Bacteria engineered to reduce hyperphenylalaninemia

Abstract

This invention provides compositions and treatment methods for reducing hyperphenylalaninemia. [Solution] A genetically modified bacterium is provided, comprising: a) one or more genes encoding phenylalanine ammonia lyase (PAL), which are operably linked to a promoter that is not naturally associated with the PAL gene and can be directly or indirectly induced; b) one or more genes encoding a phenylalanine transporter, which are not naturally associated with the phenylalanine transporter gene and can be operably linked to a promoter that is directly or indirectly induceable; and c) one or more genes encoding mutant fumarate and nitrate reductase (FNR), which are not naturally associated with the FNR gene and can be operably linked to a promoter that is directly or indirectly induceable.

Description

[Technical Field]

[0001] This application is based on U.S. Provisional Patent Application No. 62 / 256,05, filed on November 16, 2015. Application No. 2, and PCT application number PCT / US2016 / filed on May 13, 2016 Claiming priority rights over 032562, and the contents thereof, by reference, are the whole of those. This is incorporated herein. [Background technology]

[0002] This disclosure relates to hyperphenylalaninemia. This invention relates to compositions and therapeutic methods for reducing infant feeding. Genetically modified bacteria that can reduce hyperphenylalaninemia in substances In certain embodiments, the compositions and methods disclosed herein are high in phenylalanine. It can be used to treat diseases associated with hememia, such as phenylketonuria.

[0003] Phenylalanine is an essential amino acid primarily found in food proteins. Typologically, a small amount is used for protein synthesis, and the rest is phenylalanine hydroxy In enzyme pathways requiring the enzyme (PAH) and cofactor tetrahydrobiopterin It is hydroxylated to tyrosine. Hyperphenylalaninemia is toxic and causes brain damage. This is a group of diseases associated with excessive levels of phenylalanine that can potentially cause [unspecified symptoms]. Onset hyperphenylalaninemia is caused by mutations in the PAH gene and / or blockade of cofactor metabolism. This is caused by a deficiency in PAH activity resulting from the disruption of the system.

[0004] Phenylenketonuria (PKU) is a high-risk condition caused by mutations in the PAH gene. PKU is a severe form of phenylalaninemia. PKU is the most common congenital metabolic disorder worldwide. It is an autosomal recessive genetic disorder that is classified as occurring in 1 out of every 3,000 births. It has affected approximately 13,000 patients in the United States. Over 400 different P AH gene mutations have been identified (Hoeks et al., 2009). Phenyl in the blood Alanine (Phe) accumulation can cause serious damage to the central nervous system in children and adults. It is possible. 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 necessary for growth. This means that patients with PKU are supplied with just enough phenylalanine for growth. Along with amino acid supplements, medical foods and PHE-free protein supplements are also available. This means that it is still present. 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. It is necessary. Generally, treatment from birth reduces brain damage and intellectual disability (H Oeks et al., 2009; Sarkissian et al., 1999). However, Tan Protein-restricted diets must be carefully monitored, and essential amino acids and vitamins must be consumed. It must be supplemented during the process. Furthermore, using low-protein foods means that they However, this presents a challenge because it is more expensive than its unmodified counterpart, which is a high-protein alternative. (Vockley et al., 2014).

[0006] Growth retardation is common in children with PKU who continue 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 years). Excessive levels of phenylalanine in the blood that can freely cross the blood-brain barrier. Nin is a neurological disorder, behavioral disorder (e.g., hyperirritability, fatigue), and / or physical symptoms. For example, it may cause convulsions, skin rashes, and moldy odors. (International guidelines) While a lifelong dietary restriction of phenylalanine is recommended, this is difficult. It is widely considered unrealistic (Sarkissian et al., 1999), "P Continuous efforts to overcome the greatest challenges in living with KU - lifelong low pH "Adherence to e-dietary guidelines is required" (Macleod et al., 2010).

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

[0008] The enzyme phenylalanine ammonia lyase (PAL) converts phenylalanine into a non-toxic substance. It can be metabolized to Bell's ammonia and trans cinnamic acid. Unlike PAHs, PAL does not require THB cofactor activity to metabolize phenylalanine. Research is being conducted on oral enzyme therapy using PAL, but "PAL is affordable and available in sufficient quantities." "Since it was unavailable, research was not continued, not even on humans or animals." (Sarkis) Sian et al., 1999). The pegylated form of recombinant PAL (PEG-PAL) is also available by injection. It is currently under development as a possible treatment method. However, most patients who receive PEG-PAL... Which subjects suffer from injection site reactions and / or develop antibodies against this therapeutic enzyme? (Longo et al., 2014). Therefore, high-phenylalanine compounds containing PKUs. Effective, reliable, and / or long-term treatment for diseases related to blood pressure There is a significant and unmet need for this. [Overview of the project] [Means for solving the problem]

[0009] In some embodiments, the present disclosure describes how phenylalanine metabolizing enzymes (PMEs) can be used to To provide genetically modified bacteria that express [something]. In some embodiments, this disclosure provides Phenylalanine ammonia lyase and / or phenylalanine hydroxylase and / or encode and express L-amino acid deaminase, leading to hyperphenylalaninemia. We provide genetically modified bacteria that can reduce [the following].

[0010] The enzyme phenylalanine ammonia lyase (PAL) converts phenylalanine into a non-toxic substance. Bell's ammonia and trans-cinnamic acid It can be metabolized. Unlike PAH, PAL metabolizes phenylalanine. It does not require THB cofactor activity. L-amino acid deaminase (LAAD) is phenyl The oxidative deamination of rualanine is catalyzed to produce phenylpyrubate, as well as trace amounts of ammonia. It generates phenyl and hydrogen peroxide. Phenylenpyruvic acid (PPA) is used in pharmaceuticals, food, and Widely used in the chemical industry, PPA is used in many chiral drugs and food additives. Starting material for synthesizing D-phenylalanine, an unprocessed intermediate in the manufacturing of a substance. Therefore, LAAD is being 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 Enylpirbate cannot 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 "logs of amino acids," this conversion affects the neurological phenotype of PKU. This demonstrates its usefulness in control.

[0011] In some embodiments, the present disclosure encodes a phenylalanine metabolizing enzyme (PME). , provides genetically modified bacteria that express Nylalanine ammonia lyase (PAL) and / or phenylalanine hydroxy Encoding an enzyme (PAH) and / or L-amino acid deaminase (L-AAD) We provide genetically modified bacteria that can express and reduce hyperphenylalaninemia. ru.

[0012] In some embodiments, genetically engineered bacteria are used to produce non-natural phenylalanine ammonia. It contains the gene encoding aliase (PAL), which in mammals encodes phenylalanine. They can be processed and reduced. In some embodiments, the manipulated bacteria , further including genes encoding phenylalanine transporters, such as PheP. In some embodiments, the manipulated bacteria also contain the gene encoding L-AAD. It is possible. Genetically modified bacteria also have biosafety and / or biological containment capabilities. For example, it incorporates a death switch, a gene guard system, and / or is related to nutritional requirements. It may contain one or more gene sequences. In some embodiments, the manipulated bacteria are antibiotic This may include substance resistance genes. The expression of any of these gene sequences is disclosed herein. It can be regulated using various promoter systems, such as any promoter system. The promoter system uses the same promoter to regulate one or more different genes. This may include using different versions of the same promoter to regulate different genes. This may include using copies and / or regulating the expression of different genes. This may include using a combination of different promoters to achieve gene development. By using different regulatory or promoter systems to control the current state, flexibility (For example, the ability to differentially control gene expression under different environmental conditions and / or genes This provides the ability to control expression over time, and also the ability to "fine-tune" gene expression. These regulations can affect gene expression and / or bacterial growth, either or all of which may be affected. It could be helpful for optimization.

[0013] In certain embodiments, the genetically modified bacteria are nonpathogenic and phenylalanine toxin It can be introduced into the gastrointestinal tract to reduce sexual levels. In certain embodiments, phenylalanyl Nin ammonia lyase and / or phenylalanine hydroxylase and / or Alternatively, L-amino acid deaminase is stably produced by genetically modified bacteria, Rabini and / or genetically modified bacteria are used in vivo and / or in vitr It is stably maintained in o. In certain embodiments, the genetically modified bacteria are phenylalanine. To increase the uptake of those proteins, the phenylalanine transporter gene is modified. The present invention also includes a pharmaceutical composition containing genetically modified bacteria, and high phenylalanine. It also provides methods to modulate and treat disorders associated with laninemia.

