after subcutaneous (SC) injection of radiolabeled phenylalanine in mice gavaged three times with SYN-PKU707. Mice in metabolic cages (3 mice / cage; 3 cages / group) were gavaged three times with SYN1780 following subcutaneous (SC) injection of heavy Phe (1, 2, and 3 hours post-injection). [Figure 54B] Figure 1 shows the recovery of absolute amounts of labeled hippuric acid in urine after subcutaneous (SC) injection of radiolabeled phenylalanine in mice gavaged three times with SYN-PKU707. Following subcutaneous (SC) injection of heavy Phe (1, 2, and 3 hours after injection), mice in metabolic cages (3 mice / cage; 3 cages / group) were gavaged three times with SYN1780. [Figure 55]

[0023] Figure 1 shows a graph of hippuric acid recovered from urine after a single dose of the PKU strain SYN-PKU707 at TO, 2, 4, 6, and 8 hours after Phe challenge. Mice were gavaged with different cell numbers (in a single gavage) as indicated. A dose-dependent increase in hippuric acid recovered from the urine of mice was observed. [Figure 56]A graph showing hippuric acid recovery from urine compares the kinetics of hippuric acid production from phenylalanine following gavage of live cells with that resulting from gavage of pure TCA. With pure TCA, a rapid decline in urinary hippuric acid recovery is observed within the first 15 minutes of collection. With the cells, hippuric acid recovery persists for at least the first 30 minutes of collection, with a decreasing downward gradient. These results indicate that the cells remain in the small intestine and produce TCA for a useful period of time. [Figure 57A] Figure 1 is a bar graph showing the change in blood phenylalanine relative to baseline 4 hours after phenylalanine exposure in PKU mice gavaged with the indicated doses of SYN-PKU708. SYN-PKU708 was effective in reducing blood phenylalanine, indicating that the cells were active in vivo. [Figure 57B] Figure 1 is a bar graph showing absolute amounts of hippuric acid in the urine 4 hours after phenylalanine exposure in PKU mice gavaged with the indicated doses of SYN-PKU708. Hippuric acid was excreted in a dose-dependent manner in the cages of SYN-PKU708-treated mice, indicating that the cells were active in vivo. [Figure 58A] Figure 1 shows the transit time of bacteria (SYN1780) after a single gavage of approximately 3e10 cfu. Mice were given a single gavage of bacteria (approximately 3 x 10e10 CFU). At each time point (15, 30, 45, and 60 minutes after gavage), animals (n=4) were euthanized and the small intestine was removed, cut into three equal parts, and flushed. The intestinal eluate was processed for serial dilution plating to determine bacterial counts. [Figure 58B] 1 is a graph showing the transit time of bacteria (SYN1780) after a single gavage of approximately 3e10 cfu. [Figure 59A]Figure showing blood phenylalanine concentrations relative to baseline 4 hours after subcutaneous (SC) injection of phenylalanine, comparing strains SYN-PKU710 and SYN-PKU708. Mice were administered a single dose of phenylalanine by subcutaneous injection of 0.1 mg per gram of body weight. At 1, 2, and 3 hours after Phe challenge, mice were gavaged with bacteria (or water) (300 μl / dose, total 3×e10 cfu / mouse). The percent reduction in deltaPhe in SYN-PKU710 and SYN-PKU708 was calculated to be 29% and 40%, respectively. [Figure 59B] Absolute hippuric acid levels up to 4 hours after subcutaneous (SC) injection of phenylalanine, comparing strains SYN-PKU710 and SYN-PKU708. [Figure 60] 1 is a bar graph showing in vitro PAL activity, as measured by the rate of TCA accumulation, in strains in which PAL expression is under the control of various inducible promoters (arabinose, IPTG (Lacl), rhamnose, Tet, temperature (CI857)). SYN-PKU707 is shown as a benchmark control. [Figure 61A]

[0023] Figure 1 is a schematic diagram of a non-limiting example of the genetic organization of a plasmid that functions as a component of the biosafety system. The biosafety plasmid system vector contains Kid toxin and an R6K minimal ori, dapA, and promoter elements that drive expression of these components. In a non-limiting example, the plasmid contains SEQ ID NO: 81. In a non-limiting example, the plasmid contains SEQ ID NO: 82. In some embodiments, bla is knocked out and replaced with one or more constructs described herein in which PAL3 and / or PheP and / or LAAD are expressed from an inducible or constitutive promoter. [Figure 61B]

[0023] Figure 1 is a schematic diagram of a non-limiting example of the genetic organization of a plasmid that functions as a component of the biosafety system. The biosafety plasmid system vector contains Kid toxin and an R6K minimal ori, thyA, and promoter elements that drive expression of these components. In a non-limiting example, the plasmid contains SEQ ID NO: 81. In a non-limiting example, the plasmid contains SEQ ID NO: 82. In some embodiments, bla is knocked out and replaced with one or more constructs described herein in which PAL3 and / or PheP and / or LAAD are expressed from an inducible or constitutive promoter. [Figure 61C] Schematic diagram of the genetic organization of the chromosomal components of the biosafety system. A construct containing low-copy Rep (Pi) and Kis antitoxin is shown. Pi (Rep) is required for replication of the plasmid component of the system, and transcription of Pi (Rep) is driven by a promoter containing a low-copy RBS. In some embodiments, the construct comprises SEQ ID NO: 89. [Figure 61D] 61C and 61D are knocked into the ThyA locus. In this system, bacteria containing the chromosomal construct and knocked-out dapA or thyA gene can grow only in the presence of the plasmid, in the absence of dap or thymidine. [Figure 62A]

[0023] Figure 1 is a schematic diagram of a non-limiting example of a PAL construct. A schematic diagram of a non-limiting example of the configuration of a construct for PAL expression under the control of a lambda CI inducible promoter is shown. The construct also provides the coding sequence for CI857, a temperature-sensitive mutant of CI. The temperature-sensitive CI repressor mutant CI857 binds tightly at 30°C but fails to bind (repress) at temperatures above 37°C. In some embodiments, the construct comprises SEQ ID NO: 101. In some embodiments, the construct is used alone. In some embodiments, the temperature-sensitive construct is used in combination with other constitutive or inducible PAL3 constructs, such as hypoxia-, arabinose-, rhamnose-, or IPTG-inducible constructs. In some embodiments, the construct allows for pre-induction and pre-loading of PAL3 and / or PheP and / or LAAD prior to in vivo administration. In some embodiments, the construct confers in vivo activity. In some embodiments, the construct is located on a plasmid, such as a low-copy or high-copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which can be either provided on a plasmid or integrated into the bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inducible promoter, e.g., hypoxia-, arabinose-, rhamnose-, or temperature-sensitive. In some embodiments, the construct is used in combination with an LAAD expression construct. In some embodiments, a temperature-sensitive system can be used to establish a conditional auxotrophy. In strains containing deltaThyA or deltaDapA, the dapA or thyA gene can be introduced into the strain under the control of a temperature-regulated promoter system. In the absence of Thy and Dap, the strain can grow only at permissive temperatures, e.g., 37°C (and no lower). [Figure 62B]

[0023] Figure 1 is a schematic diagram of a non-limiting example of a PAL construct.

[0024] Figure 1 shows a schematic diagram of a non-limiting example of the configuration of a construct for PAL expression under the control of a rhamnose-inducible promoter. For applications of the rhamnose expression system, expression from the chromosome is sufficient to activate transcription even on multicopy plasmids, eliminating the need to express larger amounts of regulatory proteins. Therefore, only the rhaP BAD promoter is cloned upstream of the gene to be expressed. In some embodiments, this construct is used alone. In some embodiments, the rhamnose-inducible construct is used in combination with other constitutive or inducible PAL3 constructs, such as hypoxia-, arabinose-, temperature-, or IPTG-inducible constructs. In some embodiments, the construct allows for pre-induction and preloading of PAL3 and / or PheP and / or LAAD prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with a hypoxia-inducible construct. In some embodiments, the construct is located on a plasmid, such as a low-copy or high-copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which can be either provided on a plasmid or integrated into the bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inducible promoter, e.g., hypoxia-, arabinose-, rhamnose-, or temperature-sensitive. In some embodiments, the construct is used in combination with a LAAD expression construct. [Figure 62C]Schematic diagram of a non-limiting example of a PAL construct. Schematic diagram of a non-limiting example of the configuration of a construct for PAL expression under the control of an arabinose-inducible promoter. The arabinose-inducible PAL3 construct comprises AraC (inverted), a region containing the arabinose-inducible promoter, and PAL3. In some embodiments, the construct is used alone. In some embodiments, the rhamnose-inducible construct is used in combination with other constitutive or inducible PAL3 constructs, such as hypoxia-, arabinose-, temperature-sensitive, or IPTG-inducible constructs. In some embodiments, the construct allows for pre-induction and pre-loading of PAL3 and / or PheP and / or LAAD prior to in vivo administration. In a non-limiting example, the construct is useful for pre-induction and is combined with a hypoxia-inducible construct. In some embodiments, the construct is located on a plasmid, such as a low-copy or high-copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which can be either provided on a plasmid or integrated into the bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inducible promoter, e.g., hypoxia-, arabinose-, rhamnose-, or temperature-sensitive. In some embodiments, the construct is used in combination with a LAAD expression construct. [Figure 63A] The genetic organization of the PssB promoter is shown. The ssB gene product protects ssDNA from degradation. SsB interacts directly with numerous enzymes involved in DNA metabolism and is thought to play a central role in the organization of nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination, and repair. The PssB promoter was cloned in front of a LacZ reporter, and β-galactosidase activity was measured. [Figure 63B]This is a bar graph showing reporter gene activity of the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and split into two separate tubes. One tube was placed in an anaerobic chamber, while the other was maintained under aerobic conditions for the duration of the experiment. At specific times, cells were analyzed for promoter induction. The PssB promoter is active under aerobic conditions and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control gene product expression so that it is expressed only under anaerobic and / or hypoxic conditions. In this case, the oxygen-inducible PssB promoter induces the expression of a repressor, which in turn represses the expression of the gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and / or hypoxic conditions. This strategy has the advantage of providing an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control the expression of thyA and / or dapA, e.g., to create a conditional auxotrophy. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and / or hypoxic conditions, dapA or thyA is optionally expressed, allowing the strain to grow in the absence of dap or thymidine. Under aerobic conditions, expression of dapA or thyA is shut off, preventing the strain from growing in the absence of dap or thymidine. Such a strategy can be used, for example, to allow bacteria to survive under anaerobic and / or hypoxic conditions, e.g., in the digestive tract, but prevent survival under aerobic conditions (a biosafety switch). [Figure 64A]

[0023] Figure 1 shows a strategy for fine-tuning expression of a Para-PAL construct by using a ribosome binding site optimization strategy. Bioinformatics tools for RBS optimization are known in the art. In one strategy, an arabinose-regulated PAL and pheP can be integrated into the chromosome to provide efficient aerobic growth and pre-induction of the strain (e.g., in a flask, fermentor, or other suitable vessel) while maintaining an integrated PfnrS-PAL and PheP to allow for strong in vivo induction. [Figure 64B]

[0023] Figure 1 shows a strategy for enabling the expression of PAL and PheP under aerobic conditions through arabinose-inducible expression of FNRS24Y. Using a ribosome binding site optimization strategy, the expression level of FnrS24Y can be fine-tuned, for example, under optimal induction conditions (appropriate amount of arabinose for full induction). Fine-tuning is achieved by selecting an appropriate RBS with an appropriate translation initiation rate. Bioinformatics tools for RBS optimization are known in the art. [Figure 65A]Schematic diagrams showing exemplary genetic organization of various expression constructs described herein. These constructs can be used alone or in combination with other PAL3 and / or PheP and / or LAAD expression constructs. A schematic diagram of a non-limiting example of the configuration of an IPTG- and hypoxia-inducible construct with a promoter-containing region containing inverted LacI and LacO sites and an FNR binding site is shown. The promoter drives expression of two open reading frames encoding PAL3 and a third open reading frame encoding PheP; the construct can be transcribed in a tricistronic message. In a non-limiting example, the construct comprises SEQ ID NO: 95. In some embodiments, this construct is useful for pre-induction under anaerobic and / or hypoxic conditions by activating the FNR promoter. In some embodiments, this construct is useful for in vivo activation by activating the FNR promoter under anaerobic and / or hypoxic conditions, such as those found in certain regions of the gastrointestinal tract. In some embodiments, the construct in the figure comprises a PAL3 sequence, which is the original PAL3 sequence of Photorhabdus chemiluminescens. In some embodiments, the PAL3 sequence is codon-optimized for expression in E. coli. In some embodiments, codon optimization is used as an additional control to fine-tune, i.e., up-regulate or down-regulate, the PAL3 level expressed from the construct. In some embodiments, the construct is located on a plasmid, e.g., a low- or high-copy plasmid. In some embodiments, the construct is used in a biosafety system, such as the system shown in Figure 61. In some embodiments, the construct is integrated into the genome at one or more of the locations described herein. [Figure 65B]Schematic diagrams showing exemplary genetic organization of various expression constructs described herein. These constructs can be used alone or in combination with other PAL3 and / or PheP and / or LAAD expression constructs. A schematic diagram of a non-limiting example of the configuration of an IPTG- and hypoxia-inducible construct with a promoter-containing region containing inverted LacI and LacO sites and an FNR binding site is shown. The construct also contains two open reading frames encoding PAL3. This construct is transcribed in a bicistronic message. In some embodiments, the construct is combined with another construct expressing PheP from another plasmid. In some embodiments, the construct is combined with a construct expressing PheP integrated into the bacterial chromosome at one or more positions. In a non-limiting example, the construct comprises SEQ ID NO: 97. In some embodiments, this construct is useful for pre-induction under anaerobic and / or hypoxic conditions by activating the FNR promoter. In some embodiments, this construct is useful for in vivo activation by activating the FNR promoter under anaerobic and / or hypoxic conditions, such as those found in certain regions of the gastrointestinal tract. In some embodiments, the construct in the figure comprises a PAL3 sequence, which is the original PAL3 sequence of Photorhabdus chemiluminescens. In some embodiments, the PAL3 sequence is codon-optimized for expression in E. coli. In some embodiments, codon optimization is used as an additional control to fine-tune, i.e., up-regulate or down-regulate, the PAL3 level expressed from the construct. In some embodiments, the construct is located on a plasmid, e.g., a low- or high-copy plasmid. In some embodiments, the construct is used in a biosafety system, such as the system shown in Figure 61. In some embodiments, the construct is integrated into the genome at one or more of the locations described herein. [Figure 65C]Figure 1 shows a schematic diagram illustrating exemplary genetic organization of various expression constructs described herein. These constructs can be used alone or in combination with other PAL3 and / or PheP and / or LAAD expression constructs. A non-limiting example of the construction of an IPTG-inducible construct with a promoter-containing region containing LacI and LacO in reverse orientation is shown. The construct also contains two open reading frames encoding PAL3 and a third open reading frame encoding PheP. This construct can be transcribed as a tricistronic message. In a non-limiting example, the construct comprises SEQ ID NO: 96. In some embodiments, the construct in the figure contains a PAL3 sequence, which is the original sequence of Photorhabdus chemiluminescens. In some embodiments, the PAL3 sequence is codon-optimized for expression in E. coli. In some embodiments, codon optimization is used as an additional control to fine-tune, i.e., up- or down-regulate, the PAL3 levels expressed from the construct. In some embodiments, the construct is located on a plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct is used in a biosafety system, such as the system shown in Figure 61. In some embodiments, the construct is integrated into the genome at one or more locations described herein. [Figure 65D]Schematic diagrams showing exemplary genetic organizations of various expression constructs described herein. These constructs can be used alone or in combination with other PAL3 and / or PheP and / or LAAD expression constructs. A non-limiting example of the construction of an IPTG-inducible construct with a promoter-containing region containing LacI and LacO in inverted orientation is shown. The construct also contains a region containing two open reading frames encoding PAL3. This construct can be transcribed as a bicistronic message. In a non-limiting example, the construct contains SEQ ID NO: 98. In some embodiments, the construct is combined with another construct expressing PheP from a separate plasmid. In some embodiments, the construct is combined with a construct expressing PheP integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct in the figure contains a PAL3 sequence, which is the original sequence of Photorhabdus chemiluminescens. In some embodiments, the PAL3 sequence is codon-optimized for expression in E. coli. In some embodiments, codon optimization is used as an additional control to fine-tune, i.e., up-regulate or down-regulate, the PAL3 levels expressed from the construct. In some embodiments, the construct is located on a plasmid, e.g., a low- or high-copy plasmid. In some embodiments, the construct is used in a biosafety system, such as the system shown in Figure 61. In some embodiments, the construct is integrated into the genome at one or more of the locations described herein. [Figure 66]Figure 1 shows a map of example integration sites within the E. coli 1917 Nissle chromosome. These sites indicate regions where circuit components can be inserted into the chromosome without disrupting expression of essential genes. A backslash ( / ) is used to indicate that the insertion occurs between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be used to create auxotrophs. In some embodiments, individual circuit components are inserted into more than one of the indicated sites. [Figure 67] Figure 1 shows three bacterial strains that constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted at different sites within the bacterial chromosome, resulting in varying degrees of brightness under fluorescent light. Unmodified E. coli Nissle (strain 4) does not produce fluorescence. [Figure 68] Figure 1 shows a graph depicting the presence of Nissle in vivo. Streptomycin-resistant Nissle was administered orally by gavage to mice without prior antibiotic treatment. After administration, fecal pellets from a total of six mice were monitored to determine the amount of administered Nissle still present in the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The lines represent the number of Nissle recovered from daily fecal samples for 10 consecutive days. [Figure 69] Figure 1 shows a bar graph depicting the retention of streptomycin-resistant Nissle over time in various compartments of the gastrointestinal tract at 1, 4, 8, 12, 24, and 30 hours after gavage. Mice were treated with approximately 10 CFU. At each time point, animals (n=4) were euthanized and the intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon were each cut into two sections. Intestinal effluent was collected, and CFU in each compartment was determined by serial dilution plating. [Figure 70A]Phenylanine concentration in SYN-PKU302 cultures over time. After 1.5 hours of growth, ATC was added to SYN-PKU302 cultures and placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. Four hours after induction, bacteria were resuspended in assay buffer containing 4 mM phenylalanine and various pH values ​​(pH range 7.25-2.25). Aliquots were removed from the cell assay every 30 minutes over a 2-hour period for phenylalanine quantification by mass spectrometry. The rate of phenylalanine degradation decreased as the pH of the assay buffer decreased in SYN-PKU302. [Figure 70B] Phenylanine concentration in SYN-PKU304 cultures over time. After 1.5 hours of growth, ATC was added to SYN-PKU304 cultures and placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. Four hours after induction, bacteria were resuspended in assay buffer containing 4 mM phenylalanine and various pH values ​​(pH range 7.25-2.25). Aliquots were removed from the cell assay every 30 minutes over a 2-hour period for phenylalanine quantification by mass spectrometry. The rate of phenylalanine degradation decreased as the pH of the assay buffer decreased in the SYN-PKU304 strain. [Figure 71] FIG. 1 shows an example schematic of the E. coli 1917 Nissle chromosome, which contains multiple mechanisms of action (MoAs). [Figure 72] FIG. 1 shows the gene organization of an example construct in which the PAL3 and pheP genes are cotranscribed under the control of an example FNR promoter (PfnrS). [Figure 73A] Figure 1 shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to an example FNR promoter (Pfnr S) and the PAL3 gene is operably linked to a strong constitutive promoter. A schematic diagram of the PAL3 gene flanked by Int5 sites in the OFF orientation (3' to 5') is shown. Any strong constitutive promoter sequence can be used. [Figure 73B]This figure shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to an example FNR promoter (Pfnr S) and the PAL3 gene is operably linked to a strong constitutive promoter. When Int5 gene expression is activated under anaerobic and / or hypoxic conditions, recombinase-mediated flipping of PAL3 to the ON orientation (5' to 3') leads to the production of PAL3 and phenylalanine metabolism. Any strong constitutive promoter sequence can be used. [Figure 74A] Figure 1 shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to the FNR promoter (PfnrS) and the gene encoding T7 RNA polymerase is operably linked to a strong constitutive promoter adjacent to recombinase sites. A schematic diagram of the T7 RNA polymerase gene flanked by Int5 sites in the OFF orientation is shown. [Figure 74B] Figure 1 shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to the FNR promoter (PfnrS) and the gene encoding T7 RNA polymerase is operably linked to a strong constitutive promoter adjacent to the recombinase site. When Int5 gene expression is activated under anaerobic and / or hypoxic conditions, the T7 RNA polymerase gene is flipped to the ON orientation. [Figure 74C] Figure 1 shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to the FNR promoter (PfnrS) and the gene encoding T7 RNA polymerase is operably linked to a strong constitutive promoter adjacent to the recombinase site. In an engineered bacterial strain containing one copy of PAL3 under the control of a T7-driven promoter (PT7), T7 RNA polymerase expression leads to the production of PAL3 and phenylalanine metabolism. [Figure 75A]

[0023] Figure 1 shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to the ParaBAD promoter (Par aBAD) and the gene encoding T7 RNA polymerase is operably linked to a strong constitutive promoter adjacent to recombinase sites.