[0014] The present invention also provides a method for measuring and monitoring phenylalanine ammonia lyase activity. Methods and treatments for genetically modified bacteria that express phenylalanine ammonia lyase This invention provides a method for measuring and monitoring therapeutic activity. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows synthetic organisms for treating disorders characterized by phenylketonuria (PKU) and hyperphenylalaninemia. [Figure 2A] This diagram illustrates the action of phenylalanine hydroxylase in phenylketonuria (PKU). [Figure 2B] This diagram illustrates the action of phenylalanine hydroxylase (PAH). [Figure 2C] This diagram illustrates the action of phenylalanine ammonia lyase (PAL). [Figure 2D] This diagram illustrates the schematic action of L-amino acid deaminase (LAAD; for example, derived from Proteus mirabilis). [Figure 3] This figure shows an exemplary synthetic organism for treating disorders characterized by phenylketonuria (PKU) and hyperphenylalaninemia. [Figure 4] This figure shows an exemplary synthetic organism for treating disorders characterized by phenylketonuria (PKU) and hyperphenylalaninemia. [Figure 5] This figure shows an exemplary synthetic organism for treating disorders characterized by phenylketonuria (PKU) and hyperphenylalaninemia. [Figure 6] For example, this figure shows the genetic makeup of one example of a construct containing the gene encoding PAL3 and the Tet promoter sequence in a high-copy plasmid, as found in SYN-PKU202 and SYN-PKU303. [Figure 7] For example, this figure shows the genetic makeup of one example of a construct containing the gene encoding PAL3 and the FNR promoter sequence in a low-copy plasmid, as included in SYN-PKU304, SYN-PKU307, SYN-PKU305, and SYN-PKU306. [Figure 8]For example, this figure shows the genetic makeup of one example of a construct containing the gene encoding PAL3 and the Tet promoter sequence in a low-copy plasmid, such as SYN-PKU302 and SYN-PKU201. [Figure 9] This figure shows the genetic makeup of one example of a construct, such as one included in SYN-PKU401, which contains cloned LAAD genes and Tet repressor genes under the control of the Tet promoter sequence. [Figure 10] This diagram shows a schematic of a pheP knock-in construct that uses recombinant DNA to insert a second copy of pheP into the Nissle lacZ gene. [Figure 11] For example, this figure shows the gene composition of one example of a construct that includes a gene encoding PheP, a gene encoding TetR, and a Tet promoter sequence for chromosomal insertion, as contained in SYN-PKU203, SYN-PKU401, SYN-PKU402, SYN-PKU302, and SYN-PKU303. [Figure 12A] This figure shows the genetic makeup of one example construct containing the PAL3 gene cloned under the control of the FNR promoter sequence in a low-copy kanamycin-resistant plasmid (pSC101 replication start site). Under anaerobic and / or hypoxic conditions, PAL3 degrades phenylalanine to non-toxic trans-cinnamic acid. [Figure 12B] This figure shows an additional copy of pheP, an endogenous E. coli high-affinity phenylalanine transporter, which is driven by the PfnrS promoter and inserted into the lacZ locus of the Nissle chromosome. [Figure 13A]This figure schematically illustrates non-limiting embodiments of the present disclosure. It shows phenylalanine-degrading components integrated into the E. coli Nissle chromosome. In some embodiments, bacterial strains that do not contain genetically modified plasmids are used to prevent plasmid conjugation in vivo. In some embodiments, multiple insertions of the PAL gene result in an increase in copy number and / or increased phenylalanine-degrading 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] A schematic diagram of a non-limiting embodiment of the present disclosure is shown, in which the *E. coli* Nissle chromosome is genetically 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 the phenylalanine transporter gene inserted into a different insertion site (lacZ). In this embodiment, the PAL gene is PAL3 obtained from *P. luminescence*, and the phenylalanine transporter gene is pheP derived from *E. coli*. In this embodiment, the strain is SYN-PKU511. [Figure 13C] A schematic diagram of one embodiment of the present disclosure shows that the *E. coli* Nissle chromosome is genetically engineered to contain five copies of PAL under the control of an oxygen level-dependent promoter (e.g., PfnrS-PAL3) inserted at different integration sites on the chromosome (malE / K, yicS / nepI, malP / T, agaI / rsmI, and cea), and one copy of the phenylalanine transporter gene under the control of an oxygen level-dependent promoter (e.g., PfnrS-pheP) inserted at a different integration site on the chromosome (lacZ). The genome is further genetically engineered to contain the thyA trophic requirement by removing the thyA gene and / or replacing it with an unrelated gene, as well as containing the kanamycin resistance gene. [Figure 14]For example, this figure shows the gene composition of one example of a non-restrictive construct that includes the gene encoding araC, the gene encoding LAAD in Proteus mirabilis, and an arabinose-inducible promoter (ParaBAD) sequence for inserting a chromosome into an endogenous arabinose operon for chromosomal integration, as contained in SYN-PKU705. [Figure 15A] This figure shows the phenylalanine concentration in samples containing bacteria expressing PAL1 from low-copy (LC;SYN-PKU101) or high-copy (HC;SYN-PKU102) plasmids, or PAL3 from low-copy (LC;SYN-PKU201) or high-copy (HC;SYN-PKU202) plasmids, which were induced with anhydrous tetracycline (ATC) and then grown in a medium supplemented with 4 mM (660,000 ng / mL) phenylalanine. Samples were removed at 0, 4, and 23 hours. Phenylalanine concentration was determined by mass spectrometry. [Figure 15B] The levels of cinnamate in the samples 4 and 23 hours after induction are shown. In the PAL3-expressing strain, the PAL3 gene is obtained from *Photorhabdus luminescent*, an enterobacterium belonging to the same taxonomic subphylum as *Escherichia coli*. [Figure 16A] This figure shows phenylalanine concentrations in samples containing bacteria expressing low-copy (LC) or high-copy (HC) plasmids PAL1 or PAL3, or bacteria containing an additional copy of pheP driven by a chromosomally integrated Tet promoter. Bacteria were induced in ATC and then grown in medium supplemented with 4 mM phenylalanine (660,000 ng / mL) until the OD600 reached 2.0. Samples were removed at 0, 2, and 4 hours after induction, and phenylalanine concentrations were determined by mass spectrometry. In particular, the additional copy of pheP accelerated the degradation of phenylalanine (4 mM) at 4 hours. [Figure 16B]This figure shows the cinnamate levels in the samples 2 and 4 hours after induction. In some embodiments, cinnamate can be used as an alternative biomarker for strain activity. Overexpression of PheP improves phenylalanine metabolism in genetically modified bacteria. The strains analyzed in this series of data are SYN-PKU101, SYN-PKU102, SYN-PKU202, SYN-PKU201, SYN-PKU401, SYN-PKU402, SYN-PKU203, SYN-PKU302, and SYN-PKU303. [Figure 17A] This figure shows a non-limiting embodiment of the PAL construct under non-inducible conditions. It shows relatively low PAL and PheP production due to the prevention of FNR dimerization and activation of PAL and / or pheP gene expression under aerobic conditions with oxygen (O2). [Figure 17B] This figure shows a non-limiting embodiment of the PAL construct under induction conditions. Under anaerobic conditions, FNR dimerizes, and the expression of PAL and pheP is induced by the FNR promoter, resulting in upregulation of PAL and pheP production (irregular curves on "PAL" and "pheP"). Arrows near one or more rectangles indicate promoters involved in driving the transcription of such gene(s) (in the direction indicated by the arrows). Arrows above each rectangle indicate the expression products of each gene. [Figure 18] This figure shows the β-galactosidase levels in samples containing bacteria possessing low-copy plasmids expressing lacZ from FNR-involved promoters (Pfnr1-5) selected from the examples of FNR promoters shown in Table 3. Using various FNR-involved promoters, a library of reporters induced under anaerobic / hypoxic conditions at various expression levels and dynamic ranges was constructed. 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] This figure shows a schematic of the lacZ gene under the control of one example of an FNR promoter (PfnrS). LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria. [Figure 19B] This figure shows FNR promoter activity as a function of β-galactosidase activity in SYN-PKU904. The genetically engineered bacterial strain, SYN-PKU904, possessing a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Standard β-galactosidase colorimetric assay values ​​are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter initiates high levels of gene expression within 1 hour under anaerobic and / or hypoxic conditions. [Figure 19C] This figure shows the time course of bacterial cell cultures expressing lacZ, both in the presence and absence of oxygen. [Figure 20A] This figure shows the levels of phenylalanine produced under aerobic conditions in wild-type Nissle samples, bacterial samples containing low-copy plasmids expressing PAL3 from the Tet promoter or FNR promoter, or bacterial samples driven by the Tet promoter and further containing one copy of pheP integrated into the chromosome. Samples were incubated in medium supplemented with ATC and 4 mM (660,000 ng / mL) phenylalanine. Samples were removed at 0, 2, 4, and 24 hours. Phenylalanine concentrations were determined by mass spectrometry. [Figure 20B]This figure shows the levels of phenylalanine produced under anaerobic and / or hypoxic conditions in samples of wild-type Nissle, bacteria containing low-copy plasmids expressing PAL3 from the Tet promoter or FNR promoter, or bacterial samples driven by the Tet promoter and further containing one copy of pheP integrated into the chromosome. Samples were incubated in medium supplemented with ATC and 4 mM (660,000 ng / mL) phenylalanine. Samples were removed at 0, 2, 4, and 24 hours. Phenylalanine concentrations were determined by mass spectrometry. These data suggest that the FNR-responsive fnrS promoter is effective in activating PAL3 expression under anaerobic conditions, similar to the tetracycline-inducible promoter. [Figure 21] This figure shows the phenylalanine concentrations in cultures of synthetic probiotic strains with and without additional pheP copies inserted into the chromosome. After 1.5 hours of growth, the cultures were placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. Four hours after induction, the bacteria were resuspended in assay buffer containing 4 mM phenylalanine. A fixed amount was taken from the cell assay every 30 minutes for 3 hours to quantify phenylalanine by mass spectrometry. The phenylalanine degradation rate was higher in strains containing additional pheP copies (SYN-PKU304 and SYN-PKU305; left) than in strains without additional pheP copies (SYN-PKU308 and SYN-PKU307; right). [Figure 22] This figure shows the trans-cinnamate concentration (PAL activity) of strains containing a single PAL3 insertion at various chromosomal locations. [Figure 23] This figure shows the trans-cinnamate concentration (PAL activity) of strains containing multiple PAL3 insertions at various chromosomal locations. [Figure 24]This figure shows the time course of phenylalanine concentration 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 induction in a phenylalanine-containing medium for 3.5 hours, 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 anaerobically (FNR)-regulated phenylalanine ammonia lyase (PAL), integrated into five chromosomal positions, and a gene encoding an anaerobically regulated high-affinity Phe transporter (pheP), integrated into the lacZ locus. [Figure 25A] This figure shows the phenylalanine concentration in cultures of SYN-PKU401, a synthetic probiotic strain containing a high-copy pUC57 plasmid with LAAD driven by a Tet-inducible promoter. Cells were grown in a shaking flask at 37°C and induced in TCA for 2 hours in the early logarithmic 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), or under microaerobic conditions, cells (1 ml) were incubated without shaking in a 1.7 ml conical tube, or anaerobically in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. A constant amount was taken from the cell assay every 30 minutes for 2 hours to quantify phenylalanine by mass spectrometry. The phenylalanine concentrations under aerobic conditions using two cell densities are shown. A and B were repeated under the same experimental conditions. The activity under aerobic conditions is approximately 50 μmol / hour / 1e9 cells. [Figure 25B]This figure shows the phenylalanine concentration in cultures of SYN-PKU401, a synthetic probiotic strain containing a high-copy pUC57 plasmid with LAAD driven by a Tet-inducible promoter. Cells were grown in a shaking flask at 37°C and induced in TCA for 2 hours in the early logarithmic 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), or under microaerobic conditions, cells (1 ml) were incubated without shaking in a 1.7 ml conical tube, or anaerobically in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. A constant amount was taken from the cell assay every 30 minutes for 2 hours to quantify phenylalanine by mass spectrometry. A and B were repeated under the same experimental conditions. This shows the phenylalanine concentration in cells grown aerobically, microaerobically, or anaerobically. [Figure 26A] This figure shows phenylalanine 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. The mice were fasted overnight (10 hours) by removing solid food, and blood samples were collected the following morning by inducing bleeding from the mandible to determine the baseline level of phenylalanine. The mice were given solid food again, and 100 microliters (5 × 10⁹ CFU) of bacteria (SYN-PKU302 or control Nissle) were force-administered 1 hour later, followed by feeding for another 2 hours. Serum phenylalanine concentrations were determined 2 hours after force-administration. [Figure 26B] This figure shows the percentage change in blood phenylalanine concentration before and after feeding, expressed as the group mean for females or males (p<0.01). [Figure 27A]This figure shows the blood phenylalanine concentrations relative to baseline after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Mice were injected with phenylalanine (0.1 mg / group mean body weight in grams), and 30 and 90 minutes later, they were force-administered 200 μL of H2O (n=30), SYN-PKU901 (n=33), or SYN-PKU303 (n=34). The blood phenylalanine concentrations 2 hours after phenylalanine injection are shown. These data indicate that oral administration of the genetically modified probiotic strain SYN-PKU303 significantly reduces blood phenylalanine levels in mice compared to mice treated with a mock (H2O) or the parent strain (SYN-PKU901) (*, p<0.05; ***, p<0.001; ****, p<0.00001). SYN-PKU303 can interfere with the intestinal recirculation of phenylalanine. [Figure 27B] This figure shows the blood phenylalanine concentrations relative to baseline after subcutaneous exposure of phenylalanine in an in vivo mouse model of PKU. Mice were injected with phenylalanine (0.1 mg / group mean body weight in grams), and 30 and 90 minutes later, they were force-administered 200 μL of H2O (n=30), SYN-PKU901 (n=33), or SYN-PKU303 (n=34). The figure shows the blood phenylalanine concentration 4 hours after phenylalanine injection. These data indicate that oral administration of the genetically modified probiotic strain SYN-PKU303 significantly reduces blood phenylalanine levels in mice compared to mice treated with a mock (H2O) or the parent strain (SYN-PKU901) (*, p<0.05; ***, p<0.001; ****, p<0.00001). SYN-PKU303 can interfere with the intestinal recirculation of phenylalanine. [Figure 28]Figure 28 shows the blood phenylalanine concentrations relative to baseline after subcutaneous exposure of phenylalanine in an in vivo mouse model of PKU. Mice were administered 200 μL of H2O (n=30), SYN-PKU901 (n=33), SYN-PKU303 (n=34), or SYN-PKU304 (n=34) orally 30 minutes and 90 minutes after injection of phenylalanine (0.1 mg / group mean body weight). 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 an intestinal recirculation model. [Figure 29A] This figure shows the blood phenylalanine concentration relative to baseline after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Mice were administered H2O, SYN-PKU901, SYN-PKU303, or SYN-PKU304 orally 30 and 90 minutes after injection of phenylalanine (0.1 mg / mean group body weight). The figure shows the blood phenylalanine concentration 2 hours after phenylalanine injection. These data indicate that oral administration of the genetically modified probiotic strains SYN-PKU303 and SYN-PKU304 significantly reduces blood phenylalanine levels in mice compared to mice treated with a mock (H2O) or the parent strain (SYN-PKU901) (*, p<0.05;**, p<0.01;***, p<0.001;** **, p<0.0001). [Figure 29B]This figure shows the blood phenylalanine concentration relative to baseline after subcutaneous phenylalanine exposure in an in vivo mouse model of PKU. Mice were administered H2O, SYN-PKU901, SYN-PKU303, or SYN-PKU304 orally 30 and 90 minutes after injection of phenylalanine (0.1 mg / group mean body weight grams). The blood phenylalanine concentration 4 hours after phenylalanine injection is shown. These data indicate that oral administration of the genetically modified probiotic strains SYN-PKU303 and SYN-PKU304 significantly reduces blood phenylalanine levels in mice compared to mice treated with a mock (H2O) or the parent strain (SYN-PKU901) (*, p<0.05;**, p<0.01;***, p<0.001;** **, p<0.0001). [Figure 29C] This figure shows the blood phenylalanine concentration relative to baseline after subcutaneous exposure of phenylalanine in an in vivo mouse model of PKU. Mice were administered H2O, SYN-PKU901, SYN-PKU303, or SYN-PKU304 orally 30 and 90 minutes after injection of phenylalanine (0.1 mg / mean group weight grams). A scatter plot of the data shown in Figure 29A is also shown. [Figure 29D] This figure shows the blood phenylalanine concentration relative to baseline after subcutaneous exposure of phenylalanine in an in vivo mouse model of PKU. Mice were administered H2O, SYN-PKU901, SYN-PKU303, or SYN-PKU304 orally 30 and 90 minutes after injection of phenylalanine (0.1 mg / mean group weight grams). A scatter plot of the data shown in Figure 29B is also shown. [Figure 30A]This figure shows the blood phenylalanine concentration relative to baseline after subcutaneous exposure of phenylalanine in an in vivo mouse model of PKU. Mice were administered 200 μL of H2O (n=12), 200 μL of SYN-PKU901 (n=12), or 100, 200, or 400 μL of SYN-PKU304 (n=12 in each dose group) orally 30 and 90 minutes after injection of phenylalanine (0.1 mg / group mean body weight). The figures show a dose-dependent decrease in blood phenylalanine levels in SYN-PKU304-treated mice compared to mice treated with a mock (H2O) or the parent strain (SYN-PKU901) (*30% decrease; p<0.05). This experiment represents one of eight studies using the same design, each demonstrating that SYN-PKU304 can interfere with intestinal recirculation of phenylalanine. [Figure 30B] This figure shows the blood phenylalanine concentration relative to baseline after subcutaneous exposure of phenylalanine in an in vivo mouse model of PKU. Mice were administered 200 μL of H2O (n=12), 200 μL of SYN-PKU901 (n=12), or 100, 200, or 400 μL of SYN-PKU304 (n=12 in each dose group) orally 30 and 90 minutes after injection of phenylalanine (0.1 mg / group mean body weight). The figures show a dose-dependent decrease in blood phenylalanine levels in SYN-PKU304-treated mice compared to mice treated with a mock (H2O) or the parent strain (SYN-PKU901) (*30% decrease; p<0.05). This experiment represents one of eight studies using the same design, each demonstrating that SYN-PKU304 can interfere with intestinal recirculation of phenylalanine. [Figure 31A] This figure shows a schematic of PKU-specific phenylalanine metabolites. It outlines the conversion of phenylalanine to phenylpyruvic acid and phenyllactic acid in the absence of functional PAHs. These metabolites can be detected by mass spectrometry or other means, as described in Examples 24-26. [Figure 31B]This figure shows a schematic of PAL-specific phenylalanine metabolites. It outlines the conversion of phenylalanine to trans-cinnamic acid by PAL3, 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] This figure shows the blood phenylalanine concentration relative to the baseline and the concentration of phenylalanine. 30 and 90 minutes after phenylalanine injection, mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at a dose of 2.9 e10 cfu / mouse. The blood phenylalanine concentration relative to the baseline is shown, and the total metabolic activity of SYN-PKU304 was calculated to be 81.2 μmol / hour, with a total decrease Δphe compared to SYN-PKU901 of 45% (P<0.05). [Figure 32B] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at a dose of 2.9 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenylalanine concentrations 0 and 4 hours after phenylalanine injection. [Figure 32C] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at a dose of 2.9 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenylpyruvate concentrations 0 and 4 hours after phenylalanine injection. [Figure 32D]This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at a dose of 2.9 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenyllactic acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 32E] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at a dose of 2.9 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood t-cinnamic acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 32F] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU304 (n=12) at a dose of 2.9 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood hippuric acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 33A]This figure shows the blood phenylalanine concentration and phenylalanine concentration relative to the baseline. 30 and 90 minutes after phenylalanine injection, mice were orally administered a total of 800 μL of H2O (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) at a dose of 3.6 e10 cfu / mouse. The blood phenylalanine concentration relative to the baseline is shown, and the total metabolic activity of SYN-PKU517 was calculated to be 39.6 μmol / hour, with a total decrease Δphe compared to SYN-PKU801 of 17% (P<0.05). [Figure 33B] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered a total of 800 μL of H2O (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) at a dose of 3.6 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenylalanine concentrations 0 and 4 hours after phenylalanine injection. [Figure 33C] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered a total of 800 μL of H2O (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) at 800 μL (3.6 e10 cfu / mouse) 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenylpyruvate concentrations 0 and 4 hours after phenylalanine injection. [Figure 33D]This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered a total of 800 μL of H2O (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) at a dose of 3.6 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenyllactic acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 33E] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered a total of 800 μL of H2O (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) at a dose of 3.6 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the t-cinnamic acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 33F] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered a total of 800 μL of H2O (n=9), SYN-PKU801 (n=12), or SYN-PKU517 (n=12) at a dose of 3.6 e10 cfu / mouse 30 and 90 minutes after phenylalanine injection. The figure also shows the blood hippuric acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 34A]This figure shows the blood phenylalanine concentration and phenylalanine concentration relative to the baseline. 30 and 90 minutes after phenylalanine injection, mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at a dose of 3.6 e10 cfu / mouse. The blood phenylalanine concentration relative to the baseline is shown, and the total metabolic activity of SYN-PKU705 was calculated to be 133.2 μmol / hour, with a total decrease Δphe compared to SYN-PKU901 of 30% (P<0.05). [Figure 34B] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at 8 μL (3.6 e10 cfu / mouse) 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenylalanine concentrations 0 and 4 hours after phenylalanine injection. [Figure 34C] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at 8 μL (3.6 e10 cfu / mouse) 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenylpyruvate concentrations 0 and 4 hours after phenylalanine injection. [Figure 34D]This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at 8 μL (3.6 e10 cfu / mouse) 30 and 90 minutes after phenylalanine injection. The figure also shows the blood phenyllactic acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 34E] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model of PKU. Mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at 8 μL (3.6 e10 cfu / mouse) 30 and 90 minutes after phenylalanine injection. The figure also shows the blood t-cinnamic acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 34F] This figure shows the absolute values ​​of phenylalanine, PKU-specific metabolites, and PAL-specific metabolites after subcutaneous exposure of PKU in an in vivo mouse model. Mice were orally administered a total of 800 μL of H2O (n=12), SYN-PKU901 (n=12), or SYN-PKU705 (n=12) at 8 μL (3.6 e10 cfu / mouse) 30 and 90 minutes after phenylalanine injection. The figure also shows the blood hippuric acid concentrations 0 and 4 hours after phenylalanine injection. [Figure 35]This figure shows phenylalanine, as well as two toxic analogs, p-fluoro-DL-phenylalanine and o-fluoro-DL-phenylalanine, which are useful for a non-targeted approach to selecting PAL enzymes with increased activity. P-fluoro-DL-phenylalanine and o-fluoro-DL-phenylalanine are incorporated into cellular proteins in place of phenylalanine, inducing cell death. These compounds are readily taken up by PheP and can serve as substrates for PAL, as shown below, and can therefore be used in gene selection and screening to identify strains with improved Phe consumption activity. Mutations that enable more efficient PAL metabolism can disrupt the incorporation of phenylalanine analogs into cellular proteins, thus allowing growth under high concentrations of the analogs. [Figure 36] This bar graph shows 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 since dietary proteins can be broken down into dipeptides and tripeptides and made available as bacterial substrates, genetically modified bacteria can be administered with food. [Figure 37] This bar graph shows the effects of various copy numbers of pheP and PAL, and the addition of further LAAD, on the phenylalanine degradation rate in vitro. The results demonstrate that increasing the copy number of PAL increases the phenylalanine degradation rate. The addition of the high-affinity transporter pheP overrides the transport restriction, resulting in higher PAL activity. The number of transporter copies does not increase the rate (PAL, not the transport (pheP), is the limiting factor). In the presence of oxygen, LAAD can degrade Phe at a very high rate. [Figure 38A]This bar graph shows the measured values ​​for characterizing the phenylalanine enteral recirculation model. PKU mice were maintained on a solid diet without Phe, and phenylalanine (0.1 mg / kg body weight) was subcutaneously injected at T=0. Blood samples were collected at the indicated times to determine the dynamics of serum phenylalanine (Phe) after injection. This is a whisker plot showing the distribution of total phenylalanine (Phe) levels in mouse blood. [Figure 38B] This bar graph shows the measured values ​​for characterizing the phenylalanine enteral recirculation model. PKU mice were maintained on a solid diet without Phe, and phenylalanine (0.1 mg / kg body weight) was subcutaneously injected at T=0. Blood samples were collected at the indicated times to determine the dynamics of serum phenylalanine (Phe) after injection. This whisker plot shows the distribution of changes in phenylalanine (Phe) levels in mouse blood from T0. [Figure 38C] This bar graph shows the measured values ​​for characterizing a phenylalanine enteral recirculation model. PKU mice were maintained on a solid diet without Phe, and phenylalanine (0.1 mg / kg body weight) was subcutaneously injected at T=0. Blood samples were collected at the indicated time points to determine the dynamics of serum phenylalanine (Phe) after injection. The graph shows that Phe levels steadily increased for at least 6 hours. [Figure 39] This graph shows the total labeled phenylalanine concentration measured during subcutaneous 13C-Phe challenge in the PKU(enu2) mouse model to determine the degree of recirculation. At T0, all mice were maintained on a Phe-free diet with low-Phe water until injection of 0.1 mg / kg of 13C6-Phe. Blood and intestinal eluate were collected at 0, 20 minutes, and 2 hours, and phenylalanine concentrations were determined by LC-MS. The occurrence of intestinal recirculation of labeled 13C-Phe was confirmed. Furthermore, when the concentration of unlabeled Phe was determined, high levels of pre-existing (unlabeled) phenylalanine were detected in the small intestine (data not shown). [Figure 40A]This graph shows the amino acid content in various compartments in wild-type and enu2- / - mice. It also shows measured amino acid content in the blood. 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] This graph shows amino acid content in various compartments in wild-type and enu2- / - mice. The measured amino acid content in the small intestine is 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 40C] This graph shows amino acid content in various compartments in wild-type and enu2- / - mice. The measured amino acid content in the large intestine is 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 41A] This graph shows the absolute concentration of phenylalanine after forced administration of strains containing low-copy PfnrS-PAL and chromosome PfnrS-pheP (SYN-PKU305) in a rapid feeding model of PKU. In this model, Enu2 mice were fed a normal solid diet until the time of administration. The diet was removed at T0. Mice were administered hourly for 3 hours (a total of 4 doses, at 0, 1, 2, and 3 hours), and blood was collected at 4 hours to determine serum Phe concentration. Probiotic (SYN-PKU305) treated mice showed a more rapid decrease in serum Phe. [Figure 41B]This graph shows the change in phenylalanine relative to baseline after forced administration of strains including low-copy PfnrS-PAL, chromosome PfnrS-pheP (SYN-PKU305) in a rapid feeding model of PKU. In this model, Enu2 mice were fed a normal solid diet until the time of administration. The diet was removed at T0. Mice were administered hourly for 3 hours (a total of 4 doses, at 0, 1, 2, and 3 hours), and blood was collected at 4 hours to determine serum Phe concentration. Probiotic (SYN-PKU305) treated mice showed a more rapid decrease 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) along with gelatin as a carrier in drinking water after a Phe challenge (5e9 cells / ml). During the experiment, Enu2 mice were fed a normal solid diet. [Figure 42B] This graph shows the effect of administering SYN-PKU305 (containing low-copy pSC101-PfnrS-PAL3 and chromosome lacZ::PfnrS-pheP) along with gelatin as a carrier in drinking water after a Phe challenge (5e9 cells / ml). During the experiment, Enu2 mice were fed a normal solid diet. [Figure 43A] This table shows that in vitro activity correlates with in vitro activity, but the decrease in deltaPhe is approximately 50%, and in vivo activity reaches its maximum. [Figure 43B] This line graph shows that in vitro activity correlates with in vitro activity, but the decrease in deltaPhe is approximately 50%, and in vivo activity reaches its maximum. [Figure 44A]This bar graph shows the trans-cinnamic acid (TCA) concentration in the blood after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were force-administered the following: a total of 750 μL of H2O (n=12), SYN-PKU901 (streptomycin-resistant Nissle; n=12; 3 × 250 μL, total 1 × 10 e11 cfu / mouse over three force-administered doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions in PAL (2XfnrS-PAL(malEK, malPT)), two chromosomal insertions in pheP (2XfnrS-pheP(lacZ, HA1 / 2)), one chromosome in LAAD (Para::LAAD), dapA nutritional requirement, and chloramphenicol resistance; n=12; 3 × 250 μL; total 1 × 10 e11 cfu / mouse over three force-administered doses). This shows serum TCA concentrations at 0 and 4 hours after phenylalanine infusion. [Figure 44B] This bar graph shows the hippuric acid concentration in the blood after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were force-administered the following: a total of 750 μL of H2O (n=12), SYN-PKU901 (streptomycin-resistant Nissle; n=12; 3 × 250 μL, total 1 × 10 e11 cfu / mouse over three force-administered doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions in PAL (2XfnrS-PAL(malEK, malPT)), two chromosomal insertions in pheP (2XfnrS-pheP(lacZ, HA1 / 2)), one chromosome in LAAD (Para::LAAD), dapA nutritional requirement, and chloramphenicol resistance; n=12; 3 × 250 μL; total 1 × 10 e11 cfu / mouse over three force-administered doses). The serum hippuric acid concentrations at 0 and 4 hours after phenylalanine infusion are shown. [Figure 44C]This bar graph shows the urinary trans-cinnamic acid (TCA) concentration after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were force-administered the following: a total of 750 μL of H2O (n=12), SYN-PKU901 (streptomycin-resistant Nissle; n=12; 3 × 250 μL, total 1 × 10 e11 cfu / mouse over three forced doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions in PAL (2XfnrS-PAL(malEK, malPT)), two chromosomal insertions in pheP (2XfnrS-pheP(lacZ, HA1 / 2)), one chromosome in LAAD (Para::LAAD), dapA nutritional requirement, and chloramphenicol resistance; n=12; 3 × 250 μL; total 1 × 10 e11 cfu / mouse over three forced doses). Urinary TCA concentrations four hours after phenylalanine injection are shown. [Figure 44D]This bar graph shows the urinary hippuric acid concentration after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. One, two, and three hours after phenylalanine injection, mice were force-administered the following: a total of 750 μL of H2O (n=12), SYN-PKU901 (streptomycin-resistant Nissle; n=12; 3 × 250 μL, total 1 × 10 e11 cfu / mouse over three force-administered doses), or 750 μL of SYN-PKU706 (containing two chromosomal insertions in PAL (2XfnrS-PAL(malEK, malPT)), two chromosomal insertions in pheP (2XfnrS-pheP(lacZ, HA1 / 2)), one chromosome in LAAD (Para::LAAD), dapA nutritional requirement, and chloramphenicol resistance; n=12; 3 × 250 μL; total 1 × 10 e11 cfu / mouse over three force-administered doses). Urinary hippuric acid concentrations four 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 the majority of TCA produced by 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] This figure shows the amount of TCA recovered 4 hours after forced oral administration of TCA (0.0125, 0.025, 0.05, or 0.1 mg / g TCA). PKU mice were force-administered 0.0125, 0.025, 0.05, or 0.1 mg / g TCA (3 mice per cage, 2 cages per group). Urine, feces, and blood were collected 4 hours after forced oral administration, and TCA and its major breakdown product, hippuric acid, were analyzed. No significant amounts of TCA or hippuric acid were detected in blood and feces (data not shown). Observations revealed that TCA was almost completely recovered 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 administration of the PKU strain. [Figure 45B]This figure shows the amount of hippuric acid recovered 4 hours after forced oral administration of TCA (0.0125, 0.025, 0.05, or 0.1 mg / g TCA). [Figure 46] This graph shows the dynamics of TCA conversion to hippuric acid over 6 hours after Phe challenge in PKU mice (enu2- / -). Animals were transferred to 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 within 4 hours. [Figure 47A] This is a schematic diagram of a non-limiting embodiment of the genetically engineered bacterial chromosome of the present disclosure. It shows a schematic diagram of the genetic makeup of an exemplary bacterial chromosome containing a phenylalanine degradation component incorporated into the *E. coli* Nissle chromosome. 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 further contains one copy of the mutant FNR transcription factor, namely FNRS24Y (Para::FNRS24Y). [Figure 47B] This is a schematic diagram of a non-limiting embodiment of the genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the gene composition of an exemplary bacterial chromosome (e.g., as seen 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 the mutant FNR transcription factor, namely FNRS24Y (Para::FNRS24Y), and one copy of LAAD inserted into the same insertion site (arabinose operon), which is transcribed via bicistronic messages from the endogenous arabinose promoter. The bacterial chromosome further contains the dapA trophic requirement (deltaDapA). [Figure 47C]This is a schematic diagram of a non-limiting embodiment of the genetically engineered bacterial chromosome of the present disclosure. A schematic diagram of the gene composition of an exemplary bacterial chromosome (e.g., as seen 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 an arabinose operon and whose expression is driven by the native Para promoter (Para::LAAD). The genome is further modified to include a deletion of the dapA gene and the inclusion of a dapA trophic requirement. [Figure 47D] This is a schematic diagram of non-limiting embodiments of the genetically engineered bacterial chromosomes of the present disclosure. A schematic diagram of the gene composition of an exemplary bacterial chromosome (e.g., as seen 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 further contains one copy of LAAD, which is inserted into an arabinose operon and whose expression is driven by the native Para promoter (Para::LAAD). The bacterial chromosome further contains two copies of IPTG-inducible PAL3 (2XLacIPAL, exo / cea and rhtC / rhtB), dapA trophochemical requirement, and all antibiotic resistance is cured. [Figure 47E] This is a schematic diagram of non-limiting embodiments of the genetically engineered bacterial chromosomes of the present disclosure. A schematic diagram of the gene composition of an exemplary bacterial chromosome (e.g., as seen in SYN-PKU712) is shown. SYN-PKU712 corresponds to SYN-PKU707, which is essentially dapA nutrient-dependent. The bacterial chromosome contains three copies of fnrSPAL3 and two copies of fnrSpheP integrated into the chromosome, along with knocked-in Para-FNRS24Y and deltadapA (DAP nutrient-dependent). SYN-PKU712 corresponds to SYN-PKU707, which is essentially dapA nutrient-dependent. [Figure 47F]This is a schematic diagram of non-limiting embodiments of the genetically engineered bacterial chromosomes of the present disclosure. A schematic diagram of the gene composition of an exemplary bacterial chromosome (e.g., as seen in SYN-PKU711) is shown. SYN-PKU711 corresponds to SYN-PKU708, which is essentially dapA nutrient-free. The bacterial chromosome contains three copies of -fnrSPAL3 and two copies of fnrSpheP integrated into the chromosome, and a knock-in Para-FNRS24Y-LAAD. [Figure 48A] This figure shows the gene composition of an exemplary construct. It shows a construct containing FNRS24Y and the reverse araC, driven by an arabinose-inducible promoter. SYN-PKU707 and SYN-PKU712 are examples of strains containing such a construct (integrated into an arabinose operon). Exemplary sequences include SEQ ID NO: 64. In some embodiments, FNRS24Y expression (from the construct shown in Figure 48A or Figure 48B) is induced as part of an oxygen bypass switch shown in Figure 50. In some embodiments, the construct in the figure 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 a bacterial chromosome at one or more locations. In a non-limiting example, the construct in the figure is knocked into an E. coli arabinose operon and driven by an endogenous arabinose promoter. [Figure 48B]This figure shows the gene composition of an exemplary construct. FNRS24Y and LAAD are shown as constructs expressed via bicistronic messaging from an arabinose-inducible promoter. AraC is transcribed in reverse. SYN-PKU708 is an example of a strain containing such a construct (integrated into an 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 an oxygen bypass switch shown in Figure 50. In some embodiments, the constructs in the figure are used in combination with hypoxia-inducible PAL3, pheP, and / or LAAD constructs. In some embodiments, the constructs are located on plasmids, e.g., low-copy or high-copy plasmids. In some embodiments, the constructs are located on plasmid components of a biosafety system. In some embodiments, the constructs are integrated into a bacterial chromosome at one or more locations. In a non-limiting example, the constructs in the figure are knocked into an E. coli arabinose operon and driven by an endogenous arabinose promoter. [Figure 49]The gene composition of an exemplary construct, including an IPTG-inducible promoter driving the expression of reversed LacI and 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-inducing and pre-loading a therapeutic strain before 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 that is active in an in vivo environment. In some embodiments, the construct is located on a plasmid, e.g., a low-copy plasmid or a 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 may be provided on a plasmid or integrated into the bacterial chromosome at one or more locations. PheP expression may be constitutive or driven by an inductive promoter, e.g., hypoxia, arabinose, or IPTG. In some embodiments, the construct is used in combination with an LAAD expression construct. In some embodiments, the construct PAL3 sequence is the original sequence derived from Phororhabdus 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 plasmid or a high-copy plasmid. In some embodiments, the construct is used in a biosafety system such as the system shown in Figures 61A, 61B, 61C, and 61D.In some embodiments, the construct is incorporated into the genome at one or more locations described herein. [Figure 50]This figure shows an "oxygen bypass switch" useful for aerobic pre-induction of strains containing one or more PME and / or Phe transporters under the control of a hypoxic FNR promoter in an in vitro culture vessel (e.g., a flask, fermenter, or other container used during cell proliferation, cell expansion, fermentation, harvesting, purification, formulation, and / or manufacturing). In some embodiments, it is desirable to preload the strain with active PME, e.g., PAL3 and / or LAAD, before administration of the strain. This can be done by pre-inducing the expression of the enzyme when growing the strain (e.g., in a flask, fermenter, or other suitable container) and preparing it for in vivo administration. In some embodiments, the strain is induced under anaerobic and / or hypoxic conditions, for example, to induce FNR promoter activity and drive the expression of one or more PME and / or Phe transporters. In some embodiments, it is desirable to prepare, preload, and pre-induce the strain under aerobic or microaerophilic conditions. This allows for more efficient growth and, in some cases, reduces the accumulation of toxic metabolites. FNRS24Y is a mutant of FNR that is more resistant to oxygen inactivation and can therefore activate the FNR promoter under aerobic conditions (see, for example, Jervis AJ, Proc Natl Acad Sci USA, March 24, 2009, Vol. 106 (No. 12): pp. 