[0024] Figure 1 shows an exemplary construct in which the Int5 recombinase gene is operably linked to the ParaBAD promoter (ParaBAD). [Figure 75B] Figure 1 shows the genetic organization of an example construct in which the Int5 recombinase gene is operably linked to the ParaBAD promoter (Par aBAD) and the gene encoding T7 RNA polymerase is operably linked to a strong constitutive promoter adjacent to the recombinase site. A schematic diagram of the T7 RNA polymerase gene flanked by Int5 in the OFF orientation is shown. When Int5 gene expression is activated under anaerobic and / or hypoxic conditions, the T7 RNA polymerase gene is flipped to the ON orientation. In genetically engineered bacterial strains containing one copy of PAL3 under the control of a T7-driven promoter, T7 RNA polymerase expression leads to the production of PAL3 and phenylalanine metabolism. [Figure 76A]

[0023] Figure 1 shows a schematic of a recombinase-based switch to activate PAL3 expression using different inducible promoters and ribosome binding sites. Recombinase expression causes recombination flipping of the PAL3 gene to the ON direction, leading to the production of PAL3 and degradation of phenylalanine. In some embodiments, the recombinase-based switch is tuned to respond to specific levels of inducer. [Figure 76B] Figure 1 shows the relationship between inducer concentration and the percentage of constructs containing PAL3 in the ON orientation. The shaded area indicates the predicted effective range of the inducer in vivo. [Figure 77A]

[0023] Figure 1 shows another non-limiting embodiment of the present disclosure in which heterologous gene expression is activated by an exogenous environmental signal. This figure depicts one embodiment of heterologous gene expression, in which in the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that binds to and activates the ParaBAD promoter (ParaB AD), inducing expression of the Tet repressor (TetR) and antitoxin. The antitoxin accumulates within the recombinant bacterial cell, while TetR prevents expression of the toxin (which is under the control of a promoter with a TetR binding site). However, in the absence of arabinose, neither the antitoxin nor TetR is expressed. In the absence of TetR to repress toxin expression, the toxin is expressed and kills the cell. This figure also shows another non-limiting embodiment of the present disclosure in which expression of an essential gene not found in the recombinant bacterium is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of essential genes under the control of the araBAD promoter, and bacterial cells cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, inducing expression of essential genes and maintaining bacterial cell viability. [Figure 77B]

[0023] Figure 1 illustrates another non-limiting embodiment of the present disclosure in which expression of a heterologous gene is activated by an exogenous environmental signal. Figure 2 illustrates another non-limiting embodiment of the present disclosure in which an antitoxin is expressed from a constitutive promoter and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that binds to and activates the araBAD promoter, inducing expression of TetR and thus preventing toxin expression. However, in the absence of arabinose, TetR is not expressed, and the toxin is expressed, ultimately outcompeting the antitoxin and killing the cell. The constitutive promoter that regulates expression of the antitoxin will be a weaker promoter than the promoter that drives expression of the toxin. The araC gene is under the control of the constitutive promoter in this circuit. [Figure 77C]

[0023] Figure 1 shows another non-limiting embodiment of the present disclosure in which heterologous gene expression is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that binds to and activates the araBAD promoter, inducing expression of the Tet repressor (TetR) and antitoxin. The antitoxin accumulates within the recombinant bacterial cell, while TetR prevents expression of the toxin (which is under the control of a promoter with a TetR binding site). However, in the absence of arabinose, neither the antitoxin nor TetR is expressed. Without TetR to repress toxin expression, the toxin is expressed and kills the cell. The araC gene is under the control of either a constitutive promoter or an inducible promoter for this circuit (e.g., the AraC promoter). [Figure 78] 1 shows one non-limiting embodiment of the present disclosure in which an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from one or more inducible promoters. The recombinase then flips the toxin gene to an active conformation, and the natural kinetics of the recombinase result in a time delay in expression of the toxin, allowing full expression of the heterologous gene. Upon expression, the toxin kills the cell. [Figure 79]

[0023] Figure 1 shows another non-limiting embodiment of the present disclosure in which an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an antitoxin, and at least one recombinase from one or more inducible promoters. The recombinase then flips the toxin gene to an active conformation, but the presence of accumulated antitoxin suppresses toxin activity. When the exogenous environmental condition or cue(s) is no longer present, expression of the antitoxin is shut off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell. [Figure 80]

[0023] Figure 1 illustrates another non-limiting embodiment of the present disclosure in which expression of a heterologous gene and at least one recombinase is activated from one or more inducible promoters by an exogenous environmental condition or one or more environmental signals. The recombinase then flips at least one excision enzyme to an active conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence and ultimately cell death. The natural dynamics of the recombinase and excision genes result in a time delay whose kinetics can be modified and optimized depending on the number and selection of essential genes to be excised, allowing cell death to occur within hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death. [Figure 81]

[0023] Figure 1 shows one non-limiting embodiment of the present disclosure in which expression of a heterologous gene and a first recombinase is activated from one or more inducible promoters by an exogenous environmental condition or one or more environmental signals. The recombinase then flips a second recombinase from its reverse orientation to an active conformation. The activated second recombinase flips the toxin gene to an active conformation, and the natural kinetics of the recombinase result in a time delay in toxin expression, allowing the heterologous gene to be fully expressed. Upon expression, the toxin kills the cell. [Figure 82]

[0023] Figure 1 illustrates one non-limiting embodiment of the present disclosure, including a plasmid stability system with a plasmid that produces both a short-lived antitoxin and a long-lived toxin. When cells lose the plasmid, the antitoxin is no longer produced and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce equal amounts of Hok toxin and short-lived Sok antitoxin. In the top panel, the cells produce equal amounts of toxin and antitoxin and are stable. In the middle panel, the cells lose the plasmid and the antitoxin begins to decay. In the bottom panel, the antitoxin completely decays and the cells die. [Figure 83]

[0023] Figure 1 illustrates the use of GeneGuard as a genetically engineered safety component. All genetically engineered DNA resides on a plasmid that can be conditionally destroyed. See, e.g., Wright et al., 2015. [Figure 84A] FIG. 1 is a schematic diagram of the wild-type clbA construct. [Figure 84B] FIG. 1 is a schematic diagram of the clbA knockout construct. [Figure 85] FIG. 1 shows a schematic representation of a secretion system based on flagellar type III secretion, in which defective flagella are used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to the N-terminal flagellar secretion signal of a native flagellar component, such that the intracellularly expressed chimeric peptide can translocate across the inner and outer membranes into the surrounding host environment. [Figure 86] Figure 1 shows a schematic of a type V secretion system for extracellular production of recombinant proteins, in which a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery, which translocates the protein across the inner membrane into the periplasm and subsequently cleaves the signal sequence. The beta-domain is recruited to the Bam complex, where it folds and inserts into the outer membrane as a beta-barrel structure. The therapeutic peptide is then threaded into the hollow pore of the beta-barrel structure in front of the linker sequence. The therapeutic peptide is released from the linker system by autocatalytic cleavage or by targeting a membrane-associated peptidase (scissors) to a complementary protease cleavage site on the linker. [Figure 87] Schematic diagram of a type I secretion system that uses HlyB (ATP-binding cassette transporter); HlyD (membrane fusion protein); and TolC (outer membrane protein), which form channels through both the inner and outer membranes, to move passenger peptides from the cytoplasm directly to the extracellular space. The C-terminal portion of HlyA, containing the secretion signal, is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of the peptide. [Figure 88]

[0023] Figure 1 shows a schematic of the outer and inner membranes of Gram-negative bacteria, as well as several targets that can be deleted to form a leaky or destabilized outer membrane, thereby facilitating the translocation of therapeutic polypeptides, such as disulfide-bond-containing therapeutic peptides derived from eukaryotic cells, into the extracellular space. Inactivating mutations in one or more genes encoding proteins that anchor the outer membrane to the peptidoglycan backbone, such as lpp, ompC, ompA, ompF, tolA, tolB, pal, and / or one or more genes encoding periplasmic proteases, such as degS, degP, and nlpl, result in a leaky phenotype. Combinations of mutations can synergistically enhance the leaky phenotype. [Figure 89] Figure 1 shows a modified type 3 secretion system (T3SS) that allows bacteria to inject secreted therapeutic proteins into the gastrointestinal lumen. An inducible promoter (small arrow, top), e.g., the FNR-inducible promoter, drives expression of a T3 secretion system gene cassette (three large arrows, top), which produces an apparatus for secreting tagged peptides from the cell. An inducible promoter (small arrow, bottom), e.g., the FNR-inducible promoter, drives expression of a regulatory factor, e.g., T7 polymerase, which then activates expression of a tagged therapeutic peptide (hexagon). [Figure 90A] 1 is a schematic diagram of the genetic organization of an exemplary circuit of the present disclosure for expressing a therapeutic polypeptide secreted using components of the flagellar type III secretion system. The therapeutic polypeptide of interest, such as PAL and / or LAAD, is assembled behind the fliC-5'UTR and driven by the native fliC and / or fliD promoters. In another embodiment, inducible promoters, such as oxygen-level-dependent promoters (e.g., FNR-inducible promoters), and promoters induced by metabolites that may or may not be naturally present in the gastrointestinal tract (e.g., exogenously added), such as arabinose, can be used. The therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium-copy plasmid) or integrated into the fliC locus (thereby deleting all or part of fliC and / or fliD). [Figure 90B] 1 is a schematic diagram of the genetic organization of an exemplary circuit of the present disclosure for expressing a therapeutic polypeptide secreted using components of the flagellar type III secretion system. The therapeutic polypeptide of interest, such as PAL and / or LAAD, is assembled behind the fliC-5'UTR and driven by the native fliC and / or fliD promoters. In another embodiment, inducible promoters, such as oxygen-level-dependent promoters (e.g., FNR-inducible promoters), and promoters induced by metabolites that may or may not be naturally present in the gastrointestinal tract (e.g., exogenously added), such as arabinose, can be used. The therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium-copy plasmid) or integrated into the fliC locus (thereby deleting all or part of fliC and / or fliD). Optionally, the N-terminal portion of FliC is included in the construct. [Figure 90C] 1 is a schematic diagram of the genetic organization of an exemplary circuit of the present disclosure for expressing a therapeutic polypeptide secreted using components of the flagellar type III secretion system. The therapeutic polypeptide of interest, such as PAL and / or LAAD, is assembled behind the fliC-5'UTR and driven by a Tet-inducible promoter. In another embodiment, inducible promoters, such as oxygen-level-dependent promoters (e.g., FNR-inducible promoters), and promoters induced by metabolites that may or may not be naturally present in the digestive tract (e.g., exogenously added), such as arabinose, can be used. The therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium-copy plasmid) or integrated into the fliC locus (thereby deleting all or part of fliC and / or fliD). Optionally, the N-terminal portion of FliC is included in the construct. [Figure 91A]Schematic diagram of the genetic organization of an exemplary circuit of the present disclosure for expression of a therapeutic polypeptide secreted via the diffusive outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypic N-terminal Sec- or Tat-dependent secretion signal that is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include Sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the engineered bacteria contain deletions in one or more of lpp, pal, tolA, and / or nlpI. Optionally, periplasmic proteases, including but not limited to degP and ompT, are also deleted, e.g., to increase polypeptide stability in the periplasm. An FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by the Tet promoter and a promoter induced by a metabolite that may or may not be naturally present in the gastrointestinal tract (e.g., that may be exogenously added), such as arabinose. [Figure 91B]Schematic diagram of the genetic organization of an exemplary circuit of the present disclosure for expression of a therapeutic polypeptide secreted via the diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypic N-terminal Sec- or Tat-dependent secretion signal that is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include Sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the engineered bacteria contain deletions in one or more of lpp, pal, tolA, and / or nlpI. Optionally, periplasmic proteases, including but not limited to degP and ompT, are also deleted, e.g., to increase polypeptide stability in the periplasm. An FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by inducible promoters, such as oxygen level-dependent promoters (e.g., FNR-inducible promoters), and promoters induced by metabolites that may or may not be naturally present in the digestive tract (e.g., that may be exogenously added), such as arabinose. [Figure 92] Schematic representation of the design-build-test cycle. The steps are: 1. Define disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotics; 5. Activate circuits in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimized circuits in animal disease models; 9. Assimilate with the microbiome; and 10. Develop understanding of in vivo pharmacokinetics (PK) and dosing regimens. [Figure 93]FIG. 1A shows a schematic of a non-limiting manufacturing method for upstream and downstream production of genetically engineered bacteria of the present disclosure. Parameters for Starter Culture 1 (SC1): loopful of glycerol stock, duration overnight, temperature 37°C, shaking at 250 rpm. FIG. 1B shows a schematic of a non-limiting manufacturing method for upstream and downstream production of genetically engineered bacteria of the present disclosure. Parameters for Starter Culture 2 (SC2): 1 / 100 dilution of SC1, duration 1.5 hours, temperature 37°C, shaking at 250 rpm. FIG. 1C shows a schematic of a non-limiting manufacturing method for upstream and downstream production of genetically engineered bacteria of the present disclosure. Parameters for the production bioreactor: Inoculation-SC2, temperature 37°C, pH setting 7.00, pH deadband 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation / gas FLO, agitation limit 300-1200 rpm, gas FLO limit 0.5-20 standard liters per minute, duration 24 hours. FIG. 1D is a schematic diagram of a non-limiting method for upstream and downstream production of genetically engineered bacteria of the present disclosure. Harvesting parameters: centrifugation at 4000 rpm for 30 minutes, wash 1× in 10% glycerol / PBS, centrifugation, resuspension in 10% glycerol / PBS. FIG. 1E is a schematic diagram of a non-limiting method for upstream and downstream production of genetically engineered bacteria of the present disclosure. Vial filling / storage parameters: aliquots of 1-2 mL, -80°C. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure relates, inter alia, to genetically engineered bacteria, pharmaceutical compositions thereof, and high phenylalanine In some embodiments, the present invention includes methods for modulating and treating disorders associated with vasodilators. Genetically engineered bacteria encode non-natural phenylalanine lyase (PAL) and a gene for processing and reducing phenylalanine in mammals. In some embodiments, the genetically engineered bacteria can In some embodiments, the gene encoding the promoter is further included. The bacterium may also contain a gene encoding L-AAD. Biosafety and / or biocontainment, e.g., kill switches, gene gases The gene may include one or more gene sequences associated with a food system, and / or an auxotrophy. Expression of these gene sequences can be achieved using any of the promoter systems disclosed herein. A variety of promoter systems can be used to regulate the expression of one or more different promoters. The same promoter can regulate different genes, and different genes can be They can be different copies of the same promoter for expression of different genes. This may involve the combined use of different promoters to regulate the By using different regulatory or promoter systems to control gene expression , flexibility (e.g., the ability to differentially regulate gene expression under different environmental conditions and / or This provides the ability to differentially regulate gene expression over time and allows for the "fine-tuning" of gene expression. Any or all of these controls may affect gene expression and / or or may help optimize bacterial growth. and the use of genetically engineered bacteria and the bacteria for the treatment and / or prevention of conditions associated with The pharmaceutical composition containing the compound can be used to metabolize phenylalanine in the body into non-toxic molecules. In certain embodiments, the present invention can be used to treat and / or prevent disorders associated with hyperphenylalaninemia. Compositions comprising genetically engineered bacteria may be used in the disclosed methods for preventing .

[0017] In order that this disclosure may be more readily understood, certain terms are first defined. These definitions The definitions should be read in light of the remainder of the disclosure and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific Terms have the same meaning as commonly understood by one of ordinary skill in the art. are described throughout the detailed description.

[0018] "Hyperphenylalaninemia", "hyperphenylalaninemia" "hyperphenylalaninemic" and "hyperphenylalaninemic" "Excess phenylalanine" refers to increased or abnormally high levels of phenylalanine in the body. are used interchangeably herein to refer to high phenylalanine. 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, or at least 12 mg / dL, at least 14 mg / dL, at least 16 mg / dL, at least a blood level of 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 (PEP) Phenyleketonuria, atypical phenylketonuria, persistent mild hyperphenylalaninemia, non-phenylalanine Phenylketonuria hyperphenylalaninemia, phenylalanine hydroxylase deficiency, Cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase These include deficiency, and Segawa disease. Affected individuals suffer from progressive and irreversible neurological deficits, Mental retardation, encephalopathy, epilepsy, eczema, slow growth, microcephaly, tremors, limb spasms, and / or hypotension They may suffer from hyperpigmentation (Leonard 2006). It may also be secondary to other conditions, such as liver disease.

[0019] "Phenylalanine ammonia lyase" and "PAL" are enzymes that convert phenylalanine to trans-phenylalanine metabolic enzymes that convert or process phenylalanine into cinnamic acid and ammonia ( trans-cinnamic acid has low toxicity and is effective in mammals. PAL is converted by liver enzymes in the blood into hippuric acid, which is secreted in the urine. PAL enzyme activity can be substituted for THB cofactors. In some embodiments, PAL does not require factor activity. In an alternative embodiment, PAL is encoded by the P gene from a eukaryotic species. In some embodiments, PAL is encoded by, but not limited to, the AL gene. , Achromobacter xyloso xidans, Pseudomonas aeruginosa ginosa), Photorhabdus lumi nescens), Anabaena variabilis ), and Agrobacterium tumefaciens (Agrobacterium tu It is encoded by the PAL gene from bacterial species, including P. mefaciens. In some embodiments, PAL is derived from the PAL gene from Anabaena variabilis. and is referred to herein as "PAL1" (Moffitt et al., 200 7 years). In some embodiments, PAL is derived from Photorhabdus luminescence. It is encoded by the PAL gene, referred to herein as "PAL3" (Will In some embodiments, PAL is produced from yeast species, e.g., Rhodotorula spp. Rhodosporidium toruloides It is encoded by the PAL gene derived from the genus (Gilbert et al., 1985). In some embodiments, the PAL is derived from a plant species, such as Arabidopsis thaliana. s thaliana) (Wanner et al. (1995). Any suitable nucleotide and amino acid sequence of PAL, or its function. Functional fragments may be used.

[0020] "Phenylalanine hydroxylase" and "PAH" are enzymes that bind to the cofactor tetrahydrobiotin. The aromatic side chain of phenylalanine is converted to tyrosine in the human body in conjunction with pterin. It is used to refer to the enzymes that catalyze hydroxylation. Human genes encoding PAHs is located on the long (q) arm of chromosome 12 between positions 22 and 24.2. The nucleic acid sequences are highly conserved among mammals. The full-length human cDNA sequence for PAH is known and widely available. It was reported in 1985 (Kwok et al., 1985). Active fragments of PAHs are also known. (e.g., Kobe et al., 1997).

[0021] "L-amino acid deaminase" and "LAAD" are enzymes that decompose L-amino acids, Stereospecific oxidative deamination of L-amino acids to produce ammonia and hydrogen peroxide For example, LAAD is the enzyme that catalyzes the phenylalanine Numerous LAAD enzymes are known in the art. Many of them belong to the genera Proteus and Providencia. encia, and Morganella, or toxins LAAD is characterized by a rapid kinetics of phenylalanine degradation (Hou et al., A ppl Microbiol Technol.2015 October;99(20):83 pp. 91~402; “Production of phenylpyruvic aci d from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis:co mparison of enzymatic and whole-cell bio transformation approaches) for most eukaryotes and protozoa. While L-amino acid deaminases in proteolytic organisms are extracellular, Proteus species LAAD has an enzymatic activity It is localized in the plasma membrane (inner membrane) facing outward into the periplasmic space where the cytoplasmic As a result, phenylalanine transport across the inner membrane into the cytoplasm is mediated by the Proteus LAAD. Phenylanine is not required for phenylalanine degradation, which is mediated by They are readily imported into the periplasm through the outer membrane without the need for transporters, improving substrate availability. Eliminates the need for a transporter that improves

[0022] In some embodiments, the genetically engineered bacteria may be, but are not limited to, Proteus sp., LAAD genes from bacterial species, including bacteria 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 LAAD encoded by the engineered bacteria faces the periplasmic space and is located in the periplasmic space. It is localized to the cell membrane where it exerts its catalytic activity.

[0023] "Phenylalanine Metabolizing Enzymes" or "PMEs" are enzymes that break down phenylalanine. The term "phenylalanine" is used to refer to any enzyme known in the art that can PMEs are limited in that they can be encoded by genetically engineered bacteria. However, phenylalanine hydroxylase (PAH), phenylalanine ammonia ase (PAL), aminotransferase, L-amino acid deaminase (L-AAD) ), and phenylalanine dehydrogenase.

[0024] Phenylanine hydroxylase, phenylalanine dehydrogenase or amino The reaction with transferase requires a cofactor, but L-AAD and PAL require an additional cofactor. In some embodiments, the enzyme is produced by genetically engineered bacteria without the need for any cofactors. The encoded PME requires a cofactor. In some embodiments, the cofactor is a genetic It may be provided simultaneously or sequentially with the administration of the genetically 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. , genetically engineered bacteria encode phenylalanine dehydrogenase. In embodiments, the genetically engineered bacteria encode an aminotransferase. In some embodiments, the PME encoded by the genetically 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's fluorogenicity. The rate of phenylalanine degradation 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, the expression of a gene that is produced by a genetically engineered bacterium. 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.

[0025] In some embodiments, the catalytic activity of the PME is dependent on oxygen levels. In this form, PMEs are catalytically active under microaerobic conditions. D catalytic activity is oxygen dependent. In some embodiments, the LAAD is grown under microaerobic conditions, e.g., under oxygen. In some embodiments of the present invention, the PME is active under hypoxic conditions, e.g., in the colon. Functions at very low levels of oxygen found in the absence of oxygen. As a typical example, PAL activity does not depend on the presence of oxygen.

[0026] In certain embodiments, the new or improved PME is a PME that is known or The recombinant proteins encoded by the genetically engineered bacteria can be identified according to the methods described herein. In some embodiments, the enzyme encoded by the genetically engineered bacterium is In some embodiments, the enzyme is a wild-type enzyme isolated from a virus, prokaryote, or eukaryote. In this study, the enzyme sequence enhances one or more specific properties of the enzyme, such as stability or catalytic activity. The amino acid sequence of the ... present invention has been further modified or mutated to enhance the amino acid sequence of the present invention.

[0027] "Phenylalanine metabolites" are those produced as a result of the breakdown of phenylalanine. Metabolites are products of the synthesis of phenylalanine by enzymes using phenylalanine as a substrate. It is a protease that acts either directly from phenylalanine or on phenylalanine metabolite substrates. In some embodiments, the protease may be produced indirectly by a different enzyme downstream in the metabolic pathway. , a phenylalanine metabolite produced by genetically engineered bacteria encoding a PME will be done.

[0028] In some embodiments, phenylalanine metabolites are derived from PAH activity, e.g., genetic They arise directly or indirectly from PAHs produced by genetically engineered bacteria. In some embodiments, the metabolite is tyrosine. Nin metabolites accumulate in the blood or urine of PKU patients due to defective PAH activity. Non-limiting examples of such PKU metabolites include phenylpyruvate and phenyl-lactam. Other examples include phenylacetate, phenylethylamine, and phenylalanine. Contains cetyl glutamine.

[0029] In some embodiments, phenylalanine metabolites protect against PAL action, e.g., genetic It arises directly or indirectly from PAL produced by genetically engineered bacteria. Non-limiting examples of such PAL metabolites are trans-cinnamic acid and hippuric acid. In some embodiments, phenylalanine metabolites protect against LAAD effects, e.g., by inhibiting the expression of phenylalanine. It arises directly or indirectly from LAAD produced by engineered bacteria. Examples of important LAAD metabolites are phenylpyruvate and phenyllactic acid.

[0030] "Phenylalanine transporter" transports phenylalanine into bacterial cells. It is used to refer to membrane transport proteins that can transport proteins (see, e.g., Pi et al., 1991). In Escherichia coli, the pheP gene is , which encodes a high-affinity phenylalanine-specific permease responsible for phenylalanine transport. (Pi et al., 1998). In some embodiments, the phenylalanine transporter Examples include, but are not limited to, Acinetobacter calcoaceticus ter calcoaceticus, Salmonella enterica by the pheP gene from bacterial species, including E. coli, E. enterica, and Escherichia coli. Other phenylalanine transporters are encoded by the aroP gene. It encodes a phosphodiesterase that transports three aromatic amino acids, including phenylalanine, with high affinity and transports Phe Aagenes, together with P, are responsible for the largest part of phenylalanine uptake. ral) amino acid permease. In addition, low levels of phenylalanine transport activity The activity of the LIV-I / LS system was traced to the activity of the two peripheral Protein-binding proteins, LIV-binding protein (LIV-I system) and LS-binding protein The branched-chain amino acid transesterification protein (LS system) and the membrane component LivHMGF In some embodiments, the phenylalanine transporter is a bacterial In some embodiments, the phenylalanine is encoded by the aroP gene from the species. The alanine transporter is a member of the LIV- and LS-binding proteins and In some embodiments, the LivHMGF gene is encoded by a LivHMGF gene derived from a bacterial species. The genetically engineered bacteria were selected from the pheP, aroP, and LIV-I / LS systems. It contains more than one phenylalanine transporter.

[0031] "Phenylalanine" and "Phe" have the formula C6H5CH2CH(NH2)COOH It is used to refer to amino acids with the following structure: phenylalanine, tyrosine, dopamine L-phenylalanine is a precursor to phenylalanine, norepinephrine, and epinephrine. It is an amino acid and the form of phenylalanine found primarily in food proteins. The isomer D-phenylalanine is found in smaller amounts in food proteins, while DL-phenylalanine Phenylalanine is a combination of both forms. It refers to one or more of the following: phenylalanine, D-phenylalanine, and DL-phenylalanine. It is possible.