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 (No. 12): pp. 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 that article are incorporated herein by reference in their entirety.) In the oxygen bypass system described above, FNRS24Y is induced by the addition of arabinose, and under aerobic conditions, it binds to and activates the FNR promoter, thereby driving the expression of PAL3 and pheP. Therefore, by utilizing the potent FNR promoter, high levels of PAL and PheP expression can be obtained in this system, allowing for efficient propagation, production, or manufacture of strains under aerobic conditions while they are effectively pre-induced and pre-loaded. Since mutant FNRS24Y is no longer expressed in the absence of arabinose, and wild-type FNR binds to the FNR promoter to drive the expression of PAL3 and PheP in vivo, the system does not interfere with or compromise in vivo activation. 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, by arabinose. Thus, in some embodiments, LAAD and FNRS24Y can be induced simultaneously. In some embodiments, FNRS24Y and LAAD are transcribed via bicistronic messaging, and their expression is driven by the arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon to enable expression from the endogenous Para promoter. In some embodiments, FNRS24Y-LAAD is knocked into the arabinose operon to enable expression from the endogenous Para promoter.In some embodiments, a LacI promoter and IPTG induction are used in the system (instead of Para and arabinose induction). In some embodiments, a rhamnose-inducible promoter is used in the system. In some embodiments, a temperature-sensitive promoter is used to drive the expression of FNRS24Y. [Figure 51] This bar graph shows 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 yields high levels of activity under aerobic conditions in 10 ml, 20 ml, or 30 ml flasks. Furthermore, under anaerobic conditions, activation in the absence of arabinose is maintained. These results indicate that the strain is efficiently pre-induced under aerobic conditions before in vivo administration. These results also suggest that anaerobic activation without arabinose, e.g., "in vivo" activation, may be conserved in this strain. [Figure 52] This bar graph shows the serum phenylalanine concentration relative to baseline after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Mice were force-administered three doses of a total of 750 μL of H2O (n=9), SYN-PKU901 (n=9), or 800 μL of SYN-PKU707 (n=9) (1 x 10 e11 cfu / mouse) 1, 2, and 3 hours after phenylalanine injection, and blood and urine were collected 4 hours after injection. The bar graph shows the serum phenylalanine concentration relative to baseline; the total metabolic activity of SYN-PKU707 was calculated as 269 μmol / hour, and the total decrease in Δphe was 49% (P<0.05) compared to SYN-PKU901 (P<0.05). [Figure 53]This bar graph shows the absolute values ​​of hippuric acid recovered from urine after subcutaneous phenylalanine challenge in an in vivo mouse model of PKU. Mice were force-administered three doses of a total of 750 μL of H2O (n=9), SYN-PKU901 (n=9), or 800 μL of SYN-PKU707 (n=9) (1 x 10 e11 cfu / mouse) 1, 2, and 3 hours after phenylalanine injection. Blood and urine were collected 4 hours after injection. The graph shows the hippuric acid concentration in the urine 4 hours after phenylalanine injection. 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] This graph shows the recovery of absolute levels of unlabeled hippuric acid in urine after subcutaneous (SC) injection of radiolabeled phenylalanine in mice that were force-administered SYN-PKU707 three times. Following subcutaneous (SC) injection of heavy phenylalanine (1, 2, and 3 hours post-injection), mice in metabolic cages (3 mice / cage; 3 cages / group) were force-administered SYN1780 three times. [Figure 54B] This graph shows the recovery of absolute levels of labeled hippuric acid in urine after subcutaneous (SC) injection of radiolabeled phenylalanine in mice that were force-administered SYN-PKU707 three times. Following subcutaneous (SC) injection of heavy phenylalanine (1, 2, and 3 hours post-injection), mice in metabolic cages (3 mice / cage; 3 cages / group) were force-administered SYN1780 three times. [Figure 55] This graph shows hippuric acid recovered from urine after a single dose of the PKU strain SYN-PKU707 at T0, 2, 4, 6, and 8 hours after the Phe challenge. As shown, different cell counts were force-administered to mice (in a single force-dose). A dose-dependent increase in hippuric acid recovered from the mice's urine was observed. [Figure 56]The graph showing hippuric acid recovery from urine compares the dynamics of hippuric acid production from phenylalanine induced by forced administration of live cells with those of hippuric acid production derived from forced administration of pure TCA. With pure TCA, a rapid decrease in hippuric acid recovery from urine is observed within the first 15 minutes after the start of recovery. In the case of the cells mentioned above, hippuric acid recovery is sustained for at least the first 30 minutes after the start of collection, and the downward slope decreases. These results indicate that the cells remain in the small intestine for a useful amount of time and produce TCA. [Figure 57A] This bar graph shows the change in blood phenylalanine levels relative to baseline 4 hours after phenylalanine exposure in PKU mice that were force-administered the indicated dose of SYN-PKU708. SYN-PKU708 was effective in reducing blood phenylalanine levels. This indicates that the cells were active in vivo. [Figure 57B] This bar graph shows the absolute amount of hippuric acid in the urine of PKU mice 4 hours after phenylalanine exposure, after force-administering the indicated dose of SYN-PKU708. Hippuric acid was excreted in a dose-dependent manner in the cages of mice treated with SYN-PKU708. This indicates that the cells were active in vivo. [Figure 58A] This graph shows the transit time of bacteria (SYN1780) after a single forced administration of approximately 3 e10 cfu. Mice were subjected to a single forced administration of bacteria (approximately 3 × 10 e10 CFU). At each time point (15, 30, 45, and 60 minutes after forced administration), the animals (n=4) were euthanized, their small intestines were removed, cut into three equal parts, and washed. The intestinal eluate was treated for serial dilution seeding to determine the bacterial count. [Figure 58B] This graph shows the transit time of bacteria (SYN1780) after a single forced administration of approximately 3e10 cfu. [Figure 59A]This figure compares the SYN-PKU710 and SYN-PKU708 strains, showing blood phenylalanine concentrations relative to baseline 4 hours after subcutaneous (SC) injection of phenylalanine. Mice were administered a single dose of phenylalanine by subcutaneous injection of 0.1 mg per gram of body weight. Bacteria (or water) were force-administered orally to the mice 1, 2, and 3 hours after the Phe challenge (300 μl / dose, total 3 Xe10 cfu / mouse). The percentage reduction in deltaPhe in SYN-PKU710 and SYN-PKU708 was calculated to be 29% and 40%, respectively. [Figure 59B] This graph shows the absolute values ​​of hippuric acid up to 4 hours after phenylalanine subcutaneous (SC) injection, comparing the SYN-PKU710 and SYN-PKU708 strains. [Figure 60] This bar graph shows in vitro PAL activity, measured by TCA accumulation rate, in strains where PAL expression is controlled by various inducible promoters (arabinose, IPTG (LacI), rhamnose, Tet, temperature (CI857)). SYN-PKU707 is shown as the benchmark control. [Figure 61A] This is a schematic diagram of a non-limiting example of the genetic makeup of a plasmid that functions as a component of a biosafety system. The biosafety plasmid system vector contains Kid toxin and R6K minimal ori, dapA and promoter elements that drive the 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 inductive or constitutive promoter. [Figure 61B]This is a schematic diagram of a non-limiting example of the genetic makeup of a plasmid that functions as a component of a biosafety system. The biosafety plasmid system vector contains Kid toxin and R6K minimal ori, thyA and promoter elements that drive the 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 inductive or constitutive promoter. [Figure 61C] This is a schematic diagram of the genetic makeup of the chromosomal components of the biosafety system. It shows a construct containing low-copy Rep(Pi) and Kis antitoxin. Pi(Rep) is required for replication of the system's plasmid components, and Pi(Rep) transcription is driven by a promoter containing low-copy RBS. In some embodiments, the construct includes SEQ ID NO: 89. [Figure 61D] This is a schematic diagram of the genetic makeup of the chromosomal components of the biosafety system. It shows constructs containing the medium copy Rep(Pi) and Kis antitoxin. Pi(Rep) is required for replication of the system's plasmid components, and the transcription of Pi(Rep) is driven by a promoter containing the medium copy RBS. In some embodiments, the construct includes SEQ ID NO: 90. When a plasmid containing functional DapA is used (as shown in Figure 61A), the chromosomal constructs shown in Figures 61C and 61D are knocked into the DapA locus. When a plasmid containing functional ThyA is used (as shown in Figure 61B), the chromosomal constructs shown in Figures 61C and 61D are knocked into the ThyA locus. In this system, bacteria containing the chromosomal constructs and the knocked-out dapA or thyA genes can only grow in the presence of the plasmid, in the absence of dap or thymidine. [Figure 62A]This is a schematic diagram of a non-limiting example of a PAL construct. It shows a non-limiting example of the composition of a construct for PAL expression under the control of a lambda CI-inducible promoter. The construct also provides the coding sequence for CI857, a temperature-sensitive mutant of CI. The temperature-sensitive CI repressor mutant CI857 binds strongly at 30°C but cannot bind (repress) at temperatures above 37°C. In some embodiments, the construct includes SEQ ID NO: 101. In some embodiments, this construct is used alone. In some embodiments, the temperature-sensitive construct is used in combination with other constitutive or inducible PAL3 constructs, e.g., hypoxia, arabinose, rhamnose, or IPTG-inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of PAL3 and / or PheP and / or LAAD before in vivo administration. In some embodiments, the construct provides in vivo activity. 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 may be provided on a plasmid or integrated into the bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inductive promoter, e.g., hypoxia, arabinose, rhamnose, or temperature sensitivity. 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 conditional nutrient requirements. In strains containing deltaThyA or deltaDapA, the dapA or thyA gene can be introduced into a strain under the control of a thermoregulatory promoter system. In the absence of Thy and Dap, the strain can only grow at an acceptable temperature, e.g., 37°C (and not lower). [Figure 62B]This is a schematic diagram of a non-limiting example of a PAL construct. It shows a non-limiting example of the composition of a construct for PAL expression under the control of a rhamnose-inducible promoter. For the application of the rhamnose expression system, it is not necessary to express larger amounts of regulatory proteins, as the expression level from the chromosome is sufficient to activate transcription even on a multicopy plasmid. 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, e.g., 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 before in vivo administration. In non-limiting examples, 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, 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 incorporated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which may be provided on a plasmid or incorporated into the bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inductive promoter, such as hypoxia, arabinose, rhamnose, or temperature sensitivity. In some embodiments, the construct is used in combination with an LAAD expression construct. [Figure 62C]This is a schematic diagram of a non-limiting example of a PAL construct. It shows a non-limiting example of the composition of a construct for PAL expression under the control of an arabinose-inducible promoter. An arabinose-inducible PAL3 construct includes AraC (reverse), 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 constructs or inducible PAL3 constructs, e.g., 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 before in vivo administration. In non-limiting examples, 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, 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 a bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a PheP construct, which may be provided on a plasmid or incorporated into a bacterial chromosome at one or more locations. PheP expression can be constitutive or driven by an inductive promoter, such as hypoxia, arabinose, rhamnose, or temperature sensitivity. In some embodiments, the construct is used in combination with an LAAD expression construct. [Figure 63A] The gene architecture of the PssB promoter is outlined. The ssB gene product protects ssDNA from degradation. SSB directly interacts with numerous enzymes involved in DNA metabolism and is thought to play a central role in the organization and processes of nuclear protein complexes involved in DNA replication (and restart), recombination, and repair. The PssB promoter was cloned before the LacZ reporter, and β-galactosidase activity was measured. [Figure 63B]This bar graph shows the reporter gene activity of the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and divided into two different tubes. One tube was placed in an anaerobic chamber, and the other was kept under aerobic conditions for the duration of the experiment. At specific times, the cells were analyzed for promoter induction. The PssB promoter is active under aerobic conditions and blocked under anaerobic conditions. This promoter can be used to express a target gene under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product 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 represses the expression of the target gene. Thus, the target gene is expressed only in the absence of the repressor, i.e., under anaerobic and / or hypoxic conditions. This strategy has the advantage that an additional level of control exists 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, for example, to create a facultative nutritional requirement. A chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and / or hypoxic conditions, dapA or thyA may be expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, the expression of dapA or thyA is blocked, and the strain cannot grow 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 gastrointestinal tract, but to prevent their survival under aerobic conditions (biosafety switch). [Figure 64A]This figure shows strategies for fine-tuning Para-PAL construct expression by using ribosome binding site optimization strategies. Bioinformatics tools for RBS optimization are known in the art. One strategy allows for the integration of arabinose-controlled PAL and pheP into the chromosome to provide efficient aerobic growth and pre-induction of the strain (e.g., in a flask, fermenter, or other suitable container) while maintaining integrated PfnrS-PAL and PheP to enable potent in vivo induction. [Figure 64B] This figure shows a strategy to enable the expression of PAL and PheP under aerobic conditions by arabinose-inducible expression of FNRS24Y. By 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 complete induction). Fine-tuning is achieved by selecting the appropriate RBS at an appropriate translation initiation rate. Bioinformatics tools for RBS optimization are known in the art. [Figure 65A]This is a schematic diagram illustrating the exemplary gene composition 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 composition of an IPTG and hypoxia-inducible construct having a promoter-containing region including inverted LacI, LacO sites, and an FNR binding site is shown. The promoter drives the expression of two open reading frames encoding PAL3 and a third open reading frame encoding PheP; the construct can be transcribed with tricistronic messages. In a non-limiting example, the construct includes SEQ ID NO: 95. In some embodiments, this construct is useful for pre-induction under anaerobic and / or hypoxic conditions by activation of the FNR promoter. In some embodiments, this construct is useful for in vivo activation by activation of the FNR promoter under anaerobic and / or hypoxic conditions, such as those found in specific regions of the gastrointestinal tract. In some embodiments, the construct in the figure includes 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., upregulate or downregulate, the PAL3 level expressed from the construct. In some embodiments, the construct is located on a plasmid, for example, 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 65B]This is a schematic diagram illustrating the exemplary gene composition 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 composition of an IPTG and hypoxia-inducible construct having a promoter-containing region including inverted LacI and LacO sites, and an FNR binding site is shown. The construct also includes 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 a bacterial chromosome at one or more locations. In a non-limiting example, the construct includes Sequence ID No. 97. In some embodiments, this construct is useful for pre-induction under anaerobic and / or hypoxic conditions by activation of the FNR promoter. In some embodiments, this construct is useful for in vivo activation by activation of the FNR promoter under anaerobic and / or hypoxic conditions, such as those found in specific regions of the gastrointestinal tract. In some embodiments, the construct in the figure includes 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., upregulate or downregulate, the PAL3 level expressed from the construct. In some embodiments, the construct is located on a plasmid, for example, 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 65C]This is a schematic diagram showing exemplary gene compositions 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 composition of an IPTG-inducible construct having a promoter-containing region containing inverted LacI, LacO is shown. The construct also has a region containing two open reading frames encoding PAL3 and a third open reading frame doing PheP. This construct can be transcribed with a tricistronic message. In the non-limiting example, the construct includes SEQ ID NO: 96. In some embodiments, the construct in the figure includes 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., upregulate or downregulate, the PAL3 levels expressed from the construct. In some embodiments, the construct is located on a plasmid, for example, 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]This is a schematic diagram illustrating the exemplary gene composition 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 composition of an IPTG-inducible construct having a promoter-containing region containing inverted LacI, LacO is shown. The construct also has a region containing two open reading frames encoding PAL3. This construct can be transcribed with a bicistronic message. In the non-limiting example, the construct includes SEQ ID NO: 98. 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 that is integrated into a bacterial chromosome at one or more locations. In some embodiments, the construct in the figure includes 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., upregulate or downregulate, the PAL3 levels expressed from the construct. In some embodiments, the construct is located on a plasmid, for example, 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 66]This figure shows a map of example insertion sites within the *E. coli* 1917Nissle chromosome. These sites indicate regions where circuit components can be inserted into the chromosome without interfering with the expression of essential genes. Backslashes ( / ) are used to indicate that insertions occur between dispersedly or convergently expressed genes. Insertions within biosynthetic genes such as thyA can be used to create trophic requirements. In some embodiments, individual circuit components are inserted into two or more of the indicated sites. [Figure 67] This figure shows three bacterial strains that constitutively express red fluorescent protein (RFP). In strains 1-3, the RFP gene is inserted at different locations within the bacterial chromosome, resulting in varying degrees of brightness under fluorescence. Unmodified E. coli Nissle (strain 4) does not produce fluorescence. [Figure 68] This graph shows the presence of Nissle in vivo. Streptomycin-resistant Nissle was force-administered orally 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 gastrointestinal tract of the mice. The bars represent the number of bacteria administered to the mice. The lines represent the number of Nissle recovered from fecal samples daily for 10 consecutive days. [Figure 69] This bar graph shows the time course of streptomycin-resistant Nissle in various compartments of the gastrointestinal tract 1, 4, 8, 12, 24, and 30 hours after forced administration. Mice were treated with approximately 10⁹ CFU, and at each time point (n=4), the animals were euthanized and the intestines, 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 eluates were collected, and the CFU of each compartment was determined by serial dilution dispersal. [Figure 70A]This figure shows the phenylalanine concentration in SYN-PKU302 culture over time. After 1.5 hours of growth, ATC was added to the SYN-PKU302 culture and placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. After 4 hours of induction, the bacteria were resuspended in assay buffers containing 4 mM phenylalanine at various pH levels (pH range 7.25 to 2.25). A fixed amount was taken from the cell assay every 30 minutes for 2 hours to quantify phenylalanine by mass spectrometry. The rate of phenylalanine degradation decreased as the pH of the assay buffer in SYN-PKU302 decreased. [Figure 70B] This figure shows the phenylalanine concentration in SYN-PKU304 culture over time. After 1.5 hours of growth, ATC was added to the SYN-PKU304 culture and placed in a Coy anaerobic chamber supplied with 90% N2, 5% CO2, and 5% H2. After 4 hours of induction, the bacteria were resuspended in assay buffers containing 4 mM phenylalanine at various pH levels (pH range 7.25 to 2.25). A fixed amount was taken from the cell assay every 30 minutes for 2 hours to quantify phenylalanine by mass spectrometry. The rate of phenylalanine degradation decreased as the pH of the assay buffer in the SYN-PKU304 strain decreased. [Figure 71] This figure shows a schematic example of the *E. coli* 1917Nissle chromosome, which contains numerous mechanisms of action (MoA). [Figure 72] This figure shows the gene composition of one example of a construct in which the PAL3 and pheP genes are simultaneously transcribed under the control of one example of the FNR promoter (PfnrS). [Figure 73A] This figure shows the gene configuration of one example of a construct in which the Int5 recombinase gene is operably linked to one example of an FNR promoter (Pfnr S) and the PAL3 gene is operably linked to a strong constitutive promoter. A schematic diagram of the PAL3 gene with the Int5 region adjacent in the OFF direction (3' to 5') is shown. Any strong constitutive promoter sequence can be used. [Figure 73B]This figure shows the gene configuration of one example construct in which the Int5 recombinase gene is operably linked to one example of an 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 in the ON direction (5' to 3') leads to PAL3 production and phenylalanine metabolism. Any strong constitutive promoter sequence can be used. [Figure 74A] This figure shows the gene configuration of one example of a construct in which the Int5 recombinase gene is operably linked to the FNR promoter (PfnrS), and the gene encoding T7RNA polymerase is operably linked to a strong constitutive promoter adjacent to the recombinase site. A schematic diagram of the T7RNA polymerase gene with the Int5 site adjacent in the OFF direction is also shown. [Figure 74B] This figure shows the gene configuration of one example construct in which the Int5 recombinase gene is operably linked to the FNR promoter (PfnrS), and the gene encoding T7RNA 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 T7RNA polymerase gene is flipped in the ON direction. [Figure 74C] This figure shows the genetic makeup of one example of a construct in which the Int5 recombinase gene is operably linked to the FNR promoter (PfnrS), and the gene encoding T7RNA polymerase is operably linked to a strong constitutive promoter adjacent to the recombinase site. In a genetically engineered bacterial strain containing one copy of PAL3 under the control of the T7-driven promoter (PT7), T7RNA polymerase expression leads to PAL3 production and phenylalanine metabolism. [Figure 75A]This figure shows the gene configuration of one example of a 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. An exemplary construct in which the Int5 recombinase gene is operably linked to the ParaBAD promoter (ParaBAD) is shown. [Figure 75B] This figure shows the genetic makeup of one example construct in which the Int5 recombinase gene is operably linked to the ParaBAD promoter (Par aBAD), and the gene encoding T7RNA polymerase is operably linked to a strong constitutive promoter adjacent to the recombinase site. A schematic diagram of the T7RNA polymerase gene with Int5 adjacent in the OFF direction is shown. When Int5 gene expression is activated under anaerobic and / or hypoxic conditions, the T7RNA polymerase gene is flipped in the ON direction. In a genetically engineered bacterial strain containing one copy of PAL3 under the control of the T7-driven promoter, T7RNA polymerase expression leads to PAL3 production and phenylalanine metabolism. [Figure 76A] This figure schematics recombinase-based switches for activating PAL3 expression using different inducible promoters and ribosome binding sites. Recombinase expression induces recombination flipping of the PAL3 gene in the ON direction, leading to PAL3 production and phenylalanine degradation. In some embodiments, the recombinase-based switch is tuned to respond to specific levels of the inducer. [Figure 76B] This figure shows the relationship between the concentration of the inducing factor and the proportion of constructs containing PAL3 in the ON direction. The shaded area indicates the predicted effective range of the inducing factor in vivo. [Figure 77A]This figure illustrates another non-limiting embodiment of the present disclosure in which heterologous gene expression is activated by an exogenous environmental signal. It depicts one embodiment of heterologous gene expression in which, in the absence of arabinose, the AraC transcription factor adopts a repressive conformation. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that activates it by binding to the ParaBAD promoter (ParaBAD), inducing the expression of the Tet repressor (TetR) and antitoxin. The antitoxin accumulates in recombinant bacterial cells, but TetR inhibits toxin expression (under the control of a promoter with a TetR binding site). However, in the absence of arabinose, neither the antitoxin nor TetR is expressed. Since TetR, which inhibits toxin expression, is absent, the toxin is expressed and kills the cells. Another non-limiting embodiment of the present disclosure is also shown in which the expression of essential genes not found in recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformational structure that represses the 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 binds to and activates the araBAD promoter, inducing the expression of essential genes and maintaining the viability of bacterial cells. [Figure 77B] This figure illustrates another non-limiting embodiment of the disclosure in which the expression of a heterologous gene is activated by an exogenous environmental signal. This figure illustrates another non-limiting embodiment of the disclosure in which the antitoxin is expressed from a constitutive promoter and the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a repressive conformation. 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 the expression of TetR and thus interfering with toxin expression. However, in the absence of arabinose, TetR is not expressed, the toxin is expressed, and it ultimately overcomes the antitoxin, killing the cell. The constitutive promoter that regulates antitoxin expression is likely to be a weaker promoter than the promoter that drives toxin expression. The araC gene is under the control of the constitutive promoter in this circuit. [Figure 77C] This figure illustrates another non-limiting embodiment of the present disclosure in which the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a repressive conformation. 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 the expression of the Tet repressor (TetR) and antitoxin. The antitoxin accumulates in recombinant bacterial cells, but TetR inhibits toxin expression (under the control of a promoter having a TetR binding site). However, in the absence of arabinose, neither the antitoxin nor TetR is expressed. Since there is no TetR to inhibit toxin expression, the toxin is expressed and kills the cells. The araC gene is under the control of either a constitutive promoter or an inductive promoter of this circuit (e.g., the AraC promoter). [Figure 78] This figure shows one non-limiting embodiment of the present disclosure, in which the expression of a heterogene and at least one recombinase is activated from one or more inducible promoters by exogenous environmental conditions or one or more environmental signals. The recombinase then inverts the toxin gene to an active conformation, and the intrinsic kinetics of the recombinase cause a time delay in toxin expression, thereby allowing the heterogene to be fully expressed. Once expressed, the toxin kills cells. [Figure 79] This figure shows another non-limiting embodiment of the present disclosure in which the expression of a heterogene, an antitoxin, and at least one recombinase is activated from one or more inducible promoters by exogenous environmental conditions or one or more environmental signals. The recombinase then inverts the toxin gene to an active conformation, but the activity of the toxin is suppressed by the presence of accumulated antitoxin. When the exogenous environmental conditions or cues are no longer present, the expression of the antitoxin is stopped. The toxin is constitutively expressed and continues to accumulate, killing the bacterial cell. [Figure 80]This figure shows another non-limiting embodiment of the present disclosure in which the expression of a heterogene and at least one recombinase is activated from one or more inducible promoters by exogenous environmental conditions or one or more environmental signals. The recombinase then inverts at least one scavenging enzyme into an active conformation. The at least one scavenging enzyme then excises one or more essential genes, leading to senescence and ultimately cell death. The intrinsic dynamics of the recombinase and scavenging genes create a time delay, and their kinetics can be modified and optimized depending on the number and selection of essential genes to be excised, allowing cell death to occur over hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death. [Figure 81] This figure shows one non-limiting embodiment of the present disclosure in which the expression of a heterogene and a first recombinase is activated from one or more inducible promoters by exogenous environmental conditions or one or more environmental signals. The recombinase then inverts a second recombinase from the reversed direction to the active conformation. The activated second recombinase inverts the toxin gene to the active conformation, and the intrinsic kinetics of the recombinase cause a time delay in toxin expression, thereby allowing the heterogene to be fully expressed. Once expressed, the toxin kills cells. [Figure 82] This figure shows one non-limiting embodiment of the present disclosure, comprising a plasmid stability system having plasmids that produce both short-lived and long-lived toxins. When a cell loses the plasmid, the antitoxin is no longer produced, and the toxin kills the cell. In one embodiment, genetically engineered bacteria produce equal amounts of Hok toxin and short-lived Sok antitoxin. In the top panel, the cell produces equal amounts of toxin and antitoxin and is stable. In the middle panel, the cell loses the plasmid, and the antitoxin begins to disintegrate. In the bottom panel, the antitoxin completely disintegrates, and the cell dies. [Figure 83]This diagram illustrates the use of GeneGuard as a genetically modified safety component. All genetically modified DNA resides in plasmids that can be conditionally disrupted. See, for example, Wright et al., 2015. [Figure 84A] This is a schematic diagram of a wild-type clbA structure. [Figure 84B] This is a schematic diagram of the clbA knockout construct. [Figure 85] This diagram illustrates a schematic secretory system based on flagellar type III secretion, in which a chimeric peptide expressed within a cell is fused to the N-terminal flagellar secretory signal of a native flagellar component through recombination, enabling the peptide to traverse the inner and outer membranes and move into the surrounding host environment. This allows the incomplete flagella to be used for the secretion of the target therapeutic peptide (star). [Figure 86] This diagram schematics a type V secretory system for the extracellular production of recombinant proteins, in which a therapeutic peptide (star) can be fused to the N-terminal secretory signal, linker, and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence leads the protein to the SecA-YEG mechanism, which moves 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 is inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then passed through 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] This is a schematic diagram of the type I secretory system, which uses HlyB (ATP-binding cassette transporter), HlyD (membrane fusion protein), and TolC (outer membrane protein) to form channels that pass through both the inner and outer membranes, thereby moving passenger peptides directly from the cytoplasm to the extracellular space. The C-terminal portion of HlyA, which contains the secretory signal, is fused to the C-terminal portion of a therapeutic peptide (star) to mediate the secretion of that peptide. [Figure 88]The diagram outlines the outer and inner membranes of Gram-negative bacteria, and shows several targets that form a leaky or destabilized outer membrane, thereby eliminating therapeutic polypeptides, such as eukaryotic cell-derived therapeutic peptides containing disulfide bonds, to facilitate their movement into the extracellular space. Inactivating mutations in one or more genes encoding proteins that tether the outer membrane to the peptidoglycan backbone, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and / or one or more genes encoding peripheral proteases, e.g., degS, degP, nlpl, result in a leaky phenotype. Combinations of mutations can synergistically enhance the leaky phenotype. [Figure 89] This diagram shows a modified type 3 secretion system (T3SS) that allows bacteria to inject secreted therapeutic proteins into the lumen of the gastrointestinal tract. An inductive promoter (small arrow, top), e.g., the FNR inductive promoter, drives the expression of the T3 secretion system gene cassette (three large arrows, top), which produces a mechanism for secreting tagged peptides from cells. Another inductive promoter (small arrow, bottom), e.g., the FNR inductive promoter, drives the expression of a regulator, e.g., T7 polymerase, which then activates the expression of the tagged therapeutic peptide (hexagon). [Figure 90A] This is a schematic diagram of the gene configuration of an exemplary circuit of this disclosure for the expression of a therapeutic polypeptide secreted using components of the flagellar type III secretory system. The therapeutic polypeptide of interest, such as PAL and / or LAAD, is assembled behind the fliC-5'UTR and driven by the innate fliC and / or fliD promoter. In another embodiment, inductive 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., can be exogenously added), such as arabinose, may be used. The therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium-copy plasmid) or incorporated into the fliC locus (thereby deleting all or part of fliC and / or fliD). [Figure 90B] This is a schematic diagram of the gene configuration of an exemplary circuit of the present disclosure for the expression of a therapeutic polypeptide secreted using components of the flagellar type III secretory system. The therapeutic polypeptide of interest, such as PAL and / or LAAD, is assembled after the fliC-5'UTR and driven by the native fliC and / or fliD promoter. In another embodiment, inductive promoters such as oxygen level-dependent promoters (e.g., FNR-inductive promoters) and promoters induced by metabolites that may or may not be naturally present in the gastrointestinal tract (e.g., can be exogenously added), such as arabinose, may be used. The therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium-copy plasmid) or incorporated into the fliC locus (thereby deleting all or part of fliC and / or fliD). If necessary, the N-terminal portion of FliC is included in the construct. [Figure 90C] This is a schematic diagram of the gene configuration of an exemplary circuit of this disclosure for the expression of a therapeutic polypeptide secreted using components of the flagellar type III secretory system. The therapeutic polypeptide of interest, such as PAL and / or LAAD, is assembled after 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 promoter) and promoters induced by metabolites that may or may not be naturally present in the gastrointestinal tract (e.g., may be exogenously added), such as arabinose, may be used. The therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium-copy plasmid) or incorporated into the fliC locus (thereby deleting all or part of fliC and / or fliD). If necessary, the N-terminal portion of FliC is included in the construct. [Figure 91A]This is a schematic diagram of the gene architecture of an exemplary circuit of this disclosure for the expression of therapeutic polypeptides secreted via the diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to an N-terminal Sec-dependent or Tat-dependent secretory signal of a prototype, which is cleaved upon secretion into the periplasmic space. Exemplary secretory tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria include deletions in one or more lpp, pal, tolA, and / or nlpI. Periplasmic proteases, including but not limited to degP and ompT as needed, are also deleted, for example, to enhance the stability of polypeptides in the periplasm. FRT-KanR-FRT cassettes are used for downstream integration. Expression is driven by a Tet promoter and promoters induced by metabolites that may or may not be naturally present in the gastrointestinal tract (e.g., may be exogenously added), such as arabinose. [Figure 91B]This is a schematic diagram of the gene architecture of an exemplary circuit of this disclosure for the expression of therapeutic polypeptides secreted via the diffusive outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototype N-terminal Sec-dependent or Tat-dependent secretory signal that is cleaved upon secretion into the periplasmic space. Exemplary secretory tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria include deletions in one or more lpp, pal, tolA, and / or nlpI. Periplasmic proteases, including but not limited to degP and ompT as they may be, are also deleted, for example, to enhance the stability of polypeptides in the periplasm. FRT-KanR-FRT cassettes are 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 gastrointestinal tract (e.g., may be exogenously added), such as arabinose. [Figure 92] This is a schematic diagram of the design-build-test cycle. The steps are as follows: 1. Define the disease pathway; 2. Identify target metabolites; 3. Design the genetic circuit; 4. Construct synthetic biotics; 5. Activate the circuit in vivo; 6. Characterize the circuit activation dynamics; 7. Optimize in vitro productivity to the disease threshold; 8. Test the optimized circuit in an animal disease model; 9. Assimilate into the microbiome; 10. Develop an understanding of in vivo pharmacokinetics (PK) and drug regimens. [Figure 93]Figure A is a schematic diagram illustrating a non-limiting production method for the upstream and downstream production of genetically modified bacteria according to this disclosure. Parameters for initial culture 1 (SC1): Platinum loopful of glycerol stock, duration overnight, temperature 37°C, shaking at 250 rpm. Figure B is a schematic diagram illustrating a non-limiting production method for the upstream and downstream production of genetically modified bacteria according to this disclosure. Parameters for initial culture 2 (SC2): 1 / 100 dilution of SC1, duration 1.5 hours, temperature 37°C, shaking at 250 rpm. Figure C is a schematic diagram illustrating a non-limiting production method for the upstream and downstream production of genetically modified bacteria according to this disclosure. Parameters for the production bioreactor: Inoculation - SC2, temperature 37°C, pH setting 7.00, pH dead zone 0.05, dissolved oxygen setting point 50%, dissolved oxygen cascade stirring / gas FLO, stirring limit 300-1200 rpm, gas FLO limit 0.5-20 standard liters per minute, duration 24 hours. Figure D is a schematic diagram illustrating a non-limiting method for the upstream and downstream production of genetically modified bacteria according to the present disclosure. Collection parameters: centrifugation at 4000 rpm for 30 minutes, washing 1 × 10% glycerol / PBS, centrifugation, and resuspension in 10% glycerol / PBS. Figure E is a schematic diagram illustrating a non-limiting method for the upstream and downstream production of genetically modified bacteria according to the present disclosure. Vial filling / storage parameters: dispensing 1-2 mL at a time, -80°C. [Modes for carrying out the invention]

[0016] This disclosure relates, in particular, to genetically modified bacteria, their pharmaceutical compositions, and high phenylalanine The invention includes methods for modulating and treating disorders associated with necrosis. In some embodiments, Genetically modified bacteria encode non-natural phenylalanine lyase (PAL). It contains genes that process and reduce phenylalanine in mammals. Yes, it is possible. In some embodiments, genetically modified bacteria are phenylalanine trans Further includes a gene encoding a porter. In some embodiments, genetically engineered Bacteria may also contain the gene that codes for L-AAD. Genetically modified bacteria may also Biosafety and / or biological containment, e.g., kill switches, genetic moths It may include one or more gene sequences related to the pedicle system and / or nutritional requirements. The expression of these gene sequences is such that as any promoter system disclosed herein It can be regulated using various promoter systems, and the promoter can be one or more different It can be the same promoter to regulate one gene, and it can regulate different genes. It can be a different copy of the same promoter, or it can be the expression of different genes. This may include using a combination of different promoters to adjust the settings. By using different regulatory or promoter systems to control gene expression , flexibility (for example, the ability to differentially control gene expression under different environmental conditions and / or This provides the ability to control gene expression in a temporally differential manner, and also allows for "fine-tuning" of gene expression. This provides the ability to "regulate" and any or all of these regulations affect gene expression and / or It may help optimize bacterial growth. It is related to hyperphenylalaninemia, including PKU. For the treatment and / or prevention of related conditions, genetically modified bacteria and said bacteria Using a pharmaceutical composition containing this material, phenylalanine in the body can be metabolized into a non-toxic molecule. In certain aspects, it is possible to treat and / or prevent disorders associated with hyperphenylalaninemia. In the methods disclosed herein for prevention, compositions containing genetically modified bacteria may be used. .