[0032] "Operably linked" refers to the expression of nucleic acid sequences, e.g., nucleic acid sequences acting in cis. The term refers to a gene encoding, for example, PAL, linked to a regulatory region sequence so as to enable the gene to Regulatory regions can induce transcription of a gene of interest and include promoter sequences, enhancers, and sensor sequences, response elements, protein recognition sites, inducible elements, promoter control regulatory elements, protein binding sequences, 5' and 3' untranslated regions, transcription start site, termination sequence It is a nucleic acid that may contain sequences, polyadenylation sequences, and introns.

[0033] An "inducible promoter" is a promoter that is operably linked to one or more genes. It refers to a regulatory region, where expression of a gene is increased in the presence of an inducer of said regulatory region.

[0034] A "directly inducible promoter" refers to a promoter that encodes a phenylalanine metabolic enzyme, e.g., PAL and a regulatory region operably linked to a gene encoding said regulatory region. In the presence of an indirectly inducible promoter, phenylalanine metabolic enzymes are expressed. "-" refers to two or more regulatory regions, e.g., a first molecule, e.g., a phenylalanine metabolic enzyme A transcriptional regulator capable of regulating a second regulatory region operably linked to a gene encoding the protein. It refers to a regulatory system comprising a first regulatory region operably linked to a gene encoding a factor. In the presence of an inducer of the first regulatory region, the second regulatory region can be activated or repressed, Activates or represses the expression of phenylalanine metabolic enzymes. Both inducible and indirectly inducible promoters are referred to as "inducible promoters." -" is included in this.

[0035] "Exogenous environmental conditions" or "environmental conditions" refer to conditions under which the promoters described herein are directly involved. The phrase refers to a setting or situation in which the engineered microorganism is or is indirectly induced. refers to environmental conditions that are external to the host but endogenous or natural to the host target environment. Thus, "exogenous" and "endogenous" refer to the effects that environmental conditions have on the mammalian body. refers to environmental conditions that are endogenous to the organism but external or exogenous to the intact microbial cell. In some embodiments, the exogenous environmental conditions are those of the mammalian digestive system. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low oxygen, microorganisms, such as the environment of the mammalian gastrointestinal tract. In some embodiments, the exogenous environmental conditions are aerobic or anaerobic conditions. or molecules or metabolites that are specific to the mammalian gastrointestinal tract in disease states, such as protease inhibitors. In some embodiments, the exogenous environmental conditions refer to the presence of a tissue-specific or or a disease-specific metabolite or molecule. In some embodiments, the exogenous environmental condition is In some embodiments, the genetically engineered microorganisms of the present disclosure are pH-dependent. In some embodiments, the genetically engineered microorganism of the present disclosure comprises an endogenous promoter. In some embodiments, the bacterium senses oxygen levels and comprises an oxygen level-dependent promoter. Different signaling pathways respond to different oxygen levels. can be induced and occur with different kinetics.

[0036] As used herein, "exogenous environmental conditions" or "environmental conditions" also refer to conditions that are manipulated. refers to the external setting or situation or environmental conditions of the microorganisms in vitro "Exogenous environmental conditions" also refer to conditions that exist during the growth, production, and manufacturing of an organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, and low-acid conditions. These conditions include conditions for growth under normal conditions and other conditions under a set oxygen concentration. , chemical and biological factors such as tetracycline, arabinose, IPTG, and rhamnose in the culture medium. Such conditions also include the presence of nutritional inducers and / or nutritional inducers. This also includes the temperature at which the microorganism is grown prior to administration. For example, when using a particular promoter system, Some temperatures allow the expression of the payload, while others do not. Temperature and medium composition influence such exogenous environmental conditions. Growth rate, induction rate of PME (e.g., PAL or LAAD), transporter (e.g., the induction rate of other regulators (e.g., FNRS24Y), as well as affecting the overall viability and metabolic activity of the strain during strain production.

[0037] An "oxygen level dependent promoter" or "oxygen level dependent regulatory region" refers to one or more It refers to a nucleic acid sequence to which one or more oxygen level-sensitive transcription factors can bind, and the corresponding transcription factors Binding and / or activation activates downstream gene expression.

[0038] Examples of oxygen level dependent transcription factors include, but are not limited to, FNR, ANR, and DNR The corresponding FNR-responsive promoter, ANR-responsive promoter, and D NR-responsive promoters are known in the art (e.g., 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.

[0039] In a non-limiting example, the promoter (PfnrS) is a promoter that is highly oxidative when the environment is low or completely oxygen-rich. E. coli Nissle fumarate and The nitrate reductase gene S (fnrS) was derived from the nitrate reductase gene (Durand and Storz, 2002). 010; Boysen et al., 2010). The PfnrS promoter is The naturally occurring global transcription factor FNR regulates anaerobic and / or hypoxic responses in the Under anaerobic and / or hypoxic conditions, FNR forms dimers and is activated. , which binds to specific sequences in the promoters of specific genes under its control, thereby However, under aerobic conditions, oxygen activates the expression of FNR dimers. It reacts with the iron-sulfur clusters present in the PfnrS complex, converting them to an inactive form. Inducible promoters are employed to modulate protein or RNA expression. In this application, PfnrS is referred to as FNRS, fnrS, FNR, and P-FNRS promoter. Interchangeable as the promoter and other such related designations denoting the promoter PfnrS. Used for. [Table 1]

[0040] As used herein, a "non-naturally occurring" nucleic acid sequence is one that is not normally found in bacteria. Additional copies of a nucleic acid sequence, e.g., an endogenous sequence, or a different species, strain, or substrain of bacteria Heterologous sequences, such as sequences derived from bacteria of the same subtype, or modified and unmodified sequences compared to unmodified sequences derived from bacteria of the same subtype. In some embodiments, a non-naturally occurring nucleic acid sequence refers to a sequence that has been modified and / or mutated. is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-naturally occurring nucleic acid sequence may be a regulatory region, promoter, gene, and / or gene cluster. In some embodiments, the gene may be one or more genes in the set. "Non-naturally occurring" refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-naturally occurring nucleic acid sequence may be present on a plasmid or chromosome. Even if multiple copies of the promoter, gene, and / or gene cassette are present in the bacterium, Often, one or more of the regulatory region, promoter, gene, and / or gene cassette The number of copies may be mutated or otherwise varied as described herein. In some embodiments, the genetically engineered bacteria may contain a gene encoding a gene encoding a gene for which the gene is to be expressed. or to contain multiple different components of a gene cassette that perform multiple different functions. Multiple copies of the same regulatory region, promoter, gene, and / or gene cassette In some embodiments, the genetically engineered bacteria of the invention are engineered to contain a directly or indirectly inducible promoter not naturally associated with said gene, e.g. For example, the FNR promoter or L operably linked to the ParaBAD promoter, which is operably linked to AAD It contains genes encoding phenylalanine metabolic enzymes.

[0041] A "constitutive promoter" refers to a promoter under the control of and / or to which it is operably linked. A promoter refers to a promoter capable of promoting the continuous transcription of a coding sequence or gene. Constitutive promoters and variants are well known in the art and include, but are not limited to: BBa_J23100, constitutive E. coli σ S promoter (e.g., osmY promoter) (International Genetically Engineered InnerGEM (Integrated Genomic Model) Standard Biological Parts Registry (Regis try of Standard Biological Parts) Name BBa_J 45992;BBa_J45993)), constitutive E. coli σ 32 promoters (e.g., h tpG heat shock promoter (BBa_J45504), constitutive E. coli σ 70 P promoter (e.g., lacq promoter (BBa_J54200; BBa_J560 15), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951 ), GlnRS promoter (BBa_K088007), lacZ promoter (BB a_K119000;BBa_K119001);M13K07 gene I promoter ( BBa_M13101); M13K07 gene II promoter (BBa_M13102 ), M13K07 gene III promoter (BBa_M13103), M13K07 gene Gene IV promoter (BBa_M13104), gene V promoter (M13K07) BBa_M13105), M13K07 gene VI promoter (BBa_M13106 ), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110), constitutive Bacillus subtilis (Bacillus subtilis btilis)σ A A promoter (e.g., promoter veg(BBa_K14301 3), promoter 43 (BBa_K143013), P liaG (BBa_K8230 00), P lepA (BBa_K823002), P veg (BBa_K823003) ), constitutive Bacillus subtilis σ B A promoter (e.g., promoter ctc(B Ba_K143010), promoter gsiB (BBa_K143011)), Salmonella Salmonella promoters (e.g., Pspv2(B) from Salmonella) Ba_K112706), Pspv derived from Salmonella (BBa_K112707), The bacteriophage T7 promoter (e.g., T7 promoter (BBa_I71207 4;BBa_I719005;BBa_J34814;BBa_J64997;BBa_ K113010;BBa_K113011;BBa_K113012;BBa_R008 5;BBa_R0180;BBa_R0181;BBa_R0182;BBa_R018 3;BBa_Z0251;BBa_Z0252;BBa_Z0253)), Bacteriof phage SP6 promoter (e.g., SP6 promoter (BBa_J64998)), and functional fragments thereof.

[0042] The "digestive tract" is responsible for the movement and digestion of food, the absorption of nutrients, and the excretion of waste products. Refers to organs, glands, ducts, and systems. In humans, the digestive tract begins at the mouth and ends at the anus. The gastrointestinal (GI) tract further includes the esophagus, stomach, small intestine, and large intestine. The GI tract also includes the spleen. The upper gastrointestinal tract includes the esophagus, stomach, and accessory organs and glands, such as the liver, gallbladder, and pancreas. The lower gastrointestinal tract includes the remainder of the small intestine, namely the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. It can be found throughout, for example, the gastrointestinal tract, particularly in the intestines.

[0043] In some embodiments, the genetically engineered bacteria are active in the gastrointestinal tract (e.g., In some embodiments, the genetically engineered cells The bacteria are active in the large intestine (e.g., express one or more PMEs). In embodiments, the genetically engineered bacteria are active in the small intestine (e.g., one or more In some embodiments, the engineered bacteria are capable of expressing a PME in the small intestine and Without wishing to be bound by theory, it is believed that the breakdown of phenylalanine The answer is that amino acid absorption, such as phenylalanine absorption, occurs in the small intestine. By preventing or reducing the uptake of phenylalanine into the blood, Elevated Phe levels and the resulting Phe toxicity can be avoided. Extensive enteric circulation between the gut and the body regulates systemic phenylalanine removal in PKU. This may be possible (e.g., as described by Chang et al. in a rat model of PKU). (Chang et al., "A new theory of enterorecirculation" ulation of amino acids and its use for d epleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria; Artif Cells Blood Substit Immobil Biotechnol. 1995; 23(1):1-21). Phenylanine derived from blood enters the small intestine. They circulate (see, for example, Figure 39) and can be cleared by active bacteria at the site. In some embodiments, the engineered bacteria are passed through the small intestine. The genetically engineered bacteria have an increased residence time in the small intestine. In some embodiments, the genetically engineered bacteria colonize the small intestine. The engineered bacteria do not colonize the small intestine. In some embodiments, the engineered bacteria In some embodiments, the genetically engineered cells have a longer residence time in the gastrointestinal tract. The fungus colonizes small intestines. The engineered bacteria do not colonize the gastrointestinal tract.

[0044] As used herein, the term "hypoxia" refers to a condition in which the level of oxygen (O2) present in the atmosphere is too low. means to refer to a level, amount, or concentration of oxygen that is lower than the level, amount, or concentration of oxygen (e.g., For example, <21% O2; <160 torr O2). Hence the term "hypoxic conditions (multiple "Hypoxic" or "hypoxic environment" means a condition containing oxygen levels lower than those present in the atmosphere. In some embodiments, the term "hypoxia" refers to the gastrointestinal tract of a mammal. , e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, It is meant to refer to the level, amount, or concentration of oxygen (O2) found in the rectum and anal canal. In some embodiments, the term "hypoxia" refers to an oxygen level of 0 to 60 mmHg ( 0 to 60 torr O2) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2 4, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg O2). means to refer to the level, amount, or concentration of, or any and all fractional increases thereof (e.g., 0.2 mmHg, 0.5 mmHg O2, 0.75 mmHg O2, 1 0.25mmHg O2, 2.175mmHg O2, 3.45mmHg O2, 3.75 mmHg O2, 4.5mmHg O2, 6.8mmHg O2, 11.35mmHg Examples of O2 include 46.3mmHg, 58.75mmHg, and others. The decimal numbers are provided here for illustrative purposes and are not meant to be limiting in any way. (It does not mean that the oxygen level is below about 60 mmHg.) In some embodiments, "hypoxia" refers to a blood pressure of The term "hypoxia" also refers to a hypoxic state of 0 to about 60 mmHg of O2. O2 level, volume, or concentration between 0mmHg and 60mmHg inclusive Degree range, e.g., 0-5mmHg O2, <1.5mmHg O2, 6-10mmHg , <8mmHg, 47-60mmHg, etc., but these exemplary ranges are are listed here for illustrative purposes and are not meant to be limiting in any way. For example, Albenberg et al., Gastroenterology, vol. 147(5) ): pp. 1055-1063 (2014); Bergofsky et al., J Clin. I nvest., vol. 41(11): p. 1971-1980 (1962); Crompto n et al., J Exp. Biol., 43:473-478 (1965); He et al. PNAS (USA), 96:4586-4591 (1999); McKeown, Br. J. Radiol., vol. 87:20130676(2014)(doi:1 0.1259 / brj.20130676). The oxygen levels found in the digestive tracts of mammals have been discussed, and these publications In some embodiments, the term "hypoxia" is found in mammalian organs or tissues other than the digestive tract, such as the urogenital tract and tumor tissue. The term "oxygen" refers to the level, amount, or concentration of oxygen (O2) in the organ or tissue in question, and refers to the amount, amount, or concentration of oxygen in the organ or tissue in question. at hypoxic or anoxic levels In some embodiments, "hypoxia" refers to partially aerobic, semi-aerobic (se microaerobic, nanoaerobic, ultraaerobic aerobic, microoxic, hypoxic, no The level or amount of oxygen (O2) present in anoxic and / or anaerobic conditions For example, Table A summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (O2) is Dissolved oxygen ("DO") refers to the level of free, non-compound oxygen (O2) present in a liquid. It is expressed as a quantity, typically in milligrams per liter (mg / L) or parts per million (ppm ; 1 mg / L = 1 ppm) or umole (1 μmol O2 = 0 022391mg / L O2) reported. Fondriest Enviro nmental, Inc., “Dissolved Oxygen,” Fundamentals of Environmental Measurement , November 19, 2013, www.fondriest.com / environme ntal-measurements / parameters / water-quali In some embodiments, "hypoxia" refers to The term refers to a level, amount, or concentration of oxygen (O2) below approximately 6.0 mg / L DO. For example, 6.0mg / L, 5.0mg / L, 4.0mg / L, 3.0mg / L, 2.0mg / L, 1.0mg / L, or 0mg / L, as well as decimals thereof, e.g. For example, 3.25mg / L, 2.5mg / L, 1.75mg / L, 1.5mg / L, 1.2 5mg / L, 0.9mg / L, 0.8mg / L, 0.7mg / L, 0.6mg / L, 0. 5mg / L, 0.4mg / L, 0.3mg / L, 0.2mg / L and 0.1mg / L DO. These illustrative decimals are provided for illustrative purposes and are by no means The oxygen level in a liquid or solution is not meant to be limited to that of air saturation. It may be reported as a percentage or as a percentage of oxygen saturation (dissolved oxygen in solution) (O2) concentration and the maximum for a solution at constant temperature, pressure, and salt concentration under stable equilibrium (ratio to dissolved oxygen) A well-oxygenated solution that contains no oxygen producers or consumers. (e.g., mixed and / or stirred solution) is 100% air saturated. In the present embodiment, the term "hypoxia" refers to an air saturation below 40%, 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% refer to air saturation This means that any and all decimal increments (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 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04% , 0.032%, 0.025%, 0.01%, etc.) and between 0 and 40% (0% and 40 Any range of oxygen saturation levels (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-10%, 5-10%, 1 0-15%, 15-20%, 20-25%, 25-30%, etc.) are listed here The example decimals and ranges provided are for illustrative purposes only and are not meant to be limiting in any way. In some embodiments, the term "hypoxia" refers to an oxygen level below 9%. Saturation, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0% O2 saturation means to refer to the sum of any and all decimal increments (e.g., 6.5%, 5 0.0%, 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 0.075%, 0.058%, 0.04%, 0.032%, 0.025%, 0.01%, etc. ) as well as any range of oxygen saturation levels between 0 and 9% (inclusive) (e.g. , 0~5%, 0.05~0.1%, 0.1~0.2%, 0.1~0.5%, 0.5~2. These include 0%, 0-8%, 5-7%, 0.3-4.2% O2, etc. The exemplary numbers and ranges are for illustrative purposes only and are not meant to be limiting in any way. It is not something that can be done.

[0045] [Table 2-1] Table A [Table 2-2]

[0046] As used herein, the term "gene" or "gene sequence" refers to a genetic It is meant to refer to a gene sequence, e.g., a nucleic acid sequence. A genetic sequence is a complete or partial gene sequence. A gene, gene sequence, or genetic sequence is a protein or It is meant to include sequences that encode a polypeptide, and also to refer to a protein or polypeptide. Genetic sequences that do not code for a gene, such as regulatory sequences, leader sequences, signal sequences, or is meant to include other non-protein coding sequences.

[0047] "Microorganism" means a microscopic, ultramicroscopic, or submicroscopic organism that is typically a single cell. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, In some embodiments, the microorganisms are selected from the group consisting of one or more of the following: or multiple therapeutic molecules or proteins ("engineered microorganisms"). In certain embodiments, the microorganisms may extract specific metabolic products or other nutrients from their environment, e.g., the digestive tract. In certain embodiments, the microorganisms are engineered to absorb and catabolize certain beneficial compounds. synthesize novel metabolites or other compounds (synthetic or naturally occurring) and then analyze them in their environment. In certain embodiments, the engineered microorganism is engineered to release the In certain embodiments, the engineered microorganism is an engineered virus.

[0048] "Non-pathogenic bacteria" means bacteria that are unable to cause disease or adverse reactions in the host. In some embodiments, non-pathogenic bacteria are gram-negative bacteria. In some embodiments, the non-pathogenic bacteria are gram-positive bacteria. Pathogenic bacteria are commensal bacteria present in the normal flora of the gastrointestinal tract. Examples of non-pathogenic bacteria include Bacillus sp., including but not limited to Bacillus sp., Bacter sp. oides), Bifidobacterium, Breviva Brevibacteria, Clostridium um), Enterococcus, Escherichia erichia, Lactobacillus, Lactococcus Lactococcus, Saccharomyces, and and Staphylococcus, e.g., Bacillus spp. Bacillus coagulans, Bacillus subtilis, Bacteria Bacteroides fragilis, Bacteroides Bacteroides subtilis, Bacteroides teta Iotaomicron (Bacteroides thetaiotaomicron), Bacteroides Bifidobacterium bifidum ), Bifidobacterium infantis (Bifidobacterium in fantis, Bifidobacterium lactis lactis, Bifidobacterium longum longum, Clostridium butyricum ricum), Enterococcus faecium um), Escherichia coli, Lactobacillus acidophilus Lactobacillus acidophilus, Lactobacillus bulga Lactobacillus bulgaricus, Lactobacillus bulgaricus Lactobacillus casei, Lactobacillus johnsonii tobacillus johnsonii), Lactobacillus paracasei (Lact obacillus paracasei, Lactobacillus plantarum (Lact obacillus plantarum, Lactobacillus reuteri (Lactob acillus reuteri, Lactobacillus rhamnosus (Lactobaci Lactobacillus rhamnosus, Lactobacillus lactis s lactis), and Saccharomyces boulardii boulardii) (Sonnenborn et al., 2009; Dinle Yici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,7 97; 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.

[0049] "Probiotics" are microorganisms that confer a health benefit to the host organism by containing adequate amounts of these microorganisms. It is used to refer to live, non-pathogenic microorganisms, such as bacteria, that can be killed. In some embodiments, the host organism is a mammal. The target is humans. Several species, strains, and / or subtypes of non-pathogenic bacteria are currently Examples of probiotic bacteria include, but are not limited to: However, Bifidobacteria, Escherichia, Lactobacillus Bacillus spp., and Saccharomyces spp., e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, E. coli strain Nissle, Lactobacillus aeruginosa dophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus Rhus plantarum, and Saccharomyces boulardii (Dinleyic i et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797 (U.S. Patent No. 6,835,376). Probiotics are variants or exogenous forms of bacteria. It may also be a natural mutant (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006). Proteic bacteria can be genetically engineered to enhance or improve a desired biological characteristic, e.g., survival rate. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be used to enhance or improve probiotic properties. The cells may be genetically engineered to produce the desired effect.

[0050] As used herein, "stably maintained" or "stable" bacteria are those that are non- It is used to refer to bacterial host cells that carry native genetic material, e.g., the PAL gene, and It is a host cell, such that the non-native genetic material is maintained, expressed, and / or propagated. They are integrated into the main genome or propagated on self-replicating extrachromosomal plasmids. The bacteria can be grown in vitro, e.g., in culture medium, and / or in vivo, e.g., in digestion. A stable bacterium can survive and / or grow in a tube. For example, a stable bacterium can carry a PAL gene. A genetic construct containing the PAL gene, whose plasmid or chromosome is stably maintained in the host cell. The host cell may be a recombinant bacterium, whereby PAL can be expressed in the host cell, and the host cell may be an in Able to survive and / or grow in vitro and / or in vivo. In this embodiment, the copy number is determined by the presence of non-native genetic material, e.g., a PAL gene or a PAH gene. In some embodiments, the copy number affects the stability of expression of the non-native gene. It affects the quality of the cells, for example, the expression level of the PAL gene or PAH gene.

[0051] As used herein, the terms "modulate" and "treat" The words and their cognates refer to diseases, disorders, and / or conditions, or In another embodiment, "modulating" refers to the improvement of at least one discernible symptom of a condition. "To" and "to treat" refer to at least one condition that is not necessarily identifiable by the patient. In another embodiment, "modulation" refers to the improvement of one measurable physical parameter. "To treat" and "to treat" include physical (e.g., stabilization of discernible symptoms), physiological (e.g., stabilization of physical parameters) or both of the disease, disorder, and / or In another embodiment, "modulating" and "inhibiting" refer to the inhibition of progression of a condition. and "treat" means to slow the progression of a disease, disorder, and / or condition, or As used herein, "prevent" refers to reversing their progression. and its cognate words mean to delay the onset or to prevent a given disease, disorder and / or or condition or symptoms associated with such disease, disorder, and / or condition This refers to reducing the risk.

[0052] Those in need of treatment include individuals who already have a particular medical condition, as well as those with a condition who are The need for treatment may include individuals who are at risk or who may eventually develop a disease. For example, the occurrence of a disease, the presence or progression of a disease, or the receptivity of a subject with a disease to treatment. It is assessed by the presence of one or more risk factors associated with the likelihood of progression. Phenylelanemia, e.g., PKU, is caused by an inherited genetic mutation with no known cure. Hyperphenylalaninemia can also occur secondary to other conditions, such as liver disease. Treating hyperphenylalaninemia is the treatment of excess phenylalanine and / or reducing or eliminating associated symptoms. , which does not necessarily involve eliminating the underlying disease.

[0053] As used herein, a "pharmaceutical composition" refers to a pharmaceutical composition containing a physiologically suitable carrier and It refers to a preparation of the genetically engineered bacteria of the present invention together with other components such as excipients.

[0054] "Physiologically acceptable carrier" and "pharmaceutically acceptable carrier" may be used interchangeably. The phrase "carrier suitable for administration" means a carrier that does not cause significant irritation to the organism and is compatible with the bioavailability of the administered bacterial compound. A carrier or diluent that does not abolish the biological activity and properties of the compound. It can be enjoyed.

[0055] The term "excipient" refers to an agent added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene Examples of surfactants include polyethylene glycol, as well as surfactants including, for example, polysorbate 20.

[0056] The terms "therapeutically effective dose" and "therapeutically effective amount" refer to an amount that is effective to prevent, delay the onset of symptoms, or to refer to the amount of a compound that results in improvement of the symptoms of a condition, e.g., hyperphenylalaninemia. A therapeutically effective amount is, for example, an amount that treats, prevents, reduces the severity of, delays the onset of, or and / or one of the diseases or conditions associated with excessive phenylalanine levels. or may be sufficient to reduce the risk of developing more than one symptom. The therapeutically effective frequency of administration can be determined by methods known in the art and described below. It can be determined.

[0057] As used herein, the term "polypeptide" refers to a polypeptide ( "polypeptides" and "polypeptides" include amide bonds (i.e., peptide bonds) "Polypeptide" refers to a molecule consisting of amino acid monomers linked in a linear fashion by The term refers to any chain(s) of two or more amino acids, and is used to generate a product of a particular length. Therefore, "peptide," "dipeptide," "tripeptide," "oligopeptide," "protein," "amino acid chain," or a chain of two or more amino acids ( Any other term used to refer to a polypeptide is within the definition of "polypeptide." and the term "polypeptide" may be used in place of or in combination with any of these terms. The term "dipeptide" may be used interchangeably. The term "tripeptide" refers to a peptide of three linked amino acids. The term "polypeptide" also refers to, but is not limited to, glycosylated, acetylated, or unglycosylated polypeptides. methylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or non-naturally occurring It is intended to refer to the products of post-expression modification of polypeptides, including modifications with unsaturated amino acids. The polypeptide may be derived from natural biological sources or by recombinant techniques. In other embodiments, the polypeptide may be produced by the genetically engineered bacteria of the invention. or produced by a virus. The polypeptides of the present invention may be composed of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 500 or more, 500 or more, 1000 or more, or 2000 or more amino acids in size A polypeptide may have a defined three-dimensional structure, but this does not necessarily A polypeptide with a defined three-dimensional structure does not necessarily have to be folded. They are said to possess no defined three-dimensional structure but can adopt a number of different conformations. A polypeptide that is unfolded is said to be unfolded. The term may also refer to an amino acid sequence corresponding to a protein or part of a protein. or non-protein sequences, e.g., regulatory peptide sequences, leader peptide sequences, selected from a signal peptide sequence, a linker peptide sequence, and other peptide sequences It may also refer to the amino acid sequence corresponding to the sequence.

[0058] An "isolated" polypeptide or fragment, variant, or derivative thereof is one in which the Refers to a polypeptide that is not found in its natural environment. No particular level of purification is required. recombinantly produced and expressed in host cells, including bacterial or mammalian cells. The isolated polypeptides and proteins may be separated, fragmented, or purified by any suitable technique. As are partially or fully purified natural or recombinant polypeptides. For purposes of the present invention, recombinant peptides, polypeptides or or protein is a compound produced by recombinant DNA technology, i.e., a polypeptide. A cell, microorganism, or mammal transformed with an exogenous recombinant DNA expression construct that Refers to peptides, polypeptides, or proteins produced from animals. Most bacterial cultures Proteins or peptides expressed in cultures typically do not contain glycans. Also included are fragments, derivatives, analogs or variants of polypeptides, and any combination thereof. Also included as polypeptides are "fragments," "variants," "derivatives," and "analogs." The term "analog" refers to a peptide having an amino acid sequence sufficiently similar to that of the original peptide. and a polypeptide having at least one or more properties of the corresponding original polypeptide. Fragments of the polypeptides of the present invention include any polypeptides that retain the polypeptide fragments. Fragments also include specific antibody or biologically active fragments or fragments of the present invention. Variants include immunologically active fragments derived from any of the polypeptides described herein. may be naturally occurring or non-naturally occurring. Variant polypeptides can be produced using mutagenesis methods known in the art. The polypeptide may contain conservative or non-conservative amino acid substitutions, deletions or additions.

[0059] Polypeptides also include fusion proteins. The term "variant" includes the original peptide or a sequence sufficiently similar to the original peptide. As used herein, the term "fusion protein" includes fusion proteins. The term refers to a chimeric protein that contains the amino acid sequences of two or more different proteins. Typically, fusion proteins are generated by well-known in vitro recombinant techniques. The fusion protein has similar structure and function (but not identical structure and function) to the individual original proteins that make up the fusion protein. and / or similar accommodation functions (but not necessarily to the same extent). and / or similar biochemical functions (but not necessarily to the same degree). and / or immunological activity (but not necessarily to the same extent) "Derivatives" include, but are not limited to, the 20 standard amino acids. The term "peptide" includes peptides containing one or more naturally occurring amino acid derivatives of two amino acids. "Similarity" between peptides is measured by comparing the amino acid sequence of one peptide with the sequence of a second peptide. The amino acids of a peptide are determined by comparing the amino acids of the two peptides. If the amino acid substitution is similar to the corresponding amino acid in the second peptide, the substitution is conservative. For more information, see Dayhoff, MO, ed., The Atlas of Proteins. n Sequence and Structure 5, National Biom Medical Research Foundation, Washington,D. C. (1978), and Argos, EMBO J.8 (1989), 779–7 For example, amino acids belonging to one of the following groups are conserved: Represents essential changes or substitutions:- Ala, Pro, Gly, Gln, Asn, Ser, Th r;-Cys, Ser, Tyr, Thr;-Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -As p, Glu.

[0060] As used herein, the term "sufficiently similar" refers to a first and second have common structural domains and / or common functional activities. A sufficient or minimum number of identical or equivalent amino acid residues are identified in comparison with a second amino acid sequence. For example, at least about 45%, at least about 5 0%, at least about 55%, at least about 60%, at least about 65%, at least about 7 0%, at least about 75%, at least about 80%, at least about 85%, at least about 9 0%, at least about 91%, at least about 92%, at least about 93%, at least about 9 4%, at least about 95%, at least about 96%, at least about 97%, at least about 9 common structural domains that are 8%, at least about 99%, or at least about 100% identical An amino acid sequence comprising: The variants are sufficiently similar to the amino acid sequences of the peptides of the invention. The variants generally retain the functional activity of the peptides of the invention. The natural and wild-type peptides are modified by multiple amino acid deletions, additions, and / or substitutions. These include naturally occurring variants of peptides that differ in amino acid sequence from those of other peptides. and may be artificially designed variants.

[0061] As used herein, the term "linker," "linker peptide," or "peptide" refers to a peptide that is a The term "tidlinker" or "linker" refers to a molecule that connects or amplifies two polypeptide sequences. Synthetic or non-natural or natural linkers that link, for example, two polypeptide domains As used herein, the term "synthetic" refers to an amino acid sequence that does not exist in the human genome. The term refers to a non-naturally occurring amino acid sequence. Exemplary linkers are described herein. Further exemplary linkers are provided in U.S. Patent Application Publication No. 20140079701. No. 6,239,999, the contents of which are incorporated herein by reference in their entirety.

[0062] As used herein, the term "codon-optimized sequence" refers to a sequence that is codon-optimized to a sequence that is a codon-optimized sequence of an existing code. Expression of a transcribed RNA molecule engineered from a sequence or transcribed from, e.g., a coding sequence to improve translation in a host cell or organism or to improve transcription of a coding sequence. Codon optimization refers to sequences designed to optimize the expression of a gene in an expression host organism, including but not limited to: A process that involves selecting codons for a coding sequence to match codon preferences. The term "codon-optimized" includes processes that are encoded by a nucleic acid molecule. to reflect the typical codon usage of the host organism without modifying the polypeptide being produced. This refers to modifying a codon in the gene or coding region of the nucleic acid molecule. Such optimization involves modifying at least one, or two or more, or a substantial number of codons in the host It involves substituting one or more codons that are more frequently used in the organism's genes. A "codon-optimized sequence" is a sequence that has been modified from an existing coding sequence or has been modified, e.g., Expression of a transcribed RNA molecule transcribed from a coding sequence, resulting in improved translation in a host cell or organism. It refers to a sequence designed to improve transcription or transcription of a coding sequence. In some embodiments, the improved transcription and / or translation is achieved by increasing the level of transcription and / or translation. In some embodiments, improving transcription and / or translation In some embodiments, the expression of a gene comprising a nucleotide sequence ... , expression levels from the construct of interest, e.g., PAL3 levels and / or PheP levels. Codon optimization is used to fine-tune the gene expression. A process that involves selecting codons for a coding sequence to conform to the codon preferences of a Many organisms have specific amino acids in their growing polypeptide chains. Bias or preference for using specific codons to encode amino acid insertions Codon preference or codon bias, differences in codon usage among organisms, is a genetic This is allowed by code degeneracy and is well documented among many organisms. The translation bias is often correlated with the translation efficiency of messenger RNA (mRNA), and The messenger RNA (mRNA) then contains, among other things, the characteristics of the codons that are translated and It is thought to depend on the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally determined by the most frequently used tRNAs in peptide synthesis. Therefore, a gene is a reflection of the codons used in a given organism based on codon optimization. The gene expression vector can be adjusted for optimal gene expression in a subject.

[0063] As used herein, the terms "secretory system" or "secreted protein" , secretion or excretion of a protein of interest or a therapeutic protein from the cytoplasm of a microorganism, e.g., a bacterial cell. Secretory systems refer to natural or non-natural secretory mechanisms that can secrete a single protein. or two or more proteins assembled in a complex, e.g., HlyBD. Non-limiting examples of secretion systems for Gram-negative bacteria include modified I Type II flagella, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type V Type II secretion system, resistance-nodulation- division (RND) multidrug efflux pumps, including various single-membrane secretion systems. Non-limiting examples of secretion systems for positive bacteria include the Sec and TAT secretion systems. In some embodiments, the protein of interest is a protein of interest or a therapeutic protein. "Secretion tags" of either RNA or peptide origin are used to target the protein to specific secretion systems. In some embodiments, the secretion system comprises a protein of interest that is secreted from the engineered bacterium. This tag can be removed before the transfection. For example, type V autocrine-mediated secretion (auto- In secretion-mediated secretion, the N-terminal peptide The secretion tag acts as a "passenger" transporter from the cytoplasm to the periplasmic compartment by the native Sec system. Furthermore, autocrine factors translocate across the outer membrane and are removed by C Terminal secretion tags can be either autocatalytic or protease-catalyzed, e.g. It can be removed by OmpT cleavage, thereby releasing the protein of interest into the extracellular environment. do.

[0064] As used herein, the term "transporter" refers to a molecule, e.g. , mechanisms for the uptake of amino acids, toxins, metabolites, and substrates from the extracellular environment into microorganisms; For example, it is meant to refer to a protein or proteins. The phenylalanine transporter takes up phenylalanine into microorganisms.

[0065] As used herein, the articles "a" and "an" shall be understood to mean "at least one" unless expressly indicated to the contrary. It should be.

[0066] The term "and / or," when used between elements in a list, means: (1) the sole (1) there is only one enumerated element; or (2) there is more than one element of the enumeration. For example, "A, B and / or C" means The choice is A alone; B alone; C alone; A and B; A and C; B and C; or A A, B, and C. The term "and / or" indicates that the following may be present in a list: may be used interchangeably with "at least one" or "one or more" of the elements stomach.

[0067] bacteria The genetically engineered bacteria of the present invention are capable of reducing excess phenylalanine. In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are symbiotic bacteria. In some embodiments, the engineered bacteria are probiotic bacteria. The bacteria are naturally occurring bacteria that have been modified or mutated to reduce or eliminate pathogenicity. In some embodiments, the non-pathogenic bacteria is a gram-negative bacterium. In some embodiments, the non-pathogenic bacteria are gram-positive bacteria. Exemplary bacteria include: Bacillus sp., Bacteroides sp., Bifidobacterium sp., Brevibacterium sp., Bacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactobacillus Tococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coccus agrans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidus Dum, Bifidobacterium infantis, Bifidobacterium lactis , Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lac Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus lactis, and Saccharomyces boulardii. In an embodiment, the genetically engineered bacterium is Bacteroides fragilis, Bacteroides Thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidus Dum, Bifidobacterium infantis, Bifidobacterium lactis , Clostridium butyricum, Escherichia coli Nissle, Lactobacillus acidophilus Lactobacillus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus Selected from the group consisting of: Lactis

[0068] In some embodiments, the genetically engineered bacteria are among the best-characterized probiotics. Enterobacteriaceae family has evolved into one of the Escherichia coli strain Nissle 1917 (E. coli), a gram-negative bacterium of the Nissle) (Ukena et al., 2007). The strain is particularly notable for its complete harmlessness. (Schultz, 2008) and GRAS (Generally Recognized as Safe) generally recognized as safe (Rei (ster et al., 2014, underlining added by the author). Nissle is an important virulence factor (e.g., E. coli α-hemolysin, P-fimbrial adhesin) It was confirmed that E. coli Ni SSLE does not possess pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, and is invasive. have not been shown to be uropathogenic (Sonnenborn et al., 2009 As early as 1917, E. coli Nissle developed Mutaflo for therapeutic use. The therapeutic effects of E. coli Nissle and its related compounds have been investigated. It is generally accepted that the efficacy and safety of , 2007).

[0069] Those skilled in the art will appreciate that the genetic modifications disclosed herein may be adapted for use in other species, strains, and subtypes of bacteria. It is understood that genes from one or more different species may be introduced into one another. For example, the PAL gene from Rhodosporidium toruloides can be introduced into Escherichia coli. It can be expressed in prokaryotes and eukaryotes (Sarkissian et al., 1999). Phenylanine ammonia-lyases from various species are known to share sequence homology (X iang and Moore, 2005 ).

[0070] The unmodified E. coli Nissle and the genetically engineered bacteria of the present invention can also be used in the gastrointestinal tract, e.g. or by protective factors in serum (Sonnenborn et al., 2009), or by administration Destruction by activation of the kill switch after a few hours or days Thus, genetically engineered bacteria may require continuous administration. In some embodiments, residence times are calculated for human subjects. Residence times can be calculated for the genetically engineered bacteria of the present invention (see, e.g., Figure 68). (This refers to lighting.)

[0071] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding PAL. wherein the PAL gene is operably linked to a directly or indirectly inducible promoter In some embodiments, the bacterium comprises a non-native PAL gene. In some embodiments, the bacterium comprises an additional copy of a naturally occurring PAL gene. In some embodiments, the promoter is not naturally associated with the PAL gene. The promoter may be any one or more of the promoters disclosed herein.

[0072] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a PAH. wherein the PAH gene is operably linked to a directly or indirectly inducible promoter In some embodiments, the bacterium comprises a non-native PAH gene. In some embodiments, the bacterium comprises an additional copy of the native PAH gene. In some embodiments, the promoter is not naturally associated with the PAH gene. The promoter may be any one or more of the promoters disclosed herein.

[0073] In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding an LAAD. The LAAD gene is driven by a directly or indirectly inducible promoter. In some embodiments, the bacterium comprises a non-native LAAD gene. In some embodiments, the bacterium contains an additional copy of the native LAAD gene. In some embodiments, the promoter is not naturally associated with the LAAD gene. In one embodiment, the promoter is any one or more of the promoters disclosed herein.

[0074] In some embodiments, the genetically engineered bacteria comprises a phenylalanine transporter. In certain embodiments, the bacterium further comprises a gene encoding phenylalanine (PheP). containing an additional copy of the native gene encoding the alanine transporter, The ranin transporter gene is driven by a directly or indirectly inducible promoter. In an alternative embodiment, the bacterium is operably linked to a non-native phenylalanine transposon. The phenylalanine transporter gene encodes a direct or indirectly inducible promoter. The term "non-naturally occurring" phenylalanine transporter encompasses several implementations. In some embodiments, the promoter is not naturally associated with the pheP gene. In this form, the same promoter controls PheP and PAL and / or PAH and / or In some embodiments, the expression of PheP is regulated. The promoter controls the expression of PAL and / or PAH and / or L-AAD. In some embodiments, the promoter controlling the expression of PheP is different from the promoter The promoter may be any one or more of the promoters disclosed herein.

[0075] In some embodiments, the PAL, PAH, LAAD, and / or pheP are activated. The operably linked promoter is inducible, either directly or indirectly, by an exogenous environmental condition. In some embodiments, the promoter is an exogenous promoter specific to the mammalian gastrointestinal tract. In some embodiments, the promoter is induced directly or indirectly by an environmental condition. The activator is directly or indirectly induced by exogenous environmental conditions specific to the mammalian small intestine. In some embodiments, the promoter is expressed in an exogenous environment specific to the large intestine of a mammal. In some embodiments, the promoter is induced directly or indirectly by a condition. are susceptible to hypoxic or anaerobic conditions and / or low oxygen conditions, such as the environment of the mammalian gastrointestinal tract. In some embodiments, the promoter is induced directly or indirectly by The presence of molecules or metabolites specific to the mammalian gastrointestinal tract, such as propionate, In some embodiments, the promoter is induced directly or indirectly by tetracycline. In some embodiments, the cyclin is induced directly or indirectly by exposure to the cyclin. The promoter is induced directly or indirectly by exposure to arabinose. In some embodiments, the promoter is activated directly or indirectly by exposure to IPTG. In some embodiments, the promoter is induced by rhamnose or other suitable promoters known to those skilled in the art. Directly or indirectly through exposure to other chemical and / or nutritional inducers of In some embodiments, the promoter is induced directly by exogenous environmental temperature. In some embodiments, the promoter is directly or indirectly regulated by IPTG or is induced directly or indirectly by exposure to other lacI-binding compounds. In some embodiments, the promoter is directly or indirectly activated by exposure to rhamnose. In some embodiments, the promoter is directly or indirectly induced by an increase in temperature. In some embodiments, the promoter is indirectly induced by a decrease in temperature. In some embodiments, the promoter is a promoter of the present invention. It is induced directly or indirectly by molecules co-administered with the genetically engineered bacteria. Such molecules include tetracycline, IPTG, arabinose, or any other molecule known to those skilled in the art. The compound may be any other chemical and / or nutritional inducer known in the art.

[0076] In some embodiments, the promoter is present in the culture vessel (e.g., a flask or fermentor). or other suitable culture vessel), in which The strains are propagated or maintained prior to in vivo administration. Non-limiting examples of such conditions include hypoxic, anaerobic, microaerobic or aerobic conditions, and other constant oxygen levels (such as those exemplified below), the presence of arabinose, IPTG, rhamnopurification or other chemical and / or nutrient compounds described herein or known in the art. In some embodiments, the conditions in the culture vessel include the presence of a specific acid inducer. Oxygen level, for example, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40-50% oxygen, 60-70%, 70-80%, 80% oxygen 90%, 90-100% oxygen, and other oxygen levels described herein; At that set point the promoter is induced either directly or indirectly.

[0077] Reduction of hyperphenylalaninemia The genetically engineered bacteria of the present invention encode a phenylalanine metabolic enzyme (PME). In some embodiments, the genetically engineered bacteria comprise a phenylalanine metabolic pathway gene. Contains the gene encoding the pro-phenylalanine metabolizing enzyme (PME) to reduce hyperphenylalaninemia can be done.

[0078] Examples of phenylalanine metabolic enzymes include, but are not limited to, phenylalanine hydroxylase Phenylanine ammonia-lyase (PAH), phenylalanine ammonia-lyase (PAL), aminotransferase enzymes, L-amino acid deaminase (L-AAD), and phenylalanine dehydrogenase Enzymes include phenylalanine hydroxylase, phenylalanine dehydrogenase, The reaction with acetyltransferase or aminotransferase requires a cofactor, but L-AAD and and PAL do not require any additional cofactors. Without wishing to be bound by theory, the cofactor The absence of the need for phenylalanine has been demonstrated by the enzyme encoded by genetically engineered bacteria. Alanine degradation depends on substrate availability and is not limited by cofactor availability. This means:

[0079] In some embodiments, the engineered bacteria comprise one or more phenylalanine hydroxylase inhibitors. In some embodiments, the gene sequence encoding the PAH polypeptide is The engineered bacteria express one or more phenylalanine ammonia-lyase (PAL) polypeptides. It contains the gene sequence encoding phenylalanine ammonia lyase (PAL; E C4.3.1.24) is the ester of L-phenylalanine with ammonia and trans-cinnamic acid. Phenylanine ammonia-lyase is an enzyme that catalyzes the reaction that converts L- It is specific for Phe and, to a lesser extent, for L-tyrosine. The reaction catalyzed is the reaction of L-phenylalanine to produce trans-cinnamic acid and ammonia. Naturally occurring, nonoxidative deamination of alanine. Unlike mammalian enzymes (PAHs) PAL is monomeric and does not require cofactors (MacDonald et al., Biochem. m Cell Biol 2007;85:273~82. A modern v (i.e., the enzyme of phenylalanine ammonia lyase). In this study, it was found that microorganisms use L-phenylalanine (LP) as the sole carbon and nitrogen source. In one embodiment, the present invention provides a catabolic pathway that allows the utilization of The genetically engineered bacteria contain the PAL gene, which converts phenylalanine to non-toxic levels. trans-cinnamic acid and ammonia. Acid (TCA) can be further converted to the TCA metabolites benzoic acid and hippuric acid (S arkissian et al., J Mass Spectrom. 2007 June;42(6) :811~7 pages;Quantitation of phenylalanine an d its trans-cinnamic,benzoic and hippuri c acid metabolites in biological fluids PAL enzyme activity was significantly increased with THB supplementation. No factor activity is required.

[0080] In some embodiments, PAL is a strain of bacteria, including but not limited to Achromobacter xylosococcus. Cydans, Pseudomonas aeruginosa, Photorhabdus luminescens, Anabaena derived from bacterial species, including Agrobacterium variabilis, and Agrobacterium tumefaciens In some embodiments, the bacterial species is Photorhabditis cerevisiae. In some embodiments, the bacterial species is Anabaena variabilis. In some embodiments, the PAL is derived from a eukaryotic species, e.g., a yeast species, a plant species. PAL proteins are encoded by the PAL gene, which occurs in the mitochondrial nucleus. Several different PAL proteins are known in the art. Genetically engineered bacteria, once the PAL gene is expressed, can produce converts more phenylalanine than unmodified bacteria of the same bacterial subtype. Genetically engineered bacteria containing L. have been shown to reduce conditions associated with hyperphenylalaninemia, including PKU. It can be used to metabolize phenylalanine in the body into a non-toxic molecule to treat conditions such as In some embodiments, the genetically engineered bacterium is Anabaena variabilis PAL ("PAL1"). In some embodiments, the genetically engineered bacteria express expressing the rhabditis luminescent PAL ("PAL3"). A typical example is shown in Table 2.