[0017] Certain terms are defined first so that this disclosure may be more easily understood. The meaning is to be understood in consideration of the remainder of this disclosure and as a person skilled in the art. It should. Unless otherwise defined, all technologies and sciences used herein The terms have the same meaning as commonly understood by those skilled in the art. Further definitions This is described throughout the detailed explanation.

[0018] "Hyperphenylalaninemia", "High "Phenylalaninemia (hyperphenylalaninemic)" and "over "Excess phenylalanine" refers to an increased or abnormally high concentration of phenylalanine in the body. In this specification, interchangeable terms are used to refer to. In some embodiments, high-phenyl The diagnostic signal for rualaninemia 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, less Both 12 mg / dL, at least 14 mg / dL, at least 16 mg / dL, and at least Blood levels of 18 mg / dL, at least 20 mg / dL, or at least 25 mg / dL It is at the phenylalanine level. When used herein, it is high phenylalanine. Diseases related to blood-related conditions include, but are not limited to, phenylketonuria, classical or typical pheno-ketonuria. Phenylketonuria, variant phenylketonuria, persistent mild hyperphenylalaninemia, non-phenylketonuria Phenylketonuria, hyperphenylalaninemia, phenylalanine hydroxylase deficiency, Cofactor deficiency, dihydropteridine reductase deficiency, tetrahydropterin sinter This includes Ze deficiency and Segawa disease. Those affected have progressive and irreversible neurological deficiencies. Intellectual disability, encephalopathy, epilepsy, eczema, hypotension, microcephaly, tremor, limb spasms, and / or low There is a possibility of developing hyperpigmentation (Leonard 2006). Hyperphenylalaninemia It can also be secondary to other conditions, such as liver disease.