[0081] In some embodiments, the engineered bacteria contain one or more LAAD polypeptides. In some embodiments, the engineered bacterium comprises a gene sequence encoding one or more A gene sequence encoding a PAL polypeptide and one or more LAAD polypeptides of LAAD involves the production of ammonia and hydrogen peroxide via an imino acid intermediate. Stereospecific oxidation, i.e., oxygen-consuming deamination of L-amino acids to α-keto acids L-AAD is a cytosolic enzyme found in snake venoms and many bacteria (Bifulco et al., 2013). Specifically, they are found in the cell membranes of bacteria from the genera Proteus, Providencia, and Morganella. L-AAD (EC 1.4.3.2) is a flavoenzyme with a dimeric structure. Each subunit contains a non-covalently bound flavin adenine dinucleotide (FAD) cofactor. Proteus mirabilis contains two types of L-AA D (Duerre and Chakrabarty 1975). It has a wide substrate specificity and is capable of reacting with keto acids of aliphatic and aromatic L-amino acids, typically L-phenylalanine. catalyzes the oxidation of guaranidin to guaranidin (GenBank: U35383.1) (Baek et al., J Ournal of Basic Microbiology 2011, 51, 12 pp. 9-135; “Expression and characterization of a second L-amino acid deaminase isola ted from Proteus mirabilis in Escherichi Other species act primarily on basic L-amino acids (GenBan k:EU669819.1). LAAD from bacterial, fungal, and plant sources can be used as a nitrogen source. involved in the utilization of L-amino acids (i.e., ammonia produced by enzymatic activity) Most eukaryotic and prokaryotic L-amino acid deaminases are membrane-bound. Except for the Proteus species derived from the LAAD, which is a synthetic type, it is secreted extracellularly. In S. bilis, L-AAD is expressed in the cells facing outward into the periplasmic space where the enzyme activity is present. It has been reported to be located in the membrane (Pelmont J et al., (1972) "L-am ino acid oxidases of Proteus mirabilis:g biochimie 54:1359~137 4 pages).

[0082] In one embodiment, the genetically engineered bacterium of the invention comprises a LAAD gene. It can convert phenylalanine to non-toxic levels of phenylpyruvate, Nylpyruvate can also be further degraded to phenyllactate, for example, by liver enzymes. Phenylepyruvate cannot cross the blood-brain barrier, leading to another potential side effect of LAAD. Reduces brain levels of phenylalanine without allowing the accumulation of potentially toxic metabolites In some embodiments, the LAAD may be derived from, but is not limited to, Proteus sp. LAAD genes from bacterial species, including bacteria from the genera Providencia and Morganella In some embodiments, the bacterial species is Proteus mirabilis. In some embodiments, the bacterial species is Proteus vulgaris. In morphology, the genetically engineered bacteria Proteus mirabilis LAAD enzyme GenBank :U35383.1. Non-limiting examples of LAAD sequences of interest are shown in Table 2. In some embodiments, the LAAD enzyme is derived from snake venom. The engineered bacteria, when the LAAD gene is expressed, are able to grow in the same way as unmodified bacteria of the same bacterial subtype under the same conditions. Therefore, genetically engineered bacteria containing LAAD convert more phenylalanine than bacteria. The bacteria are being used internally to treat conditions related to hyperphenylalaninemia, including PKU. can be used to metabolize phenylalanine into non-toxic molecules.

[0083] In some embodiments, the engineered bacteria express wild-type enzymes as they occur in nature. In some embodiments, the engineered bacteria encodes a mutation in a wild-type sequence. In some embodiments, the mutation increases the stability of the enzyme. In some embodiments, the mutation increases the catalytic activity of the enzyme. In embodiments, the genetically engineered bacteria express one or more of the proteins listed in Table 2. In some embodiments, the genetically engineered bacterium comprises a gene encoding SEQ ID NO: A gene sequence encoding one or more polypeptides comprising any one of the sequences set forth in Nos. 1 to 8. In some embodiments, the genetically engineered bacterium comprises any of the sequences of SEQ ID NOs: 1-8. Any of the following and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% ,88%,89%,90%,91%,92%,93%,94%,95%,96%,97% , 98%, or 99% identity to the gene sequence encoding the polypeptide. In some embodiments, the genetically engineered bacteria comprises one or more of the following: In some embodiments, the genetically engineered bacteria encodes multiple enzymes. In some embodiments, the genetically engineered bacteria comprises a gene encoding AH. Some embodiments encode mutant PAHs with increased stability and / or activity. In some embodiments, the engineered bacteria contain a gene encoding wild-type PAL. In embodiments, the genetically engineered bacteria have increased stability and / or activity. In some embodiments, the genetically engineered bacterium encodes a mutant PAL. In some embodiments, the genetically engineered bacteria comprise a gene encoding a type LAAD. encodes a mutant LAAD with increased stability and / or activity. Methods for screening enzymes with specific properties are known in the art and are described herein. It is described in.

[0084] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3]

[0085] The PME, e.g., PAL, LAAD, or PAH gene(s), may be genetically engineered. It may be present on a plasmid or chromosome in bacteria. In some embodiments, the PME The gene sequence(s) are expressed under the control of one or more constitutive promoter(s). In some embodiments, the PME gene is responsive to exogenous environmental conditions as described herein. The expression is under the control of a promoter that is directly or indirectly induced by the condition. In some embodiments, the PME gene encodes a molecule or metabolite specific to the mammalian gastrointestinal tract. Regulation of promoters that are induced directly or indirectly by exogenous environmental conditions, such as the presence of In one embodiment, the PME gene is expressed under hypoxic, microaerobic, or anaerobic conditions. It is expressed under the control of a promoter that is directly or indirectly induced by conditions, and Expression of genes, such as the PAL gene, is suppressed in hypoxic or anaerobic environments, such as the mammalian gastrointestinal tract. It is activated in a toxic environment.

[0086] In some embodiments, the genetically engineered bacteria contain one or more PAL polypeptide sequences. In some embodiments, the engineered bacteria comprise a gene sequence encoding one or more The gene sequence encoding the PAL polypeptide sequence is a mammalian It is induced directly or indirectly by hypoxic or anaerobic conditions, such as those in the gastrointestinal tract of bacteria. In some embodiments, the engineered bacteria encode one or more LAAD polypeptides. In some embodiments, the engineered bacteria comprise a gene sequence encoding one or more LAA The gene sequence encoding the D polypeptide is contained in the stomach, duodenum, and Oxygenated as found in the proximal intestine, including but not limited to the ileum It is induced directly or indirectly by oxygen-rich, hypoxic, or microaerobic conditions. In this embodiment, the engineered bacterium contains a gene encoding one or more PME polypeptide sequences. The gene sequence is a product of environmental factors naturally present in the mammalian digestive tract. In another embodiment, the genetically engineered bacterium is derived directly or indirectly from a mammalian host. Environmental factors that do not naturally occur in the digestive tract of mammals, such as arabinose or IPTG, encodes one or more PME gene sequences that are directly or indirectly derived from other In embodiments, the genetically engineered bacteria are those that naturally occur in the mammalian gastrointestinal tract under inflammatory conditions. The gene encodes one or more PME gene sequences that are directly or indirectly induced by environmental factors. In some embodiments, the engineered bacteria express one or more PAL polypeptides. Gene sequences encoding and one or more L-AAD polypeptides The gene sequence may be under the control of the same promoter or different copies of the same promoter. The promoter may be grown under any of the environmental conditions described herein and in accordance with the present invention. promoters that are induced directly or indirectly by exogenous environmental conditions, such as any of the promoters described in In some embodiments, the engineered bacteria contain one or more PAL polypeptides. and a gene sequence encoding one or more L-AAD polypeptides. The gene sequences are under the control of different promoters, Any of the environmental conditions described herein and any of the promoters described herein In some embodiments, the expression of a gene is induced directly or indirectly by an exogenous environmental condition. The engineered bacteria may comprise a gene sequence encoding one or more PAL polypeptides and one The gene sequence encoding the above L-AAD polypeptide is constitutively In some embodiments, the engineered bacterium is under the control of one or more promoters. A gene sequence encoding a PAL polypeptide and one or more L-AAD polypeptides The PAL gene sequence is under the control of a constitutive promoter. In some embodiments, the LAAD gene sequence is under the control of an inducible promoter. In the present invention, the engineered bacteria contain a gene sequence encoding one or more PAL polypeptides and A gene sequence encoding one or more L-AAD polypeptides, The sequence is under the control of a constitutive promoter, and the PAL gene sequence is under the control of an inducible promoter. In any of these embodiments, the bacteria are under the control of one or more Phe transporters. The gene sequence may further include a gene sequence encoding a transporter polypeptide, The sequence can be under the control of a constitutive or inducible promoter, the same or different promoter controlling the LAAD gene sequence. It can be a computer.

[0087] In other embodiments, the engineered bacteria can be directly administered in bacterial cell culture prior to in vivo administration. encodes one or more PME gene sequences that are directly or indirectly derived; i.e. In one embodiment, one or more PME gene sequences are expressed under the control of an inducible promoter, and the inducible promoter The motor drives the bacterial culture during bacterial growth in flasks, fermentors, or other culture vessels. specific molecules or metabolites provided in the nutrient, temperature, oxygen level, or other parameters In some embodiments, the engineered bacteria respond to in vitro bacterial cell culture. one or more PME gene sequences that are directly or indirectly induced prior to administration in vivo. The one or more PME gene sequences are expressed under hypoxic or anaerobic conditions. In some embodiments, the engineered bacteria are cultured in bacterial cell culture prior to in vivo administration. Encoding one or more PME gene sequences that are directly or indirectly derived; The above PME gene sequences are expressed under aerobic conditions. In some embodiments, the engineered The selected bacteria are directly or indirectly induced in bacterial cell culture prior to in vivo administration. encodes one or more PME gene sequences; the one or more PME gene sequences may be microparticles In some embodiments, the engineered bacteria are expressed under aerobic conditions in bacterial cell culture. one or more PME genes induced directly or indirectly prior to in vivo administration of one or more PME gene sequences that are expressed in the presence of arabinose In some embodiments, the engineered bacteria are administered in vivo in bacterial cell culture. encodes one or more PME gene sequences that are directly or indirectly derived from the previous; The one or more PME gene sequences are expressed in the presence of IPTG.

[0088] Bacteria have evolved transcription factors that can sense oxygen levels. The oxygen transduction pathway can be triggered by different oxygen levels and occurs with different kinetics. Level-dependent promoters are those that contain nuclear domains to which one or more oxygen-level-sensing transcription factors can bind. The binding and / or activation of the corresponding transcription factor activates downstream gene expression. In one embodiment, the PME gene is expressed under the control of an oxygen level-dependent promoter. In a more specific embodiment, the PAL gene is expressed in a hypoxic environment, such as the environment of the mammalian gastrointestinal tract. Alternatively, it is under the control of an oxygen level-dependent promoter that is activated in an anaerobic environment.

[0089] In certain embodiments, the genetically engineered bacteria comprise a fumarate and nitrate reductase regulating one or more PME gene sequence(s) expressed under the control of the factor (FNR) promoter; For example, PAL is involved. In E. coli, FNR controls the switch from aerobic to anaerobic metabolism. It is the major transcriptional activator that regulates anaerobic and / or Under hypoxic conditions, FNR activates hundreds of genes involved in adaptation to anaerobic growth. Under aerobic conditions, FNR dimerizes into an active DNA-binding protein. In some embodiments, the dimerization is prevented and the FNR nucleic acids are inactive. The sequence is inserted into a genetically engineered bacterium. In an alternative embodiment, the genetically engineered bacterium have been shown to utilize alternative oxygen level-dependent promoters, such as the ANR promoter (Ray et al., 1999). 97), PM expressed under the control of the DNR promoter ( Trunk et al., 2010 ). In some embodiments, phenylalanine metabolism is achieved through the digestive tract, e.g., including phenylalanine E, e.g., PAL. It is particularly active in low-oxygen or anaerobic environments.

[0090] Arginine deiminase and nitrate reduction in Pseudomonas aeruginosa Anaerobic-regulated (ANR) transcription factors are "physiologically inducible factors that can be induced under oxygen-limited or anaerobic conditions." required for the expression of biological functions" (Winteler et al., 1996; Sawers 1 Pseudomonas aeruginosa ANR is homologous to E. coli FNR and is The consensus FNR site (TTGAT----ATCAA) is identified by ANR and FNR. (Winteler et al., 1996). As with FNR, anaerobic conditions In this state, ANR activates a number of genes responsible for adaptation to anaerobic growth. In this state, ANR is inactive. omonas fluorescens, Pseudomonas putida nas putida), Pseudomonas syringae ringae, and Pseudomonas mendocina docina) all have functional homologs of ANR (Zimmermann et al., 1999). 91). ANR-regulated promoters, e.g., the arcDABC operon Promoters are known in the art (see, e.g., Hasegawa et al., 1998 (See

[0091] The FNR family also plays a role in the anaerobic nitrate respiration of Pseudomonas aeruginosa ( It is a transcriptional regulator required in conjunction with ANR for the transcription of GABAergic neurons (Hasegawa et al., 1998). It also contains certain dissimilatory nitrate respiration regulators (DNR) (Arai et al., 1995). For genes, the FNR binding motif is "probably recognized only by DNR" ( Hasegawa et al., 1998). It is regulated by exogenous environmental conditions and corresponding regulatory regions. Any suitable transcriptional regulator that is controlled by the transcription factor can be used. Non-limiting examples include ArcA / B, ResD / E, NreA / B / C, and AirSR, others in the field It is well known.

[0092] FNR promoter sequences are known in the art and any suitable FNR promoter may be used. Any suitable FNR promoter sequence can be used in the genetically engineered bacteria of the present invention. The promoter may be combined with any suitable PAL. Non-limiting examples include the FNR promoter. PAL sequences are provided in Table 3, and non-limiting PAL sequences are also provided herein. In some embodiments, the genetically engineered bacteria of the invention comprise SEQ ID NO: 9, SEQ ID NO: 10, ni rB1 promoter (SEQ ID NO: 11), nirB2 promoter (SEQ ID NO: 12), ni rB3 promoter (SEQ ID NO: 13), ydfZ promoter (SEQ ID NO: 14), strong The nirB promoter (SEQ ID NO: 15) fused to a ribosome binding site, a strong ribosome The ydfZ promoter (SEQ ID NO: 16) fused to the rRNA binding site, an anaerobically induced small R The NA gene fnrS (fnrS1 promoter SEQ ID NO: 9 or fnrS2 promoter promoter (SEQ ID NO: 17), the nirB promoter (SEQ ID NO: 18) fused to the crp binding site ), and fnrS (SEQ ID NO: 19) fused to a crp binding site Includes.

[0093] In some embodiments, the genetically engineered bacteria comprises SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 DNA sequences or their functions At least about 80%, at least about 85%, at least about 90%, at least about It contains one or more nucleic acid sequences that are 95%, or at least about 99%, homologous. [Table 3] [Table 4-1] [Table 4-2]

[0094] In other embodiments, one or more PMEs, e.g., PAL, are activators of transcription factors, e.g., CRP. It is expressed under the control of an oxygen level-dependent promoter fused to binding sites for C. RP (cyclic AMP receptor protein or catabolite-activating protein, or CAP) ) is less beneficial in the presence of rapidly metabolizable carbohydrates such as glucose. in bacteria by suppressing genes responsible for carbon source uptake, metabolism, and assimilation plays a key regulatory role (Wu et al., 2015). This preference for glucose is These are called glucose repression and carbon catabolite repression (Deutscher, 2002). 2008; Gorke and Stulke, 2008). In some embodiments, P ME, e.g., PAL expression, is driven by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, PAL expression is regulated by a CRP-binding site fused to the PAL gene. In these embodiments, cyclic AMP is controlled by the FNR promoter. This binding causes a conformational change in CRP. A change is induced that allows CRP to bind tightly to its binding site. CRP binding induces RNA transcription to the FNR promoter via direct protein-protein interaction. Activates transcription of PME genes, e.g., the PAL gene, by recruiting polymerase In the presence of glucose, cyclic AMP does not bind to CRP, and PMEs, such as the PAL gene, In some embodiments, the transcription of a gene is inhibited by a binding site for a transcriptional activator. The fused oxygen level-dependent promoter (e.g., FNR promoter) can be used in, for example, By adding glucose to the growth medium in vitro, sufficient amounts of glucose If present, to ensure that the PME, e.g., PAL, is not expressed under anaerobic conditions It is used for this purpose.

[0095] In another embodiment, one or more PMEs, e.g., LAADs, have expression that is modulated in the presence of glucose. Inducible transcription factors such as CRP are fused to binding sites for transcriptional activators, e.g., CRP, so that they are repressed. The expression is under the control of a competent promoter.

[0096] In some embodiments, the LAAD is under the control of the FNR promoter. In embodiments, the LAAD is under the control of a promoter that is not the FNR promoter. AAD requires oxygen to catalyze the breakdown of phenylalanine to phenylpyruvate. Therefore, under oxygenated or hypoxic conditions, the LAAD is active. It is desirable to induce LAAD expression even under these conditions.

[0097] In some embodiments, one or more PMEs, e.g., PALs and / or LAADs, , which induces responses to specific molecules or metabolites in the environment (e.g., the mammalian digestive tract). For example, the short chain fatty acid propionate is expressed under the control of an inducible promoter. It is the major microbial fermentation metabolite localized in the gastrointestinal tract ( Hosseini et al., 2011 ). In one embodiment, expression of one or more PME genes is mediated by a promoter inducible by propionate. In a more particular embodiment, PME gene expression is under the control of a mammalian gastrointestinal One or more propionate-inducible enzymes activated by the presence of propionate in In health and / or disease states, mammalian digestive Any molecule or metabolite found in the duct can be used to induce PME gene expression. Non-limiting examples include propionate, bilirubin, aspartate aminotransferase, ferase, alanine aminotransferase, blood clotting factors II, VII, IX, and and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigen and and antibodies, alpha-fetoprotein, antimitochondrial, smooth muscle, and antinuclear antibodies, iron, Transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese In an alternative embodiment, a PME, e.g., a PAL and / or LAAD gene, Expression is activated in the presence of the sugar arabinose. araBAD Under the control of the promoter In one embodiment, LAAD expression is araBAD It is under the control of a promoter. In one embodiment, expression of the LAAD occurs under aerobic or microaerobic conditions. PAL expression is araBAD In one embodiment, the gene is under the control of a promoter. PAL expression occurs under aerobic or microaerobic conditions. occurs under aerobic or hypoxic conditions, whereas LADD expression occurs under aerobic or microaerobic conditions In one embodiment, PAL expression occurs under anaerobic or hypoxic conditions and LADD expression occurs under P araBAD It is under the control of a promoter.

[0098] In some embodiments, one or more PME genes (e.g., PAL and / or LAAD genes) are induced by exposure to chemical and / or nutritional inducers. In some embodiments, the expression is under the control of a promoter comprising one or more PMEs. Genes (e.g., PAL and / or LAAD genes) are regulated by exposure to tetracycline. In some embodiments, the expression is under the control of a promoter induced by one The PME genes (e.g., PAL and / or LAAD genes) are involved in the synthesis of arabinose. In some embodiments, the gene is expressed under the control of a promoter that is induced by exposure to wherein one or more PME genes (e.g., PAL and / or LAAD genes) are Under the control of a promoter that is induced by exposure to IPTG or other LacI inducers In some embodiments, one or more PME genes (e.g., PAL and and / or LAAD genes) are promoters induced by exposure to rhamnose. In some embodiments, one or more PME genes (e.g., PA L and / or LAAD genes) are genes that are induced by exposure to tetracycline. In some embodiments, two or more PME genes ( The PAL and LAAD genes) are expressed, and each gene is driven by a different promoter, e.g. For example, under the control of any of the promoters discussed in this paragraph and elsewhere herein. is expressed.

[0099] In some embodiments, one or more PME genes, such as PAL and / or LA The AD gene is induced by a temperature shift from a non-permissive to a permissive temperature. In some embodiments, gene expression is controlled by a method known in the art. Methods, such as optimizing ribosome binding sites, engineering transcriptional regulators, and / or Further optimization is achieved by increasing the NA stability. Bioinformatics tools are known in the art.

[0100] In any of the embodiments described herein above (and elsewhere herein), However, engineered bacteria may contain gene sequences encoding one or more Phe transporters. The gene sequence may further comprise any of the promoters described herein. It can be done under your control.

[0101] In some embodiments, the genetically engineered bacteria are characterized in that PAL is expressed in the host cell. The host cell may be in vitro, e.g., in culture, and / or in vivo, e.g., For example, a stable vector carrying the PAL gene is capable of surviving and / or growing in the gastrointestinal tract. In some embodiments, the genetically engineered cell contains a plasmid or chromosome carrying the gene. The bacterium contains two or more different PAL genes. In some embodiments, the genetically engineered The bacterium contains multiple copies of the same PAL gene. In some embodiments, the PAL gene is present on a plasmid and is operably linked to a directly or indirectly inducible promoter. In some embodiments, the PAL gene is present on a plasmid and is capable of activating the PAL gene under hypoxia. The promoter is operably linked to a promoter that is induced under anaerobic or anaerobic conditions. In this form, the PAL gene is located on the chromosome and is conserved within a directly or indirectly inducible promoter. In some embodiments, the PAL gene is operably linked to a target gene. and is operably linked to a promoter that is induced under hypoxic or anaerobic conditions. In some embodiments, the PAL gene is present on a plasmid and is activated upon exposure to tetracycline. In some embodiments, the promoter is operably linked to a dew-inducible promoter. The PAL gene is present on a plasmid and is a promoter induced by exposure to arabinose. In some embodiments, the PAL gene is operably linked to a motor. A promoter present on the cytoplasm of the lacI gene and induced by exposure to IPTG or another LacI inducer. In some embodiments, the PAL gene is operably linked to a plasmid. is present and is operably linked to a promoter that is inducible by exposure to rhamnose. In some embodiments, the PAL gene is present on a plasmid and is capable of binding to tetracycline. In some embodiments, the promoter is operably linked to a promoter that is induced by exposure to light. The PAL gene is present on a plasmid and can be transformed by a temperature shift from a non-permissive to a permissive temperature. In some embodiments, the PAL gene is operably linked to an inducible promoter. The gene is present on the chromosome and is operably linked to a promoter that is induced by exposure to arabinose. In some embodiments, the PAL gene is present on a chromosome and is functionally linked to IPTG. or operably linked to a promoter that is inducible by exposure to another LacI inducer In some embodiments, the PAL gene is present on a chromosome and is responsive to exposure to rhamnose. In some embodiments, the promoter is operably linked to a promoter that is induced by dew. The PAL gene is located on the chromosome and is a promoter induced by exposure to tetracycline. In some embodiments, the PAL gene is operably linked to a PAL vector. and is operable on a promoter that is induced by a temperature shift from a non-permissive to a permissive temperature. is linked to.