[0019] "Phenylalanine ammonia lyase" and "PAL" are enzymes that convert phenylalanine into t rans - Phenylalanine metabolizing enzymes that convert or process cinnamic acid and ammonia ( It is used to refer to PME). Trans-cinnamic acid has low toxicity and is not found in mammals. It is converted into hippuric acid, which is secreted in the urine, by liver enzymes. PAL is an excess of phenyl It can replace the enzyme PAH for metabolizing rualanine. PAL enzyme activity is supplemented by THB. Factor activity is not required. In some embodiments, PAL is derived from a prokaryotic species. Encoded by the L gene. In an alternative embodiment, PAL is derived from a eukaryotic species. Encoded by the AL gene. In some embodiments, PAL is, but is not limited to Achromobacter xyloxoxidance Pseudomonas aeruginosa (xidans), Pseudomonas aeruginosa ginosa), Photorhabdus luminescence Anabaena variabilis (Anabaena nescens) ), and Agrobacterium cumefaciens Encoded by the PAL gene derived from bacterial species, including *Mefaciens*. In some embodiments, PAL is derived from the PAL gene of Anabaena variabilis. This is coded as such and referred to as "PAL1" in this specification (Moffitt et al., 200 (7 years). In some embodiments, PAL is derived from photolabus luminescence. Encoded by the PAL gene, referred to herein as "PAL3" (Will iams et al., 2005). In some embodiments, PAL is a yeast strain, e.g., Rod Rhodosporidium toruloides It is encoded by the PAL gene derived from (Gilbert et al., 1985). In some embodiments, PAL is a plant species, such as Arabidopsis thaliana. Encoded by the PAL gene derived from s thaliana (Wanner et al.) (1995). Any suitable nucleotide and amino acid sequence of PAL, or its mechanism. Functional fragments may be used.

[0020] "Phenylalanine hydroxylase" and "PAH" are cofactors of tetrahydrobio In order to produce tyrosine in the human body in combination with pterin, the aromatic side chain of phenylalanine Used to refer to enzymes that catalyze hydroxylation. Human genes encoding PAH. It is located on the long (q) arm of chromosome 12, between positions 22 and 24.2. The amino acid of PAH. Acid sequences are highly conserved among mammals. Nuclei of human and mammalian PAHs. The acid sequence is well known and widely available. The full-length human cDNA sequence for PAH is: It was reported in 1985 (Kwok et al., 1985). The active fragment of PAH is also well known. (For example, Kobe et al., 1997).

[0021] "L-amino acid deaminase" and "LAAD" are derived from their respective keto acids, and Stereospecific oxidative deamination of L-amino acids to produce ammonium compounds and hydrogen peroxide. It is used to refer to enzymes that catalyze the chemical reaction of phenylalanine. For example, LAAD is the enzyme that catalyzes the chemical reaction of phenylalanine. It catalyzes the conversion to nylvate. Numerous LAAD enzymes are known in this field. Many of them belong to the genera Proteus and Providencia. Bacteria such as encia, and Morganella, or their venom It originates from LAAD, which is characterized by its rapid reaction rate in the decomposition of phenylalanine (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 forces). Most eukaryotes and protozoa. While L-amino acid deaminases in nucleoplasms are extracellular, Proteus species LAAD is enzyme-active. It is localized in the cell membrane (inner membrane) that faces the periplasmic interstitial space where sex exists. As a result, phenylalanine transport across the inner membrane to the cytoplasm is in Proteus LAAD. Phenylanine is not required for more mediated phenylalanine degradation. It is easily incorporated into the surrounding material through the outer membrane without the need for porters, improving the availability of the substrate. Eliminate the need for beneficial transporters.

[0022] In some embodiments, genetically modified bacteria include, but are not limited to, the genus Proteus. LAAD genes derived from bacterial species, including bacteria of the genera Providencia and Morganella. This includes. In some embodiments, the bacterial species is Proteus mirabilis (Proteus (mirabilis). In some embodiments, the bacterial species is Proteus vulgaris. It is a squirrel (Proteus vulgaris). In some embodiments, genetic manipulation The LAAD encoded by the bacteria that produced it faces the periphery interstitial space, and in the periphery interstitial space It is localized to the cell membrane where catalytic activity is generated.

[0023] "Phenylalanine metabolic enzyme" or "PME" is responsible for breaking down phenylalanine. It is used to refer to an enzyme that produces any phenylalanine known in the art. Nerve-metabolizing enzymes can be encoded by genetically modified bacteria. PMEs are limited to... However, phenylalanine hydroxylase (PAH) and phenylalanine ammonia are not present. Aase (PAL), aminotransferase, L-amino acid deaminase (L-AAD ), and phenylalanine dehydrogenase are included.

[0024] Phenylalanine hydroxylase, phenylalanine dehydrogenase, or amino The reaction with the transferase requires a cofactor, but L-AAD and PAL are even more It requires no cofactors whatsoever. In some embodiments, genetically modified bacteria The coded PME requires a cofactor. In some embodiments, this cofactor is a legacy The genetically engineered bacteria are administered simultaneously with or consecutively. In other embodiments, the gene The manipulated bacteria can produce cofactors. In some embodiments, genetic engineering The bacteria encoded encode phenylalanine hydroxylase. In some embodiments, Genetically modified bacteria encode phenylalanine dehydrogenase. In this embodiment, the genetically modified bacteria encode an aminotransferase. In that embodiment, PME encoded by genetically engineered bacteria requires a cofactor. No. Although not bound by theory, the absence of a need for a cofactor is due to the enzyme The rate of phenylalanine degradation depends on substrate availability and is controlled by cofactor availability. This means it is not limited to. In some embodiments, it is produced by genetically modified bacteria. The PME produced is PAL. In some embodiments, genetically modified bacteria are used. The PME produced is LAAD. In some embodiments, genetically modified bacteria This codes the PME combination.

[0025] In some embodiments, the catalytic activity of PME depends on the oxygen level. Morphologically, PME is catalytically active under microaerophilic conditions. As a non-limiting example, LAA The catalytic activity of D is oxygen-dependent. In some embodiments, LAAD is performed under microaerobic conditions such as It is active under hypoxic conditions. In some embodiments of the present invention, PME is used, for example, in the colon. It functions in very low levels of oxygen, or in the absence of oxygen. As a typical example, PAL activity does not depend on the presence of oxygen.

[0026] In certain embodiments, a new or improved PME is known in the art or These can be identified according to the methods described herein and encoded by genetically modified bacteria. In some embodiments, the enzyme encoded by the genetically engineered bacteria is Wild-type enzyme isolated from an Virus, prokaryote, or eukaryote. Several embodiments So, the enzyme sequence enhances one or more specific properties of the enzyme, such as stability or catalytic activity. It has been further modified or mutated to add to the mixture.

[0027] "Phenylalanine metabolites" are products that are generated as a result of the breakdown of phenylalanine. This refers to metabolites. Metabolites are produced by enzymes using phenylalanine as a substrate. Acting directly from nyalanine or on phenylalanine metabolite substrates, It may be indirectly produced by different enzymes downstream of the metabolic pathway. In some embodiments, Phenylalanine metabolites are produced by genetically modified bacteria encoding PME. It will be done.

[0028] In some embodiments, phenylalanine metabolites are derived from PAH activity, for example, It arises directly or indirectly from PAHs produced by genetically engineered bacteria. In one embodiment, the metabolite is tyrosine. In some embodiments, phenylalanine is used. Nin metabolites accumulate in the blood or urine of PKU patients due to incomplete PAH activity. Non-exclusive examples of such PKU metabolites include phenylpyruvic acid and phenyl-milk. It is an acid. Other examples include phenylacetate, phenylethylamine, and phenyl It contains cetylglutamine.

[0029] In some embodiments, phenylalanine metabolites are derived from PAL activity, for example, It arises directly or indirectly from PAL produced by genetically engineered bacteria. Non-limiting examples of PAL metabolites include trans-cinnamic acid and hippuric acid. In several embodiments, phenylalanine metabolites, due to LAAD activity, for example, genes It arises directly or indirectly from LAAD produced by manipulated bacteria. Examples of LAAD metabolites are phenylpilbert and phenyllactic acid.

[0030] The "phenylalanine transporter" transports phenylalanine to bacterial cells. It is used to refer to membrane transport proteins that can perform this function (see, for example, Pi et al., 1991). (Referring to the light). In Escherichia coli, the pheP gene is It encodes a high-affinity phenylalanine-specific permease that is responsible for phenylalanine transport. (Pi et al., 1998). In some embodiments, phenylalanine transporter - is not limited to, but Acinetobacter calcoaceticus (Acinetobacter Salmonella enterica (Salmonella calcoaceticus) The pheP gene, derived from bacterial species including *E. coli* (a enterica), is used to... It is encoded by the aroP gene. Other phenylalanine transporters are encoded by the aroP gene. Encoded, it transports three aromatic amino acids, including phenylalanine, with high affinity, and Phe Along with P, Aagene is responsible for the largest portion of phenylalanine uptake. It contains amino acid permease. Furthermore, it has low levels of phenylalanine transport activity. Sex was traced down to the activity of the LIV-I / LS system, which has two peripheral systems. The protein-binding proteins are LIV-binding proteins (LIV-I system) and LS-binding proteins. Protein (LS system), and a branched-chain amino acid transporter consisting of the membrane component LivHMGF is. In some embodiments, the phenylalanine transporter is encoded by the aroP gene derived from bacterial species. In some embodiments, the phenyl alanine transporter is encoded by the LivHMGF gene derived from LIV-binding protein, LS-binding protein, and bacterial species. In some embodiments , the genetically engineered bacterium contains more than one phenylalanine transporter selected from pheP, aroP, and the LIV-I / LS system .

[0031] "Phenylalanine" and "Phe" are used to refer to the amino acid having the formula C6H5CH2CH(NH2)COOH . Phenylalanine is a precursor of tyrosine, dopamine , norepinephrine, and epinephrine. L-Phenylalanine is an essential amino acid and is the form of phenylalanine mainly found in food proteins. The stereoisomer D-phenylalanine is found in smaller amounts in food proteins, and DL-f enylalanine is a combination of both forms. Phenylalanine can refer to one or more of L-phenylalan ine, D-phenylalanine, and DL-phenylalanine .

[0032] "Operably linked" refers to a nucleic acid sequence, for example, a nucleic acid sequence that binds to a regulatory region sequence so as to enable the expression of a nucleic acid sequence that acts in cis, for example, a gene encoding PAL . The regulatory region can induce the transcription of the gene of interest and includes a promoter sequence, an enhancer sequence, a response element, a protein recognition site, an inducible element, a promoter control element, etc.​ An element, a protein-binding sequence, 5' and 3' untranslated regions, a transcription start site, a termination sequence It may be a nucleic acid containing a polyadenylation sequence and an intron.

[0033] An "inducible promoter" refers to a regulatory region operably linked to one or more genes, and the expression of the gene increases in the presence of an inducer of the regulatory region.

[0034] A "directly inducible promoter" refers to a regulatory region operably linked to a gene encoding a phenylalanine metabolic enzyme, such as PAL. In the presence of an inducer of the regulatory region, the phenylalanine metabolic enzyme is expressed. An "indirectly inducible promoter" refers to a regulatory system including a first regulatory region operably linked to a gene encoding a transcriptional regulatory factor that can regulate a second regulatory region operably linked to a gene encoding a phenylalanine metabolic enzyme, such as a second regulatory region operably linked to a gene encoding a phenylalanine metabolic enzyme. In the presence of an inducer of the first regulatory region, the second regulatory region can be activated or suppressed, thereby activating or suppressing the expression of the phenylalanine metabolic enzyme. Both directly inducible promoters and indirectly inducible promoters are included in the "inducible promoter".

[0035] "Exogenous environmental conditions" or "environmental conditions" refer to the settings or situations in which the promoters described herein are directly or indirectly induced. The term is intended to refer to environmental conditions that are external to the engineered microorganism but endogenous or natural to the host target environment. Thus, "exogenous" and "endogenous" refer to whether the environmental conditions are external or internal to the mammalian body. Although endogenous, it refers to environmental conditions that are external or exogenous to intact microbial cells. It can be used interchangeably. In some embodiments, exogenous environmental conditions affect the digestion of mammals. It is specific to the tubule. In some embodiments, exogenous environmental conditions affect the upper digestive tract of mammals. It is specific. In some embodiments, the exogenous environmental conditions are specific to the lower digestive tract of mammals. In some embodiments, the exogenous environmental conditions are specific to the small intestine of mammals. In some embodiments, exogenous environmental conditions include hypoxic and micro-toxic environments such as the digestive tract environment of mammals. These are aerobic or anaerobic conditions. In some embodiments, exogenous environmental conditions refer to health Or molecules or metabolites specific to the digestive tract of mammals in diseased conditions, for example, p This refers to the presence of lopionate. In some embodiments, exogenous environmental conditions are tissue-specific. or disease-specific metabolites or molecules. In some embodiments, exogenous environmental conditions are It is a low pH environment. In some embodiments, the genetically engineered microorganisms of this disclosure are pH-dependent. Includes a survival promoter. In some embodiments, the genetically engineered microorganisms of this disclosure , including an oxygen level-dependent promoter. In some embodiments, bacteria detect oxygen levels They possess evolved transcription factors that enable different signaling pathways depending on oxygen levels. It can be induced and occurs with different dynamics.

[0036] As used herein, “extrinsic environmental conditions” or “environmental conditions” also means “operated conditions.” This refers to the external settings, circumstances, or environmental conditions of a microorganism, and the microorganism's in vitro (in vitro) This relates to the culture conditions. "Exogenous environmental conditions" also affect the growth, production, and manufacturing of organisms. This may refer to conditions such as aerobic culture conditions, anaerobic culture conditions, and low acidity. It also includes axenic culture conditions and other conditions under the set oxygen concentration. Such conditions also include the presence of chemical and / or nutritional inducers such as tetracycline, arabinose, IPTG, rhamnose, etc. in the culture medium. Such conditions also include the temperature at which the microorganism is grown prior to in vivo administration. For example, when using a specific promoter system, one temperature allows the expression of the payload, while another temperature does not. Oxygen levels, temperature and medium composition affect such exogenous environmental conditions. Such conditions affect the growth rate, the induction rate of PME (e.g., PAL or LAAD), the induction rate of transporters (e.g., PheP) and / or other regulatory factors (e.g., FNRS24Y), as well as 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 a nucleic acid sequence to which one or more oxygen level-sensitive transcription factors can bind, and the binding and / or activation of the corresponding transcription factor activates downstream gene expression.

[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 DNR-responsive promoter are known in the art (see, for example, Castiglion e et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991 ; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003). Non-limiting examples are shown in Table 1.

[0039] In non-specific examples, the promoter (PfnrS) is used when ambient oxygen levels are low or nonexistent. E. coli Nissle fumarate and are known to be highly expressed under conditions where they are absent. Derived from the nitrate reductase gene S (fnrS) (Durand and Storz, 2 2010; Boysen et al. (PfnrS promoter to Nissle) In this context, the naturally occurring comprehensive transcription regulator FNR anaerobic and / or hypoxic It is activated under certain conditions. Under anaerobic and / or hypoxic conditions, FNR forms dimers. Under its control, it binds to a specific sequence in the promoter of a specific gene, thereby It activates their expression. However, under aerobic conditions, oxygen is present in the FNR dimer. It reacts with iron-sulfur clusters and converts them into an inert form. Thus, PfnrS Inducible promoters are employed to modulate the expression of proteins or RNA. PfnrS is used in this application as FNRS, fnrS, FNR, P-FNRS Promo Interchangeable as other such related designations indicating the promoter and promoter PfnrS It is used. [Table 1]

[0040] As used herein, “non-natural” nucleic acid sequences are not typically found in bacteria. Nucleic acid sequences, such as additional copies of endogenous sequences, or different species, strains, or substrains of bacteria. Modifications and Refers to a mutated or mutated sequence. In some embodiments, it refers to a non-natural nucleic acid sequence. This is a synthetic sequence that does not exist in nature (see, for example, Purcell et al., 2013). (This refers to) Non-natural nucleic acid sequences are regulatory regions, promoters, genes, and / or gene clusters. It may be one or more genes in the set. In some embodiments, "Non-natural" refers to two or more nucleic acid sequences that are not found in nature in the same relationship to each other. Non-natural nucleic acid sequences can be present on plasmids or chromosomes. Furthermore, any regulatory region, Even if multiple copies of the morphotor, gene, and / or gene cassette are present in the bacteria Often, one or more regulatory regions, promoters, genes, and / or gene cassettes. The number of copies may mutate or otherwise change as described herein. It may be done. In some embodiments, genetically modified bacteria are used to increase their copy number. To that end, or to include 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. They are manipulated to include. In some embodiments, the genetically engineered bacteria of the present invention are Promoters that are not naturally associated with the aforementioned genes and can be directly or indirectly induced, e.g. For example, an FNR promoter or L that is operablely linked to the gene encoding PAL Operablely connected to the ParaBAD promoter which is operable to the AAD. It contains genes that encode phenylalanine metabolic enzymes.

[0041] A "constitutive promoter" is defined as a mechanism under its control and / or which is activatably linked to it. This refers to a promoter that can promote the continuous transcription of a coding sequence or gene. Constitutive promoters and variants are well known in the art, and are not limited to them. BBa_J23100, constitutive E. coli σ S Promoter (e.g., osmY promoter) (International Genetic Engineering Organization) iGEM ​​(Ineered Machine) Standard Biological Parts Registry try of Standard Biological Parts) Name BBa_J 45992;BBa_J45993)), constitutive E. coli σ 32 promoter (for example, h tpG heat shock promoter (BBa_J45504), constitutive Escherichia coli σ 70 P romotor (for example, lacq promoter (BBa_J54200;BBa_J560 15) Escherichia coli CreABCD phosphate detection 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), M13K07 gene V promoter ( BBa_M13105), M13K07 gene VI promoter (BBa_M13106 ), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), Constitutive Bacillus subtilis (Bacillus su btilis)σ A Promoter (for example, 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 Promoter (for example, promoter ctc(B Ba_K143010), promoter gsiB(BBa_K143011), salmo Nella (Salmonella) promoter (e.g., Pspv2(B) derived from Salmonella) Ba_K112706), Pspv (BBa_K112707) derived from Salmonella, Ba Cteriophage T7 promoter (for example, 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)), bacterioff A phasing SP6 promoter (for example, SP6 promoter (BBa_J64998)), and their functional fragments are included.

[0042] The digestive tract is responsible for the movement and digestion of food, the absorption of nutrients, and the elimination of waste products. It refers to organs, glands, tubes, and systems. In humans, the digestive tract begins at the mouth and ends at the anus. The digestive tract includes the gastrointestinal (GI) tract, which further includes the esophagus, stomach, small intestine, and large intestine. The digestive tract also includes the spleen. The upper gastrointestinal tract includes the esophagus, stomach, and other organs and glands such as the liver, gallbladder, and pancreas. The lower gastrointestinal tract includes the duodenum of the small intestine. The lower gastrointestinal tract includes the rest of the small intestine, namely the jejunum and ileum. This includes the entire large intestine, namely the cecum, colon, rectum, and anal canal. Bacteria are found in the digestive tract. It can be found throughout the entire body, for example, in the gastrointestinal tract, and especially in the intestines.

[0043] In some embodiments, genetically modified bacteria are active in the digestive tract (for example) (For example, expressing one or more PMEs). In some embodiments, genetically engineered skin The bacteria are active in the large intestine (e.g., expressing one or more PMEs). In the application morphology, genetically modified bacteria are active in the small intestine (e.g., one or more). (expresses PME). In some embodiments, genetically engineered bacteria are used in the small intestine. It is active in the large intestine. I don't want to be bound by theory, but phenylalanine The solution is that amino acid absorption, such as phenylalanine absorption, occurs in the small intestine. It is very effective. By preventing or reducing the uptake of phenylalanine into the blood, This can avoid the rise in Phe levels and the resulting Phe toxicity. Extensive enteral circulation between the intestines and the body facilitates the removal of systemic phenylalanine in PKUs. This can be made possible (for example, as described by Chang et al. in a rat model of PKU). (Chang et al., "A new theory of enterorecirc 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; Vol. 23(1): pp. 1-21). Phenylanine derived from blood enters the small intestine. They circulate (see, for example, Figure 39) and can be removed by bacteria active at the site. In some embodiments, genetically modified bacteria pass through the small intestine. Genetically modified bacteria have a longer residence time in the small intestine. Several implementations In this state, genetically modified bacteria colonize the small intestine. In some embodiments, genetically modified bacteria colonize the small intestine. The manipulated bacteria do not colonize the small intestine. In some embodiments, the genetically modified bacteria In some embodiments, genetically modified cells are used. The fungus colonizes the small intestine. In some embodiments, The genetically modified bacteria do not colonize the digestive tract.

[0044] As used herein, the term “hypoxic” refers to the level of oxygen (O2) present in the atmosphere. It means a level, amount, or concentration of oxygen that is lower than (for example) For example, <21%O2; <160 torrO2). Therefore, the term "hypoxia (plural) conditions" "Possible" or "Hypoxic environment" refers to conditions that include oxygen levels lower than those present in the atmosphere. Or it refers to the environment. In some embodiments, the term "hypoxic" refers to the digestive tract of mammals. For example, the lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, This refers 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 O2 level of 0-60 mmHg. O2 from 0 to 60 torr) (for example, 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, (O2 at 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg) It means referring to the level, quantity, or concentration, and any and all decimal increases of them. Includes (for example, 0.2 mmHg, 0.5 mmHg of O2, 0.75 mmHg of O2, 1 O2 at 0.25 mmHg, O2 at 2.175 mmHg, O2 at 3.45 mmHg, 3.75 O2 at mmHg, O2 at 4.5 mmHg, O2 at 6.8 mmHg, O2 at 11.35 mmHg This includes O2 at 46.3 mmHg, O2 at 58.75 mmHg, etc., but these are illustrative examples. The decimals listed here are for illustrative purposes only and do not imply any limitation. (Not.) In some embodiments, "hypoxia" is defined as a blood pressure of approximately 60 mmHg or less. This refers to O2 (for example, O2 between 0 and approximately 60 mmHg). The term "hypoxia" also refers to 0- The level, amount, or concentration of O2 between 60 mmHg (including 0 mmHg and 60 mmHg). A range of degrees, for example, 0-5 mmHg O2, <1.5 mmHg O2, 6-10 mmHg This can refer to values ​​such as <8 mmHg, 47-60 mmHg, etc., but these are illustrative ranges. These are listed here for explanatory purposes only and are not meant to be limiting. For example, Albenberg et al., Gastroenterology, Vol. 147 (5 ): pp. 1055-1063 (2014); Bergofsky et al., J Clin. I nvest., 41(11):1971-1980 (1962); Crompto n et al., J Exp. Biol., Vol. 43: pp. 473-478 (1965); He et al., PNAS (USA), Vol. 96: pp. 4586-4591 (1999); McKeown, Br. J. Radiol., vol. 87:20130676(2014)(doi:1 See 0.1259 / brj.20130676). In each of the aforementioned documents, various The oxygen levels found in the digestive tracts of various mammalian species are being discussed, and these literatures are relevant. These are incorporated herein as a whole. In some embodiments, the term "hypoxic" is used. It is found in organs or tissues other than the digestive tract of mammals, such as the urogenital tract and tumor tissue. This refers to the level, amount, or concentration of oxygen (O2), and in the organ or tissue in question, oxygen levels decrease. At lower levels, for example, at hypoxic or anoxic levels. It exists. In some embodiments, "hypoxic" means partially aerobic, semi-aerobic (se (micro aerobic), (microaerobic), (nano aerobic) aerobic, microoxic, hypoxic, no The level and amount of oxygen (O2) present under oxygenic and / or anaerobic conditions. Or it means concentration. 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. Expressed as a quantity, typically in milligrams per liter (mg / L) or parts per million (ppm). (1 mg / L = 1 ppm) or micromoles (1 μmol O2 = 0 It has been reported at 0.022391 mg / L (O2). Fondriest Enviro nmental, Inc., “Dissolved Oxygen,” Fundamentals of Environmental Measurement , November 19, 2013, www.fondriest.com / environme ntal-measurements / parameters / water-quali ty / dissolved-oxygen / >. In some embodiments, "hypoxic" and The term refers to a level, amount, or concentration of oxygen (O2) of approximately 6.0 mg / L DO or less. This means, for example, 6.0 mg / L, 5.0 mg / L, 4.0 mg / L, 3.0 mg / L, 2.0 mg / L, 1.0 mg / L, or 0 mg / L, and their decimals, 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. 5 mg / L, 0.4 mg / L, 0.3 mg / L, 0.2 mg / L, and 0.1 mg / L DO. These example decimals are given here for illustrative purposes only and do not mean anything. This does not mean that it is limited. The oxygen level in a liquid or solution is equal to the oxygen level in the air saturation. It can 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 a constant temperature, pressure, and salt concentration under stable equilibrium. (Ratio to dissolved oxygen). A well-oxygenated solution that does not contain oxygen products or consumption products. (For example, a mixed and / or stirred solution) is 100% air saturated. In this embodiment, the term "hypoxic" means an air saturation of 40% or less, for example, 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% This refers to air saturation levels of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0%. This means any and all decimal increases (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 (including %) (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 This includes percentages such as 0-15%, 15-20%, 20-25%, 25-30%, etc. (Listed here) The example decimals and ranges given are for illustrative purposes only and are not intended to be limiting. It does not mean that. In some embodiments, the term "hypoxic" means oxygen below 9%. Saturation, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0% O2 saturation It means the sum, and any and all decimal increases of them (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. ) and any range of oxygen saturation levels between 0 and 9% (including 0% and 9%) (for example) , 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2. This includes O2 at concentrations of 0%, 0-8%, 5-7%, and 0.3-4.2%. The few examples and their scope are for illustrative purposes only and are not meant to be limiting. It is not something that can be done.

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

[0046] As used herein, the terms “gene” or “gene sequence” are defined as “genetic genes.” This refers to a genetic sequence, such as a nucleic acid sequence. It also means a gene, gene sequence, or genetic sequence. A genetic sequence is either a complete gene sequence or a partial gene sequence. It means including a sequence. A gene, gene sequence, or genetic sequence is a protein or This means that it contains a sequence that codes for a polypeptide, and also a protein or polypeptide. Genetic sequences that do not code, for example, regulatory sequences, leader sequences, signal sequences, and This means that it contains other non-protein-coding sequences.

[0047] "Microorganisms" are typically single-celled organisms that are microscopic, ultramicroscopic, or ultramicroscopic in nature. This refers to organisms or microorganisms. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, This includes certain algae and protozoa. In some embodiments, microorganisms are used to target one of the desired species. or are engineered to produce multiple therapeutic molecules or proteins ("engineered microorganisms"). ). In certain aspects, microorganisms may extract specific metabolites or other substances from their environment, for example, the digestive tract. The compounds are manipulated to absorb and catabolize them. In certain embodiments, microorganisms are used to provide specific beneficial substances. Synthesize metabolites or other compounds (synthetic or naturally occurring) and transfer them to their environment They are manipulated to be released into the manipulated particles. In certain embodiments, the manipulated microorganisms are manipulated into the particles. It is a bacterium. In certain embodiments, the manipulated microorganism is a manipulated virus.

[0048] "Non-pathogenic bacteria" are bacteria that are unable to cause disease or adverse reactions in a host. This refers to bacteria. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In one embodiment, the non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non Pathogenic bacteria are symbiotic bacteria that exist in the normal microbiome of the digestive tract. Examples of non-pathogenic bacteria include , not limited to, the genera Bacillus, Bacterodes Bifidobacterium oides, Bifidobacterium genus, Breviva Brevibacteria and Clostridium genera Enterococcus, Escherichia Lactobacillus erichia, Lactobacillus genus, Lactococcus Genus (Lactococcus), Genus (Saccharomyces), The genus Staphylococcus, for example, Bacillus cocoa Bacillus coagulans, Bacillus subtilis, Bacter Bacteroides fragilis, Bacteroides • Bacteroides subtilis, Bacteroides teta Iotaomicron (Bacteroides thetaiotaomicron), Bifidobacterium bifidum ), Bifidobacterium infantis (Bifidobacterium in Bifidobacterium fantis, Bifidobacterium lactis Bifidobacterium lactis, Bifidobacterium longum Clostridium butyricum (longum), Clostridium butyricum Enterococcus ricum, Enterococcus faecium *Escherichia coli*, *Lactobacillus acidophilus* Lactobacillus acidophilus, Lactobacillus vulgaris Lactobacillus bulgaricus, Lactobacillus cereus Lactobacillus casei, Lactobacillus johnsonii (Lac Lactobacillus johnsonii, Lactobacillus paracasei Lactobacillus paracasei, Lactobacillus plantarum Lactobacillus plantarum, Lactobacillus reuteri Lactobacillus reuteri, Lactobacillus rhamnosus (Lactobaci Lactobacillus rhamnosus, Lactobacillus lactis (Lactococcus) Saccharomyces lactis, and Saccharomyces braudii (This includes boulardii) (Sonnenborn et al., 2009; Dinnen et al.) Yici et al., 2014; U.S. Patent No. 6,835,376; U.S. Patent No. 6,203,7 (US No. 97; US No. 5,589,168; US No. 7,731,976). Natural Pathogenic bacteria may be genetically engineered to reduce or eliminate their pathogenicity.

[0049] "Probiotics" provide health benefits to host organisms that contain an appropriate amount of microorganisms. It is used to refer to living, non-pathogenic microorganisms that can be infected, such as bacteria. In some embodiments, the host organism is a mammal. The thing is human. Several species, strains, and / or subtypes of nonpathogenic bacteria are currently pro It is recognized as a biotic. Examples are not limited to probiotic bacteria. However, Bifidobacteria, Escherichia, and Lacto Bacillus and Saccharomyces genera, for example, Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli strain Nissle, Lactobacillus ascii Dofilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus This includes Russula plantarum and Saccharomyces blaudii (Dinleyic i et al., 2014; U.S. Patent No. 5,589,168; U.S. Patent No. 6,203,797 (US Patent No. 6,835,376). Probiotics are bacterial variants or atypical bacteria. Natural mutants may also be acceptable (Arthur et al., 2012; Cuevas-Ramos et al.) (2010; Olier et al., 2012; Nougayrede et al., 2006). Non-pathological Genetic bacteria are genetically engineered to enhance or improve desired biological characteristics, such as survival rate. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. It may be done. Probiotic bacteria may be used to improve or enhance the probiotic properties. They may be genetically engineered to be able to do so.