[0102] In some embodiments, the genetically engineered bacteria are capable of expressing LAAD in host cells. The host cell may be cultured in vitro, e.g., in culture, and / or in vivo. For example, a stable vector carrying the LAAD gene is capable of surviving and / or growing in the gastrointestinal tract. In some embodiments, the vector comprises a genetically engineered plasmid or chromosome maintained in the host. The resulting bacterium contains two or more different LAAD genes. The resulting bacteria contain multiple copies of the same LAAD gene. The AD gene is carried on a plasmid and is driven by a directly or indirectly inducible promoter. In some embodiments, the LAAD gene is operably linked to a plasmid. and promoters that are inducible by, for example, arabinose or tetracycline. In some embodiments, the LAAD gene is present on a chromosome. , operably linked to a directly or indirectly inducible promoter. In embodiments, the LAAD gene is present on a chromosome, e.g., arabinose or tetracycline. In some embodiments, the promoter is operably linked to a promoter that is induced by cullin. In , the LAAD gene is present on a plasmid and is inducible by exposure to tetracycline. In some embodiments, the promoter is operably linked to a promoter that encodes a LAAD gene. The gene is carried on a plasmid and operates on a promoter that is induced by exposure to arabinose. In some embodiments, the LAAD gene is operably linked to a plasmid. and acts on a promoter that is induced by exposure to IPTG or other LacI inducers. In some embodiments, the LAAD gene is operably linked to a plasmid. and is operably linked to a promoter that is inducible by exposure to rhamnose. In some embodiments, the LAAD gene is present on a plasmid and is transformed from a non-permissive temperature to a permissive temperature. The promoter is operably linked to a promoter that is induced by a temperature shift to 100°C. In embodiments, the LAAD gene is present on a plasmid and is operable under a constitutive promoter. In some embodiments, the LAAD gene is present on a plasmid and is linked to a It is operably linked to a promoter that is inducible by exposure to tracycline. In some embodiments, the LAAD gene is present on a chromosome and is activated by exposure to arabinose. In some embodiments, the promoter is operably linked to a promoter that is induced by LA. The AD gene is present on the chromosome and is expressed upon exposure to IPTG or other LacI inducers. In some embodiments, the LAAD is operably linked to an inducible promoter. The gene is located on the chromosome and is driven by a promoter that is induced by exposure to rhamnose. In some embodiments, the LAAD gene is present on a chromosome and is non-transgenic. operably linked to a promoter that is induced by a temperature shift from the permissive temperature to the permissive temperature In some embodiments, the LAAD gene is present on a chromosome and is constitutively promoted. The sensor is operably linked to the sensor.

[0103] of bacteria containing PME genes (e.g., PAL, PAH, and / or LAAD) In any of the above embodiments, the bacterium encodes one or more Phe transporters. The Phe transporter gene sequence may further comprise a gene sequence encoding the Phe transporter gene. The plasmid or chromosome may be present on a PME. The genes present can be the same or different. The gene sequence may be under the control of the same or a different promoter as the PMR gene sequence.

[0104] In some embodiments, the genetically engineered bacteria comprises an oxygen level-dependent transcriptional regulator, For example, FNR, ANR, or DNR and the corresponding promoters from different bacterial species. Non-natural oxygen level-dependent transcriptional regulators and promoters can be expressed under the same conditions. Compared to native transcriptional regulators and promoters in bacteria, hypoxic or anaerobic conditions In this environment, a gene operably linked to the promoter, such as PAL or P In certain embodiments, the non-naturally occurring oxygen level-dependent transcriptional regulator is the FNR protein from Neisseria gonorrhoeae. (See, e.g., Isabella et al., 2011). In some embodiments, The corresponding wild-type transcriptional regulator remains intact and retains wild-type activity. In alternative embodiments, the corresponding wild-type transcriptional regulator has reduced wild-type activity or is deleted or mutated to remove

[0105] In some embodiments, the genetically engineered bacteria exhibit wild-type oxygen level-dependent transcriptional regulation. Sectional factors, e.g., FNR, ANR, or DNR, and wild-type from bacteria of the same subtype The promoters include those that are mutated relative to the corresponding promoters. The promoters were found to be more efficient in hypoxic or anaerobic environments than the wild-type promoters under the same conditions. In this environment, the promoter, e.g., PA, is enhanced in binding to a wild-type transcriptional regulator. It increases transcription of a gene operably linked to L or PAH. In this state, the genetically engineered bacteria express a wild-type oxygen level-dependent promoter, e.g., F Transcriptional analysis of the NR, ANR, or DNR promoter and wild-type promoters from bacteria of the same subtype The corresponding transcriptional regulator is mutated relative to the transcriptional regulator. Factors are more potent in hypoxic or anaerobic environments than wild-type transcriptional regulators under the same conditions. to enhance binding to wild-type promoters, such as PAL or P In certain embodiments, the mutation increases transcription of a gene operably linked to AH. Different oxygen level-dependent transcriptional regulators enhance dimerization and FNR activity in amino acids and FNR proteins containing acid substitutions (see, e.g., Moore et al., 2006 ).

[0106] In some embodiments, the genetically engineered bacteria of the present invention comprise an oxygen level-sensing transcriptionally regulated They contain multiple copies of endogenous genes encoding factors, such as the FNR gene. In embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and P The genes encoding AL are present on different plasmids. The gene encoding the protein level-sensing transcription factor and the gene encoding PAL are from the same promoter. In some embodiments, the transcription factor encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene for the oxygen level-sensing transcriptional regulator is present on a chromosome. The gene encoding the IL-1 gene and the gene encoding PAL are located on different chromosomes. In some embodiments, a gene encoding an oxygen level-sensing transcriptional regulator and PAL are The encoding genes are located on the same chromosome. In some cases, this improves expression stability. To achieve this, we express an oxygen-level-sensing transcription factor under the control of an inducible promoter. In some embodiments, expression of the transcriptional regulator is increased by phenylalanine metabolism. by a promoter different from that controlling the expression of the gene encoding the enzyme In some embodiments, expression of the transcriptional regulator is regulated by a phenylalanine metabolic enzyme. In some embodiments, the transcription factor is regulated by the same promoter that regulates expression of the transcription factor. Transcriptional factors and phenylalanine metabolic enzymes are divergently transcribed from the promoter region.

[0107] In some embodiments, the genetically engineered bacteria of the present invention can be grown under the same conditions and of the same subtype. Increase blood phenylalanine by at least about 1.5 times, and by at least about 1.5 times, compared to unmodified bacteria. 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, At least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least To reduce the oxygen content of mammals by at least about 50-fold, exogenous environmental conditions, such as the hypoxic environment of the mammalian gastrointestinal tract, In some embodiments, the genetically engineered bacteria of the invention produce PAL under PAL is produced under exogenous environmental conditions, such as the hypoxic environment of the mammalian digestive tract, and Compared with unmodified bacteria of the same subtype, they increased urinary hippuric acid by at least 1.5 times, and at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, At least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, or less at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or Certain unmodified bacteria produce appreciable levels of phenylalanine. Using recombinant forms of these bacteria, In the embodiment used, PAL-mediated processing of phenylalanine occurs under exogenous environmental conditions. It can be sensed by.

[0108] In some embodiments, the genetically engineered bacteria of the invention are in vitro bacteria. PAL is produced under exogenous environmental conditions, such as culture conditions, and unmodified PAL of the same subtype is produced under the same conditions. The amount of trans-cinnamic acid in the medium is at least about 1.5 times higher than that of the bacteria, and at least about 2 times higher than that of the bacteria. times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least About 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or less Phenylanine can be added by methods known in the art, for example, It can be measured by blood sampling and mass spectrometry. In some embodiments, cinnamic acid is P AL activity is measured by methods known in the art. The activity of cinnamic acid is directly correlated with phenylalanine degradation, and in some embodiments, cinnamic acid is a Cinnamic acid can be used as a surrogate biomarker for liver enzyme activity (Figure 16B). It can be further decomposed by enzymes to hippuric acid, both of which are measured as described in Examples 24-26. As shown herein, in vivo, TCAs rapidly convert hippuric acid Hippuric acid is then accumulated in the urine. Uric acid is by far the better biomarker of phenylalanine degradation in vivo. In some embodiments, PAL expression can be achieved by methods known in the art, e.g. It is measured by measuring the phenylalanine level in the blood. Hippuric acid is measured as described in the Examples of this specification. and can be measured by methods known in the art.

[0109] In some embodiments, the genetically engineered bacteria of the present invention can be grown under the same conditions and of the same subtype. Increase blood phenylalanine by at least about 1.5 times, and at least about 2 times, compared to unmodified bacteria. times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least About 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, or less LAAD is produced to reduce the IL-1 receptor by at least about 50-fold. These bacteria lack the ability to process phenylalanine at high levels. In the embodiment used, LAAD-mediated processing of phenylalanine is regulated by exogenous environmental conditions. Phenylanine can be detected by methods known in the art, for example, by blood sampling and and mass spectrometry. Pyruvate, a breakdown product produced by LAAD, and phenylpyruvate using mass spectrometry as described in Examples 24-26. It can be measured and used as a further readout of LAAD activity.

[0110] In some embodiments, the genetically engineered bacteria of the invention can be cultured in vivo or in vivo. Under exogenous environmental conditions, such as in vitro bacterial culture conditions, two or more PMEs, e.g., P producing AL, PAH, and / or LAAD and compared with unmodified bacteria of the same subtype under the same conditions. Compared to, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, At least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least at least about 30 times, at least about 40 times, or at least about 50 times, These embodiments reduce the amount of cinnamic acid and / or increase trans-cinnamic acid in the medium. In either case, the bacterium encodes one or more Phe transporter polypeptides. The gene sequence may further comprise a gene sequence corresponding to the gene.

[0111] In some embodiments, one or more PMEs, e.g., PALs, LAADs, and / or or PAH is expressed on a low copy plasmid. The plasmid may be useful to increase the stability of expression. Low copy plasmids may be useful to reduce leakage of expression under non-inducing conditions. In some embodiments, one or more PMEs, e.g., PALs, LAADs, and / or P AH is expressed on a high copy plasmid. In some embodiments, the high copy plasmid Midi increases PME, e.g., PAL, LAAD, and / or PAH expression. may be useful in increasing phenylalanine metabolism and reducing high phenylalanine In some embodiments, the PME expressed on a high copy plasmid For example, genetically engineered bacteria containing PAL, LAAD, and / or PAH can be used to In the absence of species pheP and additional copies of native pheP, on low-copy plasmids Genes containing the same PMEs expressed, e.g., PAL, LAAD, and / or PAH Does not increase phenylalanine metabolism compared to engineered bacteria or Same PME gene(s) on high and low copy plasmids, For example, genetically engineered genes containing PAL, LAAD, and / or PAH gene(s). Bacteria were generated, e.g., PAL1 on high-copy and low-copy plasmids. Either PAL3 or PAL4 is produced, each of which is metabolized to produce similar levels of phenylalanine. Thus, in some embodiments, phenylalanine The rate-limiting step in metabolism is phenylalanine availability (see, for example, Figure 16). These embodiments increase phenylalanine transport into cells, thereby increasing phenylalanine It may be beneficial to improve lutein metabolism. Even the plasmids can almost completely remove Phe from test samples (e.g., Fig. 16A). Additionally, in some embodiments incorporating pheP, Improved stability of PAL expression while maintaining phenylalanine metabolism in transformed bacteria Use a low-copy PAL expression plasmid in combination with PBS to reduce negative selection pressure on the PBS. In an alternative embodiment, a phenylalanine transporter is used in conjunction with a high copy plasmid.

[0112] In some embodiments, the transporter increases phenylalanine degradation. For example, Proteus mirabilis LAAD is a type of enzyme whose catalytic activity occurs in the periplasm. Phenylanine is localized in the cell membrane where it is transported. Therefore, genetically engineered bacteria expressing LAAD are In this form, the transporter may not be required for phenylalanine metabolism or may not need to be improved.

[0113] In some embodiments, the PME(s), e.g., PAL, LAAD, and / or In some embodiments, the PAH gene is expressed chromosomally. may be useful for increasing the stability of expression of PME. The ME genes, e.g., the PAL, LAAD, and / or PAH genes, are genetically engineered. The vector is integrated into the bacterial chromosome at one or more integration sites in the selected bacterium. In some embodiments, a PME gene, such as PAL, LAAD, and / or PA The H gene is inserted into the following E. coli Nissle insertion sites: malE / K, insB / I , araC / BAD, lacZ, agal / rsml, thyA, and malP / T The vector is inserted into the bacterial genome at one or more of the insertion sites. Any suitable insertion site may be used. The insertion site may be located within the genome, e.g., within a region (e.g., a nutritional Genes required for survival and / or growth, such as thyA (to create a mutant strain) within active regions of the genome, such as near genome replication sites, and / or near arabinose operations. To reduce the risk of unintended transcription, such as between AraB and AraC in the It may be located anywhere between the divergent promoters. In some embodiments, the PME gene More than one copy of a gene, e.g., PAL, PAH, and / or LAAD, e.g., , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. More than one copy of a PME gene can be integrated into more than one copy of the same PME gene. There may be more than one copy of a PME gene or different PME genes.

[0114] Exemplary constructs are shown in Tables 4-13 below. Table 4 shows the Phe constructs for chromosomal insertion. The sequence of an exemplary construct containing genes encoding the P and FNR promoter sequences (SEQ ID NO: 1) number 21), the pheP sequence is underlined, and the FNR promoter sequence is in bold. Table 5 shows the results of encoding the PAL1 and FNR promoter sequences on high copy plasmids. The sequence of an exemplary construct containing the gene encoding PAL1 is shown (SEQ ID NO: 22), and the PAL1 sequence is shown below. The FNR promoter sequence is underlined and bold. Table 6 shows the FNR promoter sequence on the high copy plasmid. Sequence of an exemplary construct containing a gene encoding PAL3 and FNR promoter sequences The sequence (SEQ ID NO: 23) is shown, with the PAL3 sequence underlined and the FNR promoter sequence. Columns are in bold. Table 7 shows the PAL1 and Tet promoter sequences on high copy plasmids. The sequence of an exemplary construct containing a gene encoding the PAL1 sequence (SEQ ID NO: 24) is shown. The columns are underlined and the Tet promoter sequence is in bold. Table 8 shows the high copy number of An exemplary construct containing a gene encoding PAL3 and a Tet promoter sequence on a smid The sequence of the construct (SEQ ID NO: 25) is shown, with the PAL3 sequence underlined and the Tet promoter. The promoter sequences are in bold. Table 9 shows the PAL1 and FNR promoter sequences on the low copy plasmids. The sequence of an exemplary construct containing a gene encoding a P The AL1 sequence is underlined and the FNR promoter sequence is in bold. Contains the gene encoding PAL3 and FNR promoter sequences on a copy plasmid The sequence of an exemplary construct (SEQ ID NO: 27) is shown, with the PAL3 sequence underlined and F The NR promoter sequence is in bold. Table 11 shows the PAL1 and PAL2 promoter sequences on low copy plasmids. The sequence of an exemplary construct containing a gene encoding the Tet promoter sequence (SEQ ID NO: 28) ) is shown, the PAL1 sequence is underlined, and the Tet promoter sequence is in bold. Table 12 encodes the PAL3 and Tet promoter sequences on low copy plasmids The sequence of an exemplary construct containing the gene (SEQ ID NO: 29) is shown, with the PAL3 sequence underlined. The Tet promoter sequence is in bold. Table 13 shows the sequence of the gene encoding pheP. a gene encoding TetR, and a Tet promoter sequence for insertion into the chromosome. The sequence of an exemplary construct (SEQ ID NO: 30) containing the following sequences is shown, with the pheP sequence underlined: The TetR sequence is boxed and the FNR promoter sequence is in bold.

[0115] [Table 4] [Table 5-1] [Table 5-2]

[0116] [Table 5] [Table 6-1] [Table 6-2] [Table 6-3]

[0117] [Table 6] [Table 7-1] [Table 7-2] [Table 7-3]

[0118] [Table 7] [Table 8-1] [Table 8-2] Table 8-3

[0119] [Table 8] Table 9-1 Table 9-2 Table 9-3 Table 9-4

[0120] [Table 9] Table 10-1 Table 10-2 Table 10-3

[0121] [Table 10] Table 11-1 Table 11-2 Table 11-3 Table 11-4

[0122] [Table 11] Table 12-1 [Table 12-2] [Table 12-3] [Table 12-4]

[0123] [Table 12] [Table 13-1] [Table 13-2] [Table 13-3] [Table 13] [Table 14]

[0124] In some embodiments, the genetically engineered bacterium comprises any one of SEQ ID NOs: 21-30. In some embodiments, the gene contains one or more sequences of the gene. The engineered bacteria have at least 75% identity with any of the sequences set forth in SEQ ID NOs: 21-30, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Genes containing one or more sequences with 96%, 97%, 98%, or 99% identity Contains gene sequences.

[0125] Phenylanine transport Each of PAL1 and PAL3 is highly expressed in the genetically engineered E. coli Nissle. The expression was confirmed on both high-copy and low-copy plasmids. The compound metabolized and reduced phenylalanine to similar levels (Figure 15). The rate-limiting step in phenylalanine metabolism was phenylalanine availability (Figure 16). In some embodiments, the phenylalanine transporter is increased into the cell, thereby increasing the phenylalanine transporter. Unexpectedly, the low-copy PAL plasmid Even when expressed in combination with pheP, it almost completely removed Phe from the test sample. Furthermore, PAL expression can be stably increased while maintaining high phenylalanine metabolism (Fig. 16A). Used in conjunction with pheP to improve quality and reduce negative selection pressure on transformed bacteria It may be even more beneficial to use a low copy PAL expression plasmid as an alternative. In this form, the phenylalanine transporter is used in conjunction with a high copy number plasmid. do.

[0126] The genetically engineered bacteria furthermore lack the gene encoding the phenylalanine transporter. Phenylanine transporters improve the transport of phenylalanine into cells. The present invention provides a method for the production of a recombinant protein comprising: obtain.

[0127] PheP is a membrane transport protein that can transport phenylalanine into bacterial cells (e.g., (See, e.g., Pi et al., 1991). In some embodiments, the recombinant vectors of the invention The native pheP gene in the bacterium is unmodified. The genetically engineered bacteria of the invention contain multiple copies of the native pheP gene. In embodiments, the genetically engineered bacteria of the invention contain multiple copies of the non-native pheP gene. In some embodiments, the genetically engineered bacteria of the present invention contain promoters, inducible promoters, promoters stronger than the native promoter, e.g. GlnRS promoter or P(Bla) promoter, or a constitutive promoter In some embodiments, the pheP gene is regulated by Expression is determined by upregulation of genes encoding phenylalanine metabolic enzymes and / or transcriptional regulators. In some embodiments, the promoter is controlled by a promoter different from that controlling the expression of the gene. In this state, expression of the pheP gene is regulated by phenylalanine metabolic enzymes and / or transcriptional regulators. In some embodiments, the p The heP gene and phenylalanine metabolic enzymes and / or transcriptional regulators are In some embodiments, PheP, phenylalanine, The expression of genes encoding thrombin metabolic enzymes and transcriptional regulators is controlled by different promoters. In some embodiments, PheP, a phenylalanine metabolic enzyme, The expression of genes encoding transcription factors is controlled by the same promoter. do.

[0128] In some embodiments, the native pheP gene in the genetically modified bacterium is modified. First, one or more additional copies of the native pheP gene control the expression of PAL. The same inducible promoter that controls the expression of PAL, such as the FNR promoter, Regulation of an inducible or constitutive promoter that is different from the promoter it controls In an alternative embodiment, the native pheP gene is not modified and is inserted into the genome under Copies of the non-native pheP gene from different bacterial species express the same inducer that controls the expression of PAL. Inducible promoters, such as the FNR promoter, or promoters controlling the expression of PAL The genome is expressed under the control of an inducible promoter different from the promoter, or a constitutive promoter. It is inserted into the system.

[0129] In some embodiments, the native pheP gene in the genetically modified bacterium is modified. First, one or more additional copies of the native pheP gene are present on the plasmid, the same inducible promoter that controls expression of L, e.g., the FNR promoter, or The promoter controlling the expression of PME is either an inducible promoter or a constitutive promoter. In an alternative embodiment, the native pheP gene is present in the bacterium under the control of a promoter. Unmodified, non-native copies of the pheP gene from different bacterial species are carried on a plasmid, P The same inducible promoter that controls the expression of AL, e.g., the FNR promoter, or The promoter controlling PAL expression is either an inducible promoter different from the promoter controlling PAL expression, or a constitutive promoter. It is present in bacteria under the control of a promoter.

[0130] In some embodiments, the native pheP gene is mutagenized to express phenylalanine. Mutants showing increased pheP transport were selected and the mutagenized pheP gene was isolated. and inserted into genetically engineered bacteria (e.g., Pi et al., 1996; Pi et al., 1999 (See also 8). The phenylalanine transporter modifications described herein are It may be present on a plasmid or a chromosome.

[0131] In some embodiments, the genetically engineered bacterium is E. coli Nissle, The native pheP gene in Nissle is unmodified and contains one or more additional The native E. coli Nissle pheP gene in pea controls the expression of PAL, a phenotype identical to that of the pheP gene in pea. Possible promoters include the FNR promoter, or the promoter controlling the expression of PAL. E. coli under the control of an inducible or constitutive promoter distinct from the motor In an alternative embodiment, the Nissle gene is inserted into the Nissle genome. The native pheP gene is unmodified, and a copy of the non-native pheP gene from a different bacterium is used. However, the same inducible promoters that control the expression of PAL, such as the FNR promoter, or an inducible promoter different from the promoter controlling the expression of PAL, or It is inserted into the E. coli Nissle genome under the control of a constitutive promoter. In an embodiment, the genetically engineered bacterium is E. coli Nissle, The native pheP gene in The E. coli Nissle pheP gene is the same gene that controls the expression of PAL on the plasmid. Controls expression of an inducible promoter, such as the FNR promoter, or PAL The cells are grown under the control of an inducible promoter or a constitutive promoter, which is different from the promoter. In an alternative embodiment, the native pheP gene in E. coli Nissle is not modified, and a copy of the non-native pheP gene from a different bacterium is placed on the plasmid. The same inducible promoter that controls the expression of PAL, e.g., the FNR promoter, if or an inducible promoter different from the promoter controlling PAL expression, or a constitutive promoter. It is present in bacteria under the control of a specific promoter.

[0132] In other embodiments, a gene encoding one or more Phe transporters The (group) can be located on a plasmid or chromosome, and their gene expression is controlled by the methods described herein. The promoter can be controlled by any of the promoters disclosed, and the promoter can be a PM Be identical to or different from the promoter regulating the E gene(s) but can.

[0133] E. coli has five different transport systems for accumulating aromatic amino acids (AroP, Mtr, It has been reported that it has the aroP gene (PheP, TnaB, and TyrP). The general amino acid permease encoded by It transports aromatic amino acids with high affinity and, together with PheP, is a major player in phenylalanine uptake. Furthermore, low levels of phenylalanine accumulation are thought to play a role in the Acid transporter-deficient E. coli strain (ΔaroP ΔpheP Δmtr Δtna Δ tyrP) and traced it to the activity of the LIV-I / LS system. The / LS system is composed of two periplasmic binding proteins, the LIV binding protein (LIV-I system) and the LIV-II system. ) and LS-binding proteins (LS system), as well as a branch consisting of a membrane component, LivHMGF It is a chain amino acid transporter (Koyanagi et al., and references therein; I dentification of the LIV-I / LS System as the Third Phenylalanine Transporter in E scherichia coli K-12).

[0134] In some embodiments, the genetically engineered bacteria comprise the aroP gene. In an embodiment, the genetically engineered bacterium is E. coli Nissle, The native aroP gene in E. coli was not modified, and the native E. coli Nissle aroP gene One or more additional copies of the PME are present on a plasmid or in the chromosome. the same inducible promoter that controls the FNR promoter, or araB AD promoter, an inducible promoter different from the promoter controlling PME expression In an alternative embodiment, the gene is present in E. coli under the control of a constitutive promoter. The native aroP gene in Nissle was not modified, and non-native aroP genes from different bacteria were Copies of the roP gene are located on the plasmid or in the chromosome, and are the same genes that control the expression of the PME. Inducible promoters, such as the FNR promoter or the AraBAD promoter or an inducible promoter different from the promoter controlling the expression of the PME, or is present in bacteria under the control of a constitutive promoter.

[0135] In other embodiments, the genetically engineered bacteria may contain the same or different inducible or constitutive It contains AroP and PheP under the control of a specific promoter.

[0136] In some embodiments, the pheP gene is expressed chromosomally. In some embodiments, chromosomal expression may be useful to increase the stability of pheP expression. In some embodiments, the pheP gene is present in one or more of the genetically engineered bacteria. In some embodiments, the vector is integrated into the bacterial chromosome at a number of integration sites. The eP gene is inserted into the following insertion sites in E. coli Nissle: malE / K, insB / I, araC / BAD, lacZ, agal / rsml, thyA, and malP / T The vector is inserted into the bacterial genome at one or more of the following locations: (See, e.g., Figure 66.) The insertion site may be located within the genome, e.g., Genes required for survival and / or growth, such as thyA (to create auxotrophic strains), Within genes, within active regions of the genome such as near sites of genome replication, and / or arabinose To reduce the risk of unintended transcription, such as between AraB and AraC in the operon The promoter may be located anywhere between the two divergent promoters.

[0137] In some embodiments, the genetically engineered bacteria contain one or more Phe transporters. The Phe transporter encodes a Phe-like molecule that is directly or indirectly transported prior to in vivo administration. Pre-induced strains (e.g., flasks, fermenters, etc.) during production of the strain prior to in vivo administration. or other culture vessels in response to specific molecules or metabolites provided in the culture medium. (expressed under the control of a virion promoter).

[0138] In another embodiment, the genetically engineered bacteria contain one or more Phe transporters. The Phe transporter is induced directly or indirectly by in vivo administration. (e.g., response conditions in the exogenous in vivo environment (e.g., the gastrointestinal tract) , or expressed under the control of an inducible promoter for a specific molecule or metabolite. In some embodiments, the promoter is a molecule specific to the gut or a molecule specific to hypoxia. In some embodiments, the bacterial strain is induced by chemical and / or nutritional It is administered in combination with an inducer.

[0139] In some embodiments, the genetically engineered bacteria have multiple mechanisms of action and / or one In certain embodiments, the bacterium contains different auxotrophies on the chromosome. Inclusion sites (e.g., malE / K, yicS / nepI, malP / T, agaI / rs An oxygen level-dependent promoter (e.g., P f nrS -PAL3) and five copies of PAL under its control, with different integrations on the chromosome. An oxygen level dependent promoter (e.g., P fnrS One copy of the phenylalanine transporter gene is under the control of In a more particular embodiment, the bacterium is genetically engineered to contain a kanamycin resistance gene. The thyA gene was further engineered to contain the thyA auxotrophy. It is deleted and / or replaced with an unrelated gene.

[0140] Oxygen level-independent inducible promoter In some embodiments, the genetically engineered bacteria are induced via an arabinose induction system. The genes for arabinose metabolism include the PAraBAD promoter and the α-amyloid promoter. Organized into a single operon, AraBAD, controlled by the motor. The D (or Para) promoter satisfies the criteria for an inducible expression system. AD likely functions through dual regulation of AraC, which functions as both an inducer and a repressor. Because of its regulatory role, it provides tighter control of payload gene expression than many other systems. Furthermore, the level of expression based on ParaBAD allows for fine-tuning of the expression level of the payload. The L-arabinose concentration can be adjusted over a wide range to regulate the The cell population exposed to subsaturating L-arabinose concentrations showed a 2% increase in the number of induced and non-induced cells. The L-arabinose transporter is divided into two subgroups, and the type of subgroup is determined by the determined by individual cell-to-cell differences in availability (Zhang et al., Devel opment and Application of an Arabinose-I nducible Expression System by Facilitati ng Inducer Uptake in Corynebacterium glu tamicum; Appl.Environ.Microbiol. August 2012 78, No. 16, pp. 5831-5838). Alternatively, induced release from ParaBad Currently, as described herein, the ribosome binding site (RBS) is optimized to control or The expression level can be adjusted or fine-tuned. Exemplary constructs (control of arabinose-inducible promoters) The construct for PAL expression below is shown in Figure 62C.

[0141] In one embodiment, one or more PMEs (e.g., PALs and / or LAADs), and and / or Phe transporters (e.g., PheP), and / or transcriptional regulators Expression of the α- and β-actin-dependent nucleotides (e.g., FNRS24Y) is driven by one or more arabinose-inducible promoters. In one embodiment, expression of PAL is driven directly or indirectly by arabinose. In one embodiment, Phe is driven directly or indirectly by an inducible promoter. Expression of P is driven directly or indirectly by an arabinose-inducible promoter In one embodiment, expression of the LAAD is directed by an arabinose-inducible promoter. In one embodiment, expression of FNRS24Y is driven indirectly by arabinose induction. It is driven directly or indirectly by an inducible promoter.

[0142] In another embodiment, one or more arabinose-inducible promoters are Drives expression of stimulant messages. Arabinose-induced bicist A ronic message is sent to one or more PMEs (e.g., PAL or LAAD) and / or or one or more Phe transporters (e.g., PheP) and / or one or more Such bicistronic molecules may include transcriptional regulators (e.g., FNRS24Y). Non-limiting examples of messages include PAL and PheP, PAL and LAAD, PAL and FNRS24Y, PheP and LAAD, PheP and and FNRS24Y, LAAD and FNRS24Y. Sage also contains two transcripts of the same gene, e.g., PAL-PAL, LAAD-LA AD, PheP-PheP and / or FNRSY24S-FNRSY24S Non-limiting examples of bicistronic messages are described herein, e.g., the sequence Para-FNRS24Y-LAAD is included in number 73.

[0143] In one embodiment, one or more arabinose-inducible promoters are tricistronic. Arabinose-induced tricistronic message expression The page may include one or more PMEs (e.g., PALs or LAADs) and / or one or more Phe transporters (e.g., PheP) and / or one or more transcriptional regulators ( For example, FNRS24Y). Such tricistronic messages The genes can be, for example, (1) transcription of three messages of the same gene; (2) transcription of one of the first genes; (3) three different genes; It may contain a transcription of one message. or LAAD) and / or PheP in any combination with a tricistronic Non-limiting examples of tricistronic messages include For example, PAL-PAL-PheP, as contained in SEQ ID NO: 95 (bold), is Included.

[0144] In one embodiment, the one or more arabinose-inducible promoters are Arabinose-induced multicistronic A message may be one or more PMEs (e.g., PAL or LAAD) and / or one one or more Phe transporters (e.g., PheP) and / or one or more transcriptional regulators Such multicistronic genes can include a factor (e.g., FNRS24Y). Messages can be, for example, (1) transcription of several messages of one and the same gene; one or more messages of one gene and one or more messages of a second gene; and (3) transcription of one or more messages from one or more different genes (DN Placed on A like beads on a string ). One or more PMEs (e.g., PAL or LAAD) and / or one or more Ph e transporter (e.g., PheP) and / or one or more regulatory factors (e.g., Any combination of FNRS24Y) can be included in a multicistronic message.

[0145] In some embodiments, the arabinose-inducible promoter is a promoter that is regulated by one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g. , PheP) and / or transcriptional regulators (e.g., FNRS24Y) in vivo In some embodiments, the expression of the nucleotide sequence is useful for expression or is induced during in vivo expression. , one or more PMEs and / or Phe transporters (e.g., PheP), and and / or the expression of transcriptional regulators (e.g., FNRS24Y) is regulated by one or more arabinose-induced It is driven in vivo directly or indirectly by an inducible promoter. In some embodiments, the promoter is co-injected with the genetically engineered bacteria of the invention. It is induced directly or indirectly by a given molecule, for example, arabinose.

[0146] In some embodiments, one or more PMEs (e.g., PAL and / or LAAD) ) and / or Phe transporters (e.g., PheP) and / or transcriptional regulation Expression of factors (e.g., FNRS24Y) can be monitored in vitro prior to in vivo administration. During the growth, preparation or manufacture of the strain, the expression of the present invention is regulated by one or more arabinose-inducible promoters. In some embodiments, the promoter is driven directly or indirectly by an arabinose-inducible promoter. The motors are activated during cultivation, e.g., growth in a flask, fermenter, or another suitable culture vessel. In some embodiments, expression is induced to bind the bacteria to the payload prior to administration. by molecules, e.g., arabinose, added to bacterial cultures to preload them with The promoter may be directly or indirectly induced. In some embodiments, the promoter is induced by an arabino In some embodiments, the Arabidopsis-induced culture is grown aerobically. The North-induced cultures are grown anaerobically.

[0147] In one embodiment, the arabinose-inducible promoter is a second promoter (e.g., one or more PMEs (e.g., PA) in conjunction with a second constitutive or inducible promoter L and / or LAAD) and / or Phe transporters (e.g., PheP ) and / or a transcriptional regulator (e.g., FNRS24Y) to drive expression of the construct. In some embodiments, two promoters are located proximally in the construct to control its expression. an arabinose-inducible promoter drives expression under a first set of exogenous conditions; The second promoter drives expression under a second set of exogenous conditions. The first and second conditions represent two consecutive culture conditions (i.e., flask, fermentor, or During the preparation of cultures in other suitable culture vessels, e.g., arabinose and IPT In another non-limiting example, the first induction condition can be a culture condition (e.g., The second inducing condition may be an in vivo condition, e.g., including the presence of arabinose. Such in vivo conditions include hypoxia, microaerobic or anaerobic conditions, gastrointestinal metabolism, and These include the presence of metabolic products and / or metabolic products administered in combination with the bacterial strain. In some embodiments, one or more arabinose promoters regulate the expression of the same gene sequence. In conjunction with the FNR promoter driving one or more PMEs (e.g., PAL and / or or LAAD) and / or Phe transporters (e.g., PheP) and / or or drive the expression of a transcriptional regulator (e.g., FNRS24Y).

[0148] In some embodiments, the arabinose-inducible promoter is a promoter as described herein. Low copy plasmid or high copy plasmid or Biosafety System Plus From the mid, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., F In some embodiments, the arabinose-inducible promoter drives expression of the α-amyloid β-glucanase (Aβ) gene. The promoter expresses one or more PMEs (e.g., PAL and and / or LAAD) and / or Phe transporters (e.g., PheP) and and / or drive expression of a transcriptional regulator (e.g., FNRS24Y). The sites are described herein.

[0149] In some embodiments, one or more PMEs (e.g., PAL and / or LAAD) ) and / or Phe transporters (e.g., PheP) and / or transcriptional regulation A factor (e.g., FNRS24Y) was knocked into the arabinose operon, resulting in the expression of the native arabinose In some embodiments, FNRS24 is driven by a North-inducible promoter. Y was knocked into the arabinose operon and driven by the native arabinose-inducible promoter. In some embodiments, FNRS24Y-LAAD is driven by arabinose. It is knocked into peron and driven by the native arabinose-inducible promoter.

[0150] In some embodiments, the genetically engineered bacteria has a sequence similar to any of SEQ ID NO: 67. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The arabinose-inducible construct further comprises a gene encoding AraC, which is One or more PMEs (e.g., PAL or LAAD), and / or one or more Phe transporter (e.g., PheP), and / or one or more regulatory factors (e.g., In some embodiments, the gene is divergently transcribed from the same promoter as the gene for the FNRS24Y gene. The genetically engineered bacteria may have at least 80%, 81%, or 90% identity with any of the sequences of SEQ ID NO: 66. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity In some embodiments, the genetically engineered bacterium comprises one or more gene sequences that A polypeptide encoded by any of the sequences of SEQ ID NO: 66 and at least 80% ,81%,82%,83%,84%,85%,86%,87%,88%,89%,90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% It includes one or more gene sequences that encode polypeptides having identity.

[0151] In some embodiments, the genetically engineered bacteria is inducible by a rhamnose induction system. The rhaBAD gene contains one or more gene sequences capable of activating the rhaBAD promoter. The rhaP BAD promoter is organized into a single operon controlled by It is regulated by two activators, RhaS and RhaR, and the corresponding genes are rha It belongs to a single transcription unit that is transcribed in the opposite direction to BAD. Under this condition, RhaR binds to the rhaP RS promoter, and RhaR and RhaS are produced. RhaS then activates rhaP BAD and rh together with L-rhamnose. aPT binds to the T promoter and activates transcription of the structural gene. AraC is supplied, In contrast to the arabinose system, which is transcribed divergently in the gene sequence, the rhamnose expression system In some cases, the expression of the chromosome is sufficient to activate transcription even on multicopy plasmids. Because the expression level is sufficient, there is no need to express more regulatory proteins. In this case, only the rhaP BAD promoter is cloned upstream of the gene to be expressed. Full induction of rhaBAD transcription also suppresses CRP, a key regulator of catabolic repression. Alternatively, inducible expression from rhaBAD requires the binding of the rhaBAD-cAMP complex. As described herein, the ribosome binding site (RBS) can be optimized to control or fine-tune the ribosome binding site (RBS). An exemplary construct is shown in Figure 62B (control of a rhamnose-inducible promoter) Constructs for PAL expression are shown below.

[0152] In one embodiment, one or more PMEs (e.g., PALs and / or LAADs), and and / or Phe transporters (e.g., PheP), and / or transcriptional regulators Expression of the rhamnose-inducible promoters (e.g., FNRS24Y) is driven by one or more rhamnose-inducible promoters. In one embodiment, expression of PAL is driven directly or indirectly by rhamnose induction. In one embodiment, the PheP promoter is driven directly or indirectly by a promoter. Expression is driven directly or indirectly by a rhamnose-inducible promoter. In embodiments, expression of LAAD is directly or indirectly driven by a rhamnose-inducible promoter. In one embodiment, expression of FNRS24Y is driven directly by a rhamnose-inducible promoter. In a non-limiting example, PAL expression is driven directly or indirectly by a For example, by a construct containing a rhamnose-inducible PAL such as that contained in SEQ ID NO: 106. It is driven by

[0153] In another embodiment, one or more rhamnose-inducible promoters are Rhamnose-induced bicistronic message expression. A check message may be sent to one or more PMEs (e.g., PAL or LAAD) and / or One or more Phe transporters and / or one or more transcriptional regulators (e.g., F NRS24Y), as described above for arabinose induction.

[0154] In one embodiment, one or more rhamnose-inducible promoters are Drives expression of Sage, a tricistronic message inducible by rhamnose may be administered with one or more PMEs (e.g., PAL and / or LAAD) and / or Phe Transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS 24Y). Rhamnose-induced tricistronic messages may include: One or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporter and / or one or more transcriptional regulators (e.g., FNRS24Y). This is the same as that described above for arabinose induction.

[0155] In one embodiment, the one or more rhamnose-inducible promoters are multicistronic. Rhamnose-induced multicistronic message expression Sage may be used with one or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or one or more regulators (e.g., For example, FNRS24Y). A message may be sent to one or more PMEs (e.g., PAL or LAAD) and / or one one or more Phe transporters and / or one or more transcriptional regulators (e.g., FN RS24Y), as described above for arabinose induction.

[0156] In some embodiments, the rhamnose-inducible promoter comprises one or more PMEs (PA L or LAAD) and / or one or more Phe transporters (e.g., Phe P) and / or the in vivo expression of one or more regulatory factors (e.g., FNRS24Y) In some embodiments, the expression of the nucleotide sequence of the present invention is useful in vivo or is induced during in vivo expression. One or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporter (e.g., PheP) and / or one or more regulatory factors (e.g., FN RS24Y) is directly expressed in vivo by one or more rhamnose-inducible promoters. In some embodiments, the promoter is directly or indirectly driven by the gene of the present invention. Direct or indirect binding by molecules co-administered with the engineered bacteria, e.g., rhamnose. It is directly induced.

[0157] In some embodiments, one or more PMEs (e.g., PALs or LAADs) and and / or one or more Phe transporters (e.g., PheP) and / or one or more The above regulators (e.g., FNRS24Y) were tested in vitro before in vivo administration. During the growth, preparation or production of the strain in In some embodiments, the promoter is driven directly or indirectly by a rhamnose-inducible promoter. The microbial cells are grown during cultivation, e.g., in a flask, fermenter, or another suitable culture vessel. In some embodiments, expression is induced to prime the bacteria with the payload prior to administration. Promoter-like activity can be achieved by preloading the bacterial culture with a molecule, e.g., rhamnose. The activator is induced directly or indirectly. In some embodiments, the activator is induced by rhamnose. The induced culture is grown aerobically. In some embodiments, the culture is induced with rhamnose. The derived cultures are grown anaerobically.

[0158] In one embodiment, the rhamnose-inducible promoter is a second promoter (e.g., In conjunction with one or more PMEs (e.g., PAL, or LAAD) and / or one or more Phe transporters (e.g., PheP ) and / or expression of a construct containing one or more regulatory elements (e.g., FNRS24Y) In some embodiments, two promoters are located proximally in the construct, and the The rhamnose-inducible promoter drives expression of the construct under a first set of exogenous conditions. and a second promoter drives expression under a second set of exogenous conditions. In the example, the first and second conditions are two consecutive culture conditions (i.e., flask, During the preparation of the culture in a fermenter or other suitable culture vessel, e.g., rhamnose and In another non-limiting example, the first induction condition can be a culture condition: For example, the second inducing condition may include the presence of rhamnose, and the second inducing condition may be in vivo conditions. Such in vivo conditions may include hypoxic, microaerobic, or anaerobic conditions. The presence of gastrointestinal metabolites and / or the metabolic products administered in combination with the bacterial strain In some embodiments, one or more rhamnose promoters are located in the same gene. In association with the FNR promoter driving expression of the sequence, one or more PMEs and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., F NRS24Y) expression.

[0159] In some embodiments, the rhamnose-inducible promoter is a low-copy promoter as described herein. -Plasmid or high copy plasmid or biosafety system plasmid? et al., one or more PMEs (e.g., PAL or LAAD) and / or one or more Ph e transporter (e.g., PheP) and / or one or more regulatory factors (e.g., In some embodiments, the rhamnose-inducible promoter drives expression of FNRS24Y. The promoter expresses one or more PMEs (e.g., PAL or or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or drive expression of one or more regulatory factors (e.g., FNRS24Y). Suitable insertion sites are described herein.

[0160] In some embodiments, the genetically engineered bacteria comprises any of the sequences of SEQ ID NO: 107. and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, or 99% identity.

[0161] In some embodiments, the genetically engineered bacteria are isopropyl β-D-1-thiogas Lactopyranoside (IPTG)-inducible system or other systems that induce transcription from the Lac promoter It contains one or more gene sequences that are inducible via the compound IPTG. Allolactose, a lactose metabolite, activates transcription of In contrast to allolactose, the sulfur atom in IPTG is a non-hydrolyzable This creates a degradable chemical bond, preventing IPTG from decomposing and maintaining a constant concentration. It binds to the repressor and allosterically transfers the tetrameric repressor from the lac operator (lacI), thereby allowing transcription of the genes in the lac operon. Since IPTG is not metabolized by E. coli, its concentration remains constant and Lac The promoter-controlled expression rate is tightly controlled both in vivo and in vitro. Since other transport pathways are also involved, IPTG uptake is controlled by the lactose permease. Inducible expression from PLac is independent of the ribosomal pathway, as described herein. It can be controlled or fine-tuned by optimizing the binding site (RBS). Alternatively, other compounds that inactivate LacI can be used in place of IPTG.

[0162] In one embodiment, one or more PMEs (e.g., PALs and / or LAADs) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., For example, expression of FNRS24Y is driven directly by one or more IPTG-inducible promoters. In one embodiment, expression of PAL is driven directly or indirectly by an IPTG-inducible promoter. In one embodiment, expression of PheP is driven directly or indirectly by a In one embodiment, the promoter is driven directly or indirectly by an IPTG-inducible promoter. In this study, expression of LAAD was directly or indirectly driven by an IPTG-inducible promoter. In one embodiment, expression of FNRS24Y is driven by an IPTG-inducible promoter. Directly or indirectly driven. Contains PAL under the control of an IPTG-inducible promoter Non-limiting examples of constructs (e.g., as contained in SEQ ID NO: 74) (see, e.g., Figure 60) ).

[0163] In another embodiment, one or more IPTG-inducible promoters are IPTG-induced bicistronic gene expression A message may be one or more PMEs (e.g., PAL or LAAD) and / or one one or more Phe transporters (e.g., PheP) and / or one or more transcriptional regulators The gene may contain a factor (e.g., FNRS24Y), as described above for arabinose induction. In one embodiment, the one or more IPTG-inducible promoters are the same as those in IPTG-induced tricistronic gene expression drives the expression of tricistronic messages. A check message may be sent to one or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or one or more transcription factors Regulatory factors (e.g., FNRS24Y) can be included, as described above with respect to arabinose induction. In one embodiment, the one or more IPTG-inducible promoters are the same as those described above. Multicistronic messages are driven by IPTG-induced A stochastic message is sent by one or more PMEs (e.g., PAL or LAAD) and and / or one or more Phe transporters (e.g., PheP) and / or one or more The transcriptional regulators may include the above transcription factors (e.g., FNRS24Y), which are involved in arabinose induction. and is the same as above.

[0164] In some embodiments, an IPTG-inducible promoter is a promoter that is regulated by one or more PMEs (e.g., , PAL and / or LAAD) and / or Phe transporters (e.g., P heP) and / or transcriptional regulator (e.g., FNRS24Y) expression in vivo In some embodiments, the 1 one or more PMEs and / or Phe transporters (e.g., PheP) and / or Expression of a transcription factor (e.g., FNRS24Y) is regulated by one or more IPTG-inducible promoters. They are driven in vivo directly or indirectly by motors. In some embodiments, the promoter is linked to a molecule, e.g., a gene encoding a promoter, that is co-administered with the genetically engineered bacterium of the present invention. For example, it is induced directly or indirectly by IPTG.

[0165] In some embodiments, one or more PMEs (e.g., PAL and / or LAAD) ) and / or Phe transporters (e.g., PheP) and / or transcriptional regulation Expression of factors (e.g., FNRS24Y) can be monitored in vitro prior to in vivo administration. directly driven by one or more IPTG-inducible promoters during the growth, preparation or production of the strain In some embodiments, the IPTG-inducible promoter is directly or indirectly driven. induced during culture, e.g., during growth in a flask, fermenter, or another suitable culture vessel. In some embodiments, expression is induced to preload the bacteria with the payload prior to administration. The promoter is activated by a molecule, e.g., IPTG, that is added to the bacterial culture to activate it. In some embodiments, the gene is induced by IPTG. In some embodiments, the culture is induced with IPTG. The cultures are grown anaerobically.

[0166] In one embodiment, the IPTG-inducible promoter is a second promoter (e.g., a second In combination with one or more PMEs (e.g., PAL and and / or LAAD) and / or Phe transporters (e.g., PheP) and The construct drives expression of a gene encoding the nucleotide sequence of the nucleotide sequence of interest and / or a transcriptional regulator (e.g., FNRS24Y). In some embodiments, two promoters are located proximally in the construct, and the expression of the construct an IPTG-inducible promoter drives expression under a first set of exogenous conditions; and the second promoter drives expression under a second set of external conditions. In the above, the first and second conditions are two consecutive culture conditions (i.e., flask, fermenter). or other suitable culture vessels during the preparation of the culture, e.g., arabinose and In another non-limiting example, the first induction condition can be a culture condition (e.g., IPTG). and the second induction condition may be an in vivo condition (including the presence of IPTG). Such in vivo conditions may include hypoxic, microaerobic or anaerobic conditions, The presence of gastrointestinal metabolites and / or metabolites administered in combination with the bacterial strain In some embodiments, one or more IPTG-inducible promoters are located in the same gene. In conjunction with the FNR promoter, which drives expression of the sequence, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or drive expression of transcriptional regulators (e.g., FNRS24Y).

[0167] In some embodiments, the IPTG-inducible promoter is a low copy promoter as described herein. From plasmids or high copy plasmids or biosafety system plasmids , one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS2 In some embodiments, the IPTG-inducible promoter drives expression of the 4Y gene. From the construct integrated into the bacterial chromosome, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or Exemplary insertion sites are described herein. It is written in the book.

[0168] In some embodiments, the genetically engineered bacteria has a sequence similar to any of SEQ ID NO: 76. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% ,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% In some embodiments, the sequence of the gene is a sequence of the gene having 99% identity to the sequence of the gene of the present invention. , the IPTG-inducible construct further contains a gene encoding lacI, the lacI gene One or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporter (e.g., PheP) and / or one or more regulatory factors (e.g., FN In some embodiments, the gene is divergently transcribed from the same promoter as the gene The engineered bacteria have at least 80%, 81%, 82% identity with any of the sequences of SEQ ID NO: 75. ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity In some embodiments, the genetically engineered bacterium comprises one or more gene sequences. Identifies at least 80%, 81%, or more identical to a polypeptide encoded by any of the sequences of SEQ ID NO:75 ,82%,83%,84%,85%,86%,87%,88%,89%,90%,91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity It comprises one or more gene sequences encoding a polypeptide having the

[0169] In some embodiments, the genetically engineered bacteria expresses a tetracycline-inducible system It contains one or more inducible gene sequences. First system (Tight control of gene expression in mammalian cells by tetracycline-responsi ve promoters., Gossen M & Bujard H. PNAS, 1 June 15, 1992; Vol. 89(12): 5547-51) is a tetracycline known as fu: in the presence of tetracycline, from a tet-inducible promoter The tetracycline-controlled transactivator (tTA) reduces the expression of tetR. to the C-terminal domain of VP16 (virion protein 16) from herpes simplex virus. In the absence of tetracycline, the tTA te The tR portion binds to the tetO sequence in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR and binds to tTA. Second, the presence of tetracycline prevents the ATP from binding to the tetO sequence, resulting in induction rather than repression. A reverse Tet repressor (rTetR) based on the presence of Tet receptors has been developed. The activator rtTA (reverse tetracycline-controlled transactivator) is The tetracycline-on system was created by fusing tR with VP16. Also known as the tTA-dependent system.

[0170] In one embodiment, one or more PMEs (e.g., PALs and / or LAADs) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., For example, expression of FNRS24Y is driven by one or more tetracycline-inducible promoters. In one embodiment, expression of PAL is driven directly or indirectly by tetracycline. In one embodiment, the P Expression of heP is driven directly or indirectly by a tetracycline-inducible promoter. In one embodiment, expression of the LAAD is driven by a tetracycline-inducible promoter. In one embodiment, expression of FNRS24Y is driven directly or indirectly by It is driven directly or indirectly by a tracycline-inducible promoter.

[0171] In another embodiment, one or more tetracycline-inducible promoters are It drives the expression of tetracycline-induced vasopressin (VPS) ... A cistronic message is sent via one or more PMEs (e.g., PAL or LAAD) and and / or one or more Phe transporters (e.g., PheP) and / or The gene may contain one or more transcriptional regulators (e.g., FNRS24Y), and may be arabinose-induced In one embodiment, one or more tetracycline-induced A cis-promoter drives expression of the tricistronic message. The tricistronic messages induced by one or more PMEs (e.g., PA L or LAAD) and / or one or more Phe transporters (e.g., Phe P) and / or one or more transcriptional regulators (e.g., FNRS24Y). The methods are the same as those described above for arabinose induction. In one embodiment, one or more A tetracycline-inducible promoter drives expression of multicistronic messages Tetracycline-induced multicistronic messages consist of one or more PME (e.g., PAL or LAAD) and / or one or more Phe trans a promoter (e.g., PheP) and / or one or more transcriptional regulators (e.g., FNR S24Y), as described above for arabinose induction.

[0172] In some embodiments, the tetracycline-inducible promoter comprises one or more PMEs. (PAL and / or LAAD) and / or Phe transporters (e.g., P heP) and / or transcriptional regulator (e.g., FNRS24Y) expression in vivo In some embodiments, the 1 one or more PMEs and / or Phe transporters (e.g., PheP) and / or The expression of one or more tetracycline-induced transcription factors (e.g., FNRS24Y) They are driven in vivo directly or indirectly by inducible promoters. In one embodiment, the promoter is co-administered with the genetically engineered bacteria of the invention. It is induced directly or indirectly by molecules such as tetracycline.

[0173] In some embodiments, one or more PMEs (e.g., PAL and / or LAAD) ) and / or Phe transporters (e.g., PheP) and / or transcriptional regulation Expression of factors (e.g., FNRS24Y) can be monitored in vitro prior to in vivo administration. During the growth, preparation or manufacture of the strain, one or more tetracycline-inducible promoters In some embodiments, the tetracycline-induced The inducible promoter may be introduced during cultivation, for example in a flask, fermentor, or another suitable culture vessel. In some embodiments, expression is induced to promote growth of the bacteria prior to administration. Molecules added to bacterial cultures for preloading with erode, e.g., tetracycline In some embodiments, the promoter is induced directly or indirectly by the In some cases, tetracycline-induced cultures are grown aerobically. In this embodiment, the tetracycline-induced culture is grown anaerobically.

[0174] In one embodiment, the tetracycline-inducible promoter is a second promoter (e.g., one or more PMEs (e.g., a second constitutive or inducible promoter) in combination with PAL and / or LAAD) and / or Phe transporters (e.g., Ph eP) and / or a construct containing a transcriptional regulator (e.g., FNRS24Y) In some embodiments, two promoters are located proximally in the construct, and the construct The tetracycline-inducible promoter drives expression of the construct under a first set of exogenous conditions. a first promoter drives expression under a first set of exogenous conditions, and a second promoter drives expression under a second set of exogenous conditions. In a limiting example, the first and second conditions are two consecutive culture conditions (i.e., During the preparation of the culture in a lab, fermenter or other suitable culture vessel, e.g., In another non-limiting example, the first induction condition can be erythrocytic leukemia (e.g., erythrocytic leukemia) or ...). is a culture condition (e.g., including the presence of tetracycline), and the second induction condition is in v Such in vivo conditions may include hypoxic, microaerophilic or Anaerobic conditions, the presence of gastrointestinal metabolites, and / or the bacterial strains administered in combination with In some embodiments, one or more tetracycline promoters are included. In cooperation with the FNR promoter, one or more PMEs drive the expression of the same gene sequence. (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., driving the expression of transcription factors (e.g., PheP) and / or transcriptional regulators (e.g., FNRS24Y) do.

[0175] In some embodiments, the tetracycline-inducible promoter is a promoter as described herein. Low copy plasmid or high copy plasmid or Biosafety System Plus From the mid, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., F In some embodiments, the tetracycline-inducible promoter drives expression of NRS24Y. The promoter is expressed by one or more PMEs (e.g., PA) from a construct integrated into the bacterial chromosome. L and / or LAAD) and / or Phe transporters (e.g., PheP ) and / or transcriptional regulators (e.g., FNRS24Y). The insertion sites are described herein.

[0176] In some embodiments, the genetically engineered bacteria comprises the bolded sequence of SEQ ID NO: 39 (te t promoters in bold) and at least 80%, 81%, 82%, 83%, or 84% %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 95%, 96%, 97%, 98%, or 99% identity to one or more gene sequences In some embodiments, the tetracycline-inducible construct comprises a sequence encoding AraC. AraC further comprises a gene encoding one or more PMEs (e.g., PAL or LAA) D), and / or one or more Phe transporters (e.g., PheP), and / or or divergently transcribed from the same promoter as one or more regulatory elements (e.g., FNR24Y). In some embodiments, the genetically engineered bacterium comprises the italicized sequence of SEQ ID NO: 39. at least 80%, 8 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical In some embodiments, the genetically engineered bacteria comprises one or more gene sequences having the desired properties. is the italicized sequence of SEQ ID NO: 39 (the Tet repressor is in italics) and a polypeptide encoded by any of the %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 94%, 95%, 96%, 97%, 98%, or 99% identity It comprises one or more gene sequences encoding a peptide.

[0177] In some embodiments, the genetically engineered bacteria comprise a gene encoding a gene encoding a gene for which expression is regulated by a temperature-sensitive mechanism. The thermoregulator contains one or more gene sequences that are controlled by external chemicals or special This has the advantage that strong transcriptional regulation can be achieved without using a culture medium (e.g., Nemani et al., Magnetic nanoparticle hyperthermia ind. uced cytosine deaminase expression in mi croencapsulated E. coli for enzyme-prodr ug therapy; J Biotechnol. 2015, June 10; Vol. 203 :32-40 and references therein). Temperature-regulated protein expression using the pL and / or pR phage lambda promoters Currently, it has been used to engineer recombinant bacterial strains. The gene of interest was cloned into the heat-labile mutant cI857 replica of bacteriophage λ. At temperatures below 37°C, cI857 is efficiently regulated by the pR promoter. Binds to the oL or oR domain of the motor and blocks transcription by RNA polymerase At temperatures above 37°C, the functional cI857 dimer is destabilized, resulting in the formation of oL or Binding to the oR DNA sequence is eliminated, and mRNA transcription is initiated. An exemplary construct is shown in Figure 1. Inducible expression from ParaBad is shown in Fig. 62A. This can be controlled or further fine-tuned by optimization of the RBS.

[0178] In one embodiment, one or more PMEs (e.g., PALs and / or LAADs) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., For example, expression of FNRS24Y is driven directly or by one or more temperature-regulated promoters. In one embodiment, expression of PAL is indirectly driven by a temperature-regulated promoter. In one embodiment, expression of PheP is driven directly or indirectly by a temperature-regulated process. In one embodiment, the expression of the LAAD is driven directly or indirectly by a motor. , driven directly or indirectly by a temperature-regulated promoter. Expression of NRS24Y is driven directly or indirectly by a temperature-regulated promoter Constructs in which PAL expression is induced at 37°C and 42°C but not at lower temperatures A non-limiting example of is included in SEQ ID NO: 101 (see, e.g., Figures 60 and 62A).

[0179] In another embodiment, the one or more temperature-regulated promoters are one or more bicistronic Inducible by temperature, e.g., 37°C and / or 42°C. The bicistronic message to be sent may be one or more PMEs (e.g., PAL or LA AD) and / or one or more Phe transporters (e.g., PheP) and / or or one or more transcriptional regulators (e.g., FNRS24Y), In one embodiment, one or more temperature-regulated promoters The promoter drives the expression of the tricistronic message. and / or tricistronic messages induced by 42°C contain one or more PMEs. (e.g., PAL or LAAD) and / or one or more Phe transporters ( PheP) and / or one or more transcriptional regulators (e.g., FNRS24Y) and the same as described above for arabinose derivatization. One or more temperature-regulated promoters drive the expression of multicistronic messages Thermoregulatory-induced multicistronic messages are transmitted to more than one PM. E (e.g., PAL or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or one or more transcriptional regulators (e.g., FNRS24Y ), as described above for arabinose derivatization.

[0180] In some embodiments, the temperature-regulated promoter comprises one or more PMEs (e.g., PAs). L and / or LAAD) and / or Phe transporters (e.g., PheP ) and / or transcriptional regulators (e.g., FNRS24Y) in vivo or is induced during in vivo expression. In some embodiments, one or more PME and / or Phe transporter (e.g., PheP) and / or translocation Expression of transcription factors (e.g., FNRS24Y) is driven by one or more temperature-regulated promoters. In some embodiments, the promoter is driven directly or indirectly in vivo by The promoter is activated by a molecule, e.g., temperature, that is co-administered with the genetically engineered bacteria of the invention. It is induced directly or indirectly by

[0181] In some embodiments, one or more PMEs (PALs and / or LAADs) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., For example, FNRS24Y expression is associated with in vitro growth and development prior to in vivo administration. Driven directly or indirectly by one or more temperature-regulated promoters during preparation or manufacturing In some embodiments, the production of one or more PMEs and / or PheP is blocked. This can be advantageous in a temperature regulation system by controlling the temperature to a lower temperature, e.g. This can be done by growing the strain at 0°C. The temperature can then be increased to 37°C and / or 42°C. Expression can be induced by increasing the temperature to 0°C. Thus, the temperature-regulated promoter can be used during cultivation, e.g., in a flask, fermentor, or another suitable culture vessel. In some embodiments, the induction is performed in a culture medium at a temperature between 37°C and 42°C. The culture is grown aerobically. In some embodiments, the culture is grown at a temperature between 37°C and 42°C. The induced cultures are grown anaerobically.

[0182] In one embodiment, the temperature-regulated promoter is a second promoter (e.g., In conjunction with one or more PMEs (e.g., constitutive or inducible promoters) PAL and / or LAAD) and / or Phe transporters (e.g., Expression of constructs containing PheP) and / or transcriptional regulators (e.g., FNRS24Y) In some embodiments, two promoters are located proximally in the construct, driving the A temperature-regulated promoter drives expression under a first set of exogenous conditions. A second promoter drives expression under a second set of exogenous conditions. In this study, the first and second conditions were defined as two consecutive culture conditions (i.e., flask, During the preparation of the culture in the fermenter or other suitable culture vessel, e.g., temperature regulation and In another non-limiting example, the first induction condition can be a culture condition, e.g., , the permissive temperature, and the second inducing condition can be an in vivo condition. In vivo conditions include hypoxia, microaerobic or anaerobic conditions, the presence of gastrointestinal metabolites, and / or metabolites administered in combination with the bacterial strain. In this state, one or more temperature-regulated promoters are linked to the FNR promoters driving the expression of the same gene sequence. In collaboration with the promoter, one or more PMEs (e.g., PAL and / or LAAD) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS24Y) expression.

[0183] In some embodiments, the temperature regulated promoter is a low copy number as described herein. One of the following: mid or high copy plasmid or biosafety system plasmid or more PME (e.g., PAL and / or LAAD) and / or Phe trans transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS24Y) In some embodiments, the temperature-regulated promoter is integrated into the bacterial chromosome. From the embedded construct, one or more PMEs (e.g., PAL and / or LAAD) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS24Y). Exemplary insertion sites are described herein. do.

[0184] In some embodiments, the genetically engineered bacteria comprises any of the sequences of SEQ ID NO: 103. and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 In some embodiments, the sequence comprises one or more gene sequences that share at least 99% or 100% identity. The temperature-regulated construct further contains the gene encoding the mutant cI857 repressor. and / or one or more PMEs (e.g., PAL or LAAD), and / or one one or more Phe transporters (e.g., PheP), and / or one or more regulators thereof (e.g., FNR24Y) are divergently transcribed from the same promoter. In this embodiment, the genetically engineered bacteria have at least 80% identity with any of the sequences of SEQ ID NO: 102. ,81%,82%,83%,84%,85%,86%,87%,88%,89%,90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% In some embodiments, the gene is a genetically engineered gene. The bacterium may express at least one polypeptide encoded by any of the sequences of SEQ ID NO: 102. Also 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or It contains one or more gene sequences encoding polypeptides with 99% identity.

[0185] In some embodiments, the genetically engineered bacteria is driven by a PssB promoter. It contains one or more gene sequences that are indirectly induced through a system that drives the Pssb promoter. The promoter is active under aerobic conditions and is turned off under anaerobic conditions.

[0186] This promoter can be used to express a gene of interest under aerobic conditions. The promoter of the gene product may also be expressed under anaerobic conditions so that the gene product is only expressed under anaerobic conditions. In this case, the oxygen-inducible PssB protease can be used to tightly control the expression of PssB. The motor induces the expression of a repressor that suppresses the expression of the gene of interest. , the gene of interest is expressed only in the absence of a repressor, i.e., under anaerobic conditions. This strategy has the advantage of providing an additional level of control for improved fine tuning and tighter control. Figure 63A shows a schematic of the genetic organization of the PssB promoter.

[0187] In one embodiment, one or more PMEs (e.g., PALs and / or LAADs) and and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., For example, expression of FNRS24Y is under the control of one or more PssB promoters. In one embodiment, expression of PAL is indirectly regulated by one or more repressors. It is indirectly regulated by a repressor expressed under the control of the PssB promoter on In one embodiment, expression of PheP is under the control of one or more PssB promoters. In one embodiment, expression of LAAD is indirectly regulated by a repressor expressed by , indirectly by a repressor expressed under the control of one or more PssB promoters In one embodiment, expression of FNRS24Y is regulated by one or more PssB promoters. It is indirectly regulated by a repressor expressed under the control of

[0188] In another embodiment, induction of the RssB promoter is achieved by one or more bicistronic The induced bicistronic message drives the expression of one or more P ME (e.g., PAL or LAAD) and / or one or more Phe transporters - (e.g., PheP) and / or one or more transcriptional regulators (e.g., FNRS24 Y), as described above for arabinose derivatization. In this form, induction of the RssB promoter regulates the expression of one or more tricistronic messages. The tricistronic message being induced is the expression of one or more PMEs (e.g. PAL or LAAD) and / or one or more Phe transporters (e.g. , PheP) and / or one or more transcriptional regulators (e.g., FNRS24Y) and are the same as those described above for arabinose derivatization. Induction of the ssB promoter indirectly regulates the expression of one or more multicistronic messages. Arabinose-induced multicistronic messages drive expression of One or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporter (e.g., PheP) and / or one or more transcriptional regulators (e.g., FNRS24Y), which are the same as those described above for arabinose induction. do.

[0189] In some embodiments, induction of the RssB promoter is achieved by one or more PMEs (e.g., , PAL and / or LAAD), and / or Phe transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS24Y) in vivo In some embodiments, induction of the RssB promoter indirectly drives expression. During the in vitro propagation, preparation, or manufacturing of the strain prior to administration in vivo, One or more PMEs (e.g., PAL and / or LAAD), and / or Phe transfer transporters (e.g., PheP) and / or transcriptional regulators (e.g., FNRS24 In some embodiments, the RssB promoter is induced to indirectly drive expression of the RssB promoter. The conditions are provided in a culture, e.g., a flask, fermentor, or another suitable culture vessel. will be done.

[0190] In some embodiments, the PssB promoter is regulated by one or more PMEs (e.g., PAL and / or LAAD), and / or Phe transporters (e.g., PheP ), and / or expression of a transcriptional regulator (e.g., FNRS24Y), as described herein. Low copy or high copy plasmids or biosafety system plasmids In some embodiments, the PssB promoter is indirectly driven from one or more PME (e.g., PAL ...

Claims

1. a) one or more genes encoding phenylalanine ammonia lyase (PAL) having at least 90% identity to any one of the sequences of SEQ ID NOs: 1 to 4, wherein the genes are operably linked to an inducible promoter selected from a temperature-regulated promoter, an IPTG-inducible promoter, a rhamnose-inducible promoter, and a PssB promoter; b) one or more genes encoding a non-naturally occurring phenylalanine transporter, the genes being operably linked to an inducible promoter not naturally associated with the phenylalanine transporter gene.

2. 2. The gene expression system of claim 1, further comprising one or more genes encoding L-amino acid deaminase (LAAD), wherein the genes are operably linked to an inducible promoter not naturally associated with the LAAD gene.

3. The gene expression system of claim 1 or 2, wherein the gene encoding the PAL is operably linked to an IPTG-inducible promoter.

4. The gene expression system according to any one of claims 1 to 3, further comprising a gene encoding a LacI repressor.

5. (a) the promoter operably linked to the gene encoding the PAL and the promoter operably linked to the gene encoding the phenylalanine transporter are separate copies of the same promoter; (b) the gene encoding the PAL and the gene encoding the phenylalanine transporter are operably linked to the same copy of the same promoter.

6. (a) the gene encoding the LAAD is operably linked to a promoter that is different from the promoter operably linked to the gene encoding the PAL; and / or (b) The gene expression system according to any one of claims 2 to 5, wherein the gene encoding the phenylalanine transporter is operably linked to a promoter that is different from a promoter that controls expression of the gene encoding the PAL and / or the gene encoding the LAAD.

7. The gene expression system according to any one of claims 1 to 6, wherein the promoter operably linked to the gene encoding the phenylalanine transporter is selected from a promoter that is induced under hypoxic or anaerobic conditions; a temperature-regulated promoter; and a promoter that is induced by arabinose, IPTG, tetracycline, or rhamnose.

8. The gene expression system of any one of claims 2 to 7, wherein the promoter operably linked to the gene encoding the LAAD is selected from a promoter that is induced under hypoxic or anaerobic conditions; a temperature-regulated promoter; and a promoter that is induced by arabinose, IPTG, tetracycline, or rhamnose.

9. The gene expression system according to any one of claims 2 to 8, wherein the gene encoding the phenylalanine transporter and / or the gene encoding the LAAD is under the control of a promoter inducible by arabinose, IPTG, tetracycline, or rhamnose.

10. The gene expression system according to any one of claims 1 to 9, wherein the phenylalanine transporter is PheP.

11. The gene expression system according to any one of claims 1, 2, 5 to 10, wherein the temperature-regulated promoter is induced at a temperature between 37°C and 42°C.

12. The gene expression system according to any one of claims 1, 2, 5 to 11, wherein the temperature-regulated promoter is a lambda CI inducible promoter.

13. The gene expression system of any one of claims 1, 2, 5 to 12, further comprising one or more genes encoding temperature-sensitive CI repressor mutants.

14. 14. The gene expression system of claim 13, wherein the temperature-sensitive CI repressor mutant is CI857 having the polypeptide sequence of SEQ ID NO:

105.

15. The gene expression system according to any one of claims 1 to 14, wherein the gene encoding the phenylalanine transporter and / or the gene encoding the LAAD is under the control of an FNR-responsive promoter; or a promoter inducible by arabinose, IPTG, tetracycline, or rhamnose.

16. 15. The gene expression system of claim 13 or 14, wherein the gene encoding the temperature-sensitive CI repressor mutant and the gene encoding LAAD are under the control of the same promoter.

17. 17. The gene expression system according to any one of claims 1 to 16, wherein the PAL is derived from Anabaena variabilis (PAL1) or Photorhabdus luminescens (PAL3).

18. The gene expression system according to any one of claims 1 to 17, wherein the phenylalanine transporter is PheP.

19. A genetically engineered cell comprising the gene expression system of any one of claims 1 to 18.

20. The gene encoding a phenylalanine transporter, the gene encoding PAL, and / or the gene encoding LAAD is (a) located on a chromosome in said cell; 20. The cell of claim 19, wherein (b) the gene is located on a plasmid in the cell.

21. A pharmaceutically acceptable composition comprising an expression construct or cell according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. 22. The composition of claim 21 formulated for oral administration.

23. 23. The composition of claim 21 or 22 for use in a method for reducing hyperphenylalaninemia or treating a disease associated with hyperphenylalaninemia.

24. 24. The composition of claim 23, wherein the disease is selected from the group consisting of phenylketonuria, classic or typical phenylketonuria, atypical phenylketonuria, persistent mild hyperphenylalaninemia, non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase deficiency, Segawa disease, and liver disease.

25. 21. Use of an expression construct or cell according to any one of claims 1 to 20 in the manufacture of a medicament for reducing hyperphenylalaninemia or for treating a disease associated with hyperphenylalaninemia.

26. 26. The use of claim 25, wherein the disease is selected from the group consisting of phenylketonuria, classic or typical phenylketonuria, atypical phenylketonuria, persistent mild hyperphenylalaninemia, non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin synthase deficiency, Segawa disease, and liver disease.

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