[0050] As used herein, “stable” or “stable” bacteria are non It is used to refer to natural genetic material, such as bacterial host cells that possess the PAL gene, and It resides so that the non-natural genetic material is retained, expressed, and / or multiplied. It is either integrated into the main genome or replicates on self-replicating extrachromosomal plasmids. Bacteria can be found in vitro, for example in culture media, and / or in vivo, for example in digestion. They can survive and / or grow within the tube. For example, stable bacteria possess the PAL gene. A gene set containing the PAL gene, in which the plasmid or chromosome is stably maintained in the host cell. It may also be a substitute bacterium, thereby allowing PAL to be expressed in the host cell, and the host cell is in They can survive and / or grow in vitro and / or in vivo. In this embodiment, the copy number is determined by non-natural genetic material, such as the PAL gene or PAH gene. This affects the stability of offspring expression. In some embodiments, the copy number is a non-natural gene. It affects the quality, 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 their related terms. This refers to the improvement of at least one identifiable symptom. In another embodiment, "modulate The terms "to do" and "to treat" are not always distinguishable by the patient, at least Both refer to an improvement in a single measurable physical parameter. In another embodiment, "modulation "To treat" and "to heal" refer to physical (e.g., stabilization of identifiable symptoms) and physiological treatments. (For example, stabilization of physical parameters) or both of these, diseases, disorders, and / or Or it refers to inhibiting the progression of a state. In another embodiment, it means "modulating" "To treat" means to slow the progression of a disease, disorder, and / or condition, or This refers to reversing the progression of those events. In this specification, it is used to mean "prevent". And its cognate words mean delaying the onset of a given disease, disorder and / or or suffer from a condition or such disease, disorder, and / or symptoms associated with the condition It refers to reducing the amount of waste.

[0052] Those requiring treatment include individuals who already have a specific medical condition, and those who have the condition. This may include individuals that have a squirt or are ultimately susceptible to the disease. The need for treatment is, for example, For example, the onset of a disease, the presence or progression of a disease, or the treatment of a person with a disease. It is assessed by the presence of one or more risk factors associated with the possibility of pregnancy. Phenylalaninemia, such as PKU, is a congenital gene mutation for which there is no known cure. This is caused by hyperphenylalaninemia, which can also occur as a complication of other conditions, such as liver disease. It is possible that... Treating hyperphenylalaninemia involves treating the excess phenylalanine This may include reducing or eliminating the symptoms and / or related symptoms. It is not necessarily required to eliminate the underlying disease.

[0053] As used herein, “pharmaceutical composition” means a physiologically appropriate carrier and / or refers to the preparation of the genetically modified bacteria of the present invention with other components such as excipients.

[0054] "Physiologically acceptable carriers" and "pharmaceutically acceptable carriers" may be used interchangeably. The term "carrier" refers to a carrier that does not cause significant irritation to organisms and does not cause the bacterial compound administered to them to survive. This refers to a carrier or diluent that does not impair the physical activity and properties of the material. Additives are included in these terms. Born.

[0055] The term "excipient" is used to describe an ingredient added to a pharmaceutical composition to further facilitate the administration of the active ingredient. It refers to an inert substance. Examples include, but are not limited to, calcium bicarbonate and calcium phosphate. Um, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oil, polyethylene The material contains lenglycol and surfactants, such as polysorbate 20.

[0056] The terms "therapeutic effective dose" and "therapeutic amount" refer to the prevention, delay, and prevention of the onset of symptoms. This refers to the amount of compound that brings about improvement in a condition, for example, the symptoms of hyperphenylalaninemia. It is used. The therapeutically effective dose is, for example, to treat, prevent, reduce the severity of, or delay the onset of the disease. one of the diseases or conditions associated with excessive phenylalanine levels It may be sufficient to reduce the risk of developing multiple symptoms. The therapeutically effective dose, and The therapeutic frequency of administration is known in the art and is determined by the method described below. It can be determined.

[0057] As used herein, the term "polypeptide" means "polypeptide ( Includes "(singular)" and "polypeptides (plural)", and an amide bond (i.e., a peptide bond) This refers to a molecule composed of amino acid monomers linked in a linear fashion. (Polypeptide) The term refers to any chain(s) of two or more amino acids, and the product of a specific length. It does not refer to "peptide", "dipeptide", "tripeptide", "Oligopeptides," "proteins," "amino acid chains," or chains of two or more amino acids ( Any other term used to refer to (plural) "polypeptide" falls within the definition of "polypeptide". The term "polypeptide" is included and may be used in place of or interchange with any of these terms. The term "dipeptide" refers to two linked amino acids. This refers to a peptide. The term "tripeptide" refers to a peptide consisting of three linked amino acids. It refers to cytoplasm. The term "polypeptide" also refers to glycosylated, acetylated, but is not limited to acetylated polypeptides. Chillation, phosphorylation, amidation, derivatization, proteolytic cleavage, or naturally occurring This term is intended to refer to the products of post-expression modifications of polypeptides, including modifications by certain amino acids. Polypeptides may be derived from natural biological sources or by recombinant technology. It may be produced. In other embodiments, the polypeptide is the genetically engineered bacteria of the present invention. or produced by a virus. The polypeptide of the present invention has 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 In sizes of 1 or more, 500 or more, 1000 or more, or 2000 or more amino acids It may exist. Polypeptides may have a defined three-dimensional structure, but not necessarily this. It does not need to have such a structure. A polypeptide with a defined three-dimensional structure is folded. It is said to have a defined three-dimensional structure, but it can adopt a large number of different conformations. Polypeptides are referred to as unfolded polypeptides. ("Peptide" or "polypeptide") The term can also refer to the amino acid sequence corresponding to a protein or a part of a protein. Or, a non-protein sequence, for example, a regulatory peptide sequence, a leader peptide sequence, Selected from signal peptide sequences, linker peptide sequences, and other peptide sequences. It may also refer to the amino acid sequence corresponding to the sequence.

[0058] "Isolated" polypeptide or its fragment, variant, or derivative is Refers to polypeptides that do not exist in the natural environment. No specific level of purification is required. However, it is produced by recombination expressed in host cells, including bacteria or mammalian cells. The polypeptides and proteins are separated, fragmented, or separated by any appropriate technique. As is the case with partially or fully purified natural or recombinant polypeptides. For the purposes of this invention, recombinant peptides, polypeptides are considered isolated. Proteins are produced using recombinant DNA technology, that is, polypeptides. Cells, microorganisms, or mammals transformed by exogenous recombinant DNA expression constructs. Refers to peptides, polypeptides, or proteins produced from animals. Most bacterial cultures Proteins or peptides expressed in nutrients typically do not contain glycans. Polypeptide fragments, derivatives, analogs or variants, and any combination thereof It is also included as a polypeptide. "Fragment," "Variant," "Derivative," and "Category" The term "analog" refers to a molecule that has an amino acid sequence that is sufficiently similar to the amino acid sequence of the original peptide. It includes a polypeptide that possesses at least one or more properties of the corresponding original polypeptide. The polypeptide fragment of the present invention contains any polypeptide to be retained. Includes fragments and deletion fragments. Fragments may also contain specific antibodies or bioactive fragments or the details described herein. Contains immunologically active fragments derived from any polypeptide described in the book. Variant It may or may not be naturally occurring. Barriers that do not exist naturally The variant can be produced using mutagenesis methods known in the field. Lipeptides may contain conserved or non-conserved amino acid substitutions, deletions, or additions.

[0059] Polypeptides also include fusion proteins. When used herein, "ba The term "riant" refers to a sequence containing the original peptide or a sequence that is sufficiently similar to the original peptide. Contains a fusion protein. When used herein, the term "fusion protein" is used in this specification. The term refers to a chimeric protein that contains the amino acid sequences of two or more different proteins. Typical example: Typically, fusion proteins are generated from well-known in vitro recombination techniques. The fusion protein has a structure and function similar to the individual original proteins that make up the fusion protein (however (They do not necessarily have to be the same degree), and / or similar adjustment functions (but not necessarily (These do not necessarily have to be to the same degree), and / or similar biochemical functions (but not necessarily the same). (They do not need to be the same degree) and / or immunological activity (but not necessarily the same degree) (It is not necessary) may have. "Derivatives" are not limited to, but include 20 standard amino acids. Contains a peptide containing one or more naturally occurring amino acid derivatives of an acid. The "similarity" between peptides is determined by comparing the amino acid sequence of one peptide with the sequence of a second peptide. It is determined by comparison. The amino acids of one peptide are determined to be identical or conserved. In the case of amino acid substitution, it is similar to the corresponding amino acid of the second peptide. Conservative position For translation, see Dayhoff, MO, ed., The Atlas of Protei n Sequence and Structure 5, National Biom Medical Research Foundation, Washington,D. C. (1978), and Argos, EMBO J.8 (1989), 779-7 This includes the items listed on page 85. For example, amino acids belonging to one of the following groups are preserved. Represents existential change or substitution: -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” means the first and second The amino acid sequences of the following have a common structural domain and / or common functional activity: Compared to the second amino acid sequence, a sufficient or minimum number of identical or equivalent amino acid residues This refers to the first amino acid sequence it contains. 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 containing is defined herein as sufficiently similar. Preferably The variant is sufficiently similar to the amino acid sequence of the peptide of the present invention. Ants generally retain the functional activity of the peptide of the present invention. Variants are one or more This involves the deletion, addition, and / or substitution of multiple amino acids, resulting in the natural and wild-type peptides. It contains peptides with different amino acid sequences. These are naturally occurring variants. And may also be artificially designed variants.

[0061] As used herein, "linker," "linker peptide," or "peptide" may be used. The term "tidlinker" or "linker" refers to the connection of two polypeptide sequences or Linking, for example, linking two polypeptide domains, synthetic or non-natural or natural This refers to an amino acid sequence that does not exist in the original. When used herein, the term "synthetic" is used in this specification. The term refers to an amino acid sequence that does not exist in nature. Exemplary linkers are described herein. Further exemplary linkers are provided in U.S. Patent Application Publication No. 20140079701. The contents thereof are incorporated in their entirety herein by reference.

[0062] As used herein, the term “codon-optimized sequence” refers to an existing code Expression of transcription RNA molecules modified from sequences, or, for example, transcribed from coding sequences. To improve translation in host cells or organisms, or to improve the transcription of coding sequences. This refers to sequences designed to achieve certain characteristics. Codon optimization is not limited to, but is relevant to the expression of the host organism. The process involves selecting codons for a code sequence to match the codon preference. This includes the process. The term "codon-optimized" is encoded by nucleic acid molecules. To reflect the typical codon usage frequency of the host organism without modifying the polypeptide. This refers to modifying codons in the gene or coding region of the nucleic acid molecule in question. Such optimization involves at least one, two or more, or a considerable number of codons, in the host This involves substituting with one or more codons that are more frequently used in the genes of an organism. A "codon-optimized sequence" is a sequence that has been modified from an existing code sequence, or, for example, Expression of transcription RNA molecules transcribed from coding sequences modifies translation in host cells or organisms. This refers to an array designed to improve or enhance the transcription of a code array. In one embodiment, the improvement of transcription and / or translation is the rate of transcription and / or translation. This includes increasing the bell. In some embodiments, it improves transcription and / or translation. This includes reducing the level of transcription and / or translation. In some embodiments, , expression levels from the target construct, e.g., PAL3 level and / or PheP level Codon optimization is used to fine-tune the expression. Codon optimization involves the expression host organism. The process involves selecting codons in a code sequence to match the codon preference. This includes, but is not limited to, certain elements in the growth polypeptide chain. Many organisms have certain elements in their growth polypeptide chains. Bias or preference for using specific codons to encode mino acid insertions This indicates that codon preference or codon bias, or differences in codon usage frequency between organisms, are genetic. It is permissible due to the degeneracy of cryptography and is well documented among many organisms. Translation bias often correlates with the translation efficiency of messenger RNA (mRNA), and then... Furthermore, the messenger RNA (mRNA) is particularly important for the characteristics of the codons that are translated. This is thought to depend on the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in cells is generally most frequently used in peptide synthesis. It is a reflection of the codons used. Therefore, genes are a given life based on codon optimization. It can be regulated for optimal gene expression in an organism.

[0063] As used herein, the terms “secretory system” or “secretory protein” are used in this specification. , the secretion or excretion of the target protein or therapeutic protein from the cytoplasm of microorganisms, such as bacteria. This refers to natural or unnatural secretory mechanisms that can produce secretions. The secretory system contains a single protein. Or, a complex, for example, two or more proteins that are constructed in HlyBD It may include quality. Non-limiting examples of secretory systems for Gram-negative bacteria include modified I Type II flagella, Type I (e.g., hemolysin secretory system), Type II, Type IV, Type V, Type VI, and V Type II secretion system, resistance-nodulation- The division (RND) is a multidrug efflux pump, which includes various single-membrane secretory systems. Non-limiting examples of secretory systems for positive bacteria include the Sec and TAT secretory systems. In some embodiments, the target protein is the target protein or therapeutic protein. A "secretion tag" of either RNA or peptide origin to induce a specific secretion system Includes. In some embodiments, the secretory system secretes the target protein from the manipulated bacteria. This tag can be removed beforehand. For example, type V autocrine-mediated secretion (auto- In secretion-mediated secretion, the N-terminal peptide The secretory tag is a "passenger" tag that travels from the cytoplasm to the periplasmic compartment via the natural Sec system. It is removed during the translocation of ptide. Furthermore, when autocrine factors migrate across the outer membrane, C Terminal secretory tags are either autocatalyzed or protease-catalyzed, for example. It can be removed by OmpT cleavage, thereby releasing the target protein into the extracellular environment. ru.

[0064] As used herein, the term "transporter" refers to a molecule, for example Mechanisms for taking up amino acids, toxins, metabolites, substrates, etc., from the extracellular environment into microorganisms. For example, it refers to proteins (plural is possible). For example, phenylalanine proteins such as PheP. Nin transporters take phenylalanine into microorganisms.

[0065] As used herein, the articles “a” and “an” are used as follows: Unless explicitly stated otherwise, it is understood to mean "at least one". It should be done.

[0066] The phrase "and / or" is used between elements in an enumeration, (1) single (1) Only one of the enumerated elements exists, or (2) There are more than one of the enumerated elements. It is intended to mean one of the following: 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 This indicates that B, C, and C may be the options. The phrase "and / or" is used in the enumeration. It may be used interchangeably with "at least one" or "one or more" of the elements. stomach.

[0067] bacteria The genetically modified bacteria of the present invention can reduce excess phenylalanine. In some embodiments, the genetically modified bacteria are non-pathogenic bacteria. Morphologically, genetically modified bacteria are symbiotic bacteria. In some embodiments, genetic engineering The bacteria produced are probiotic bacteria. In some embodiments, genetically modified The bacteria were modified or mutated to reduce or eliminate pathogenicity. These are naturally pathogenic bacteria. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. Exemplary bacteria include: Not limited to, but including the genera Bacillus, Bacterodes, Bifidobacterium, and Brevibacterium. Teria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactobacillus Tococcus, Saccharomyces, and Staphylococcus genera, for example, Bacillus coccus Aglans, Bacillus subtilis, Bacteroides fragilis, Bacteroides... Subtilis, Bacteroides tetaiotaomicron, Bifidobacterium bifi Bifidobacterium dum, Bifidobacterium infantis, Bifidobacterium lactis Bifidobacterium longum, Clostridium butyricum, Enterococcus • Faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus Tobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus Tobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Includes Lactobacillus lactis and Saccharomyces boudii. Specific fruits In terms of application methods, genetically modified bacteria include Bacteroides fragilis and Bacteroides... Tethaeotaomicron, Bacteroides subtilis, Bifidobacterium bifi Bifidobacterium dum, Bifidobacterium infantis, Bifidobacterium lactis Clostridium butyricum, E. coli Nissle, Lactobacillus acidophilus Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus Selected from the group consisting of Lactis.

[0068] In some embodiments, genetically engineered bacteria are the most characteristic probiotics. Enterobacteriaceae, which has evolved into one of the staghorn fossils Nissle 1917 (E. coli) is a Gram-negative bacterium. (Nissle) (Ukena et al., 2007). The strain is particularly notable for its complete harmlessness. (Schultz, 2008), GRAS (Generally Recognized as Safe) (Rei (ster et al., 2014, underlining added by the author). Genome sequencing of E. coli Nissle identifies important virulence factors (e.g., E. coli α-hemolysin, P-fimbriae adhesion factor). It was confirmed that it was missing (Schultz, 2008). Furthermore, E. coli Ni SSLE does not possess pathogenic adhesion molecules, does not produce any enterotoxins or cytotoxins, and is invasive. It has been shown that it is not urinary tract pathogenic (Sonnenborn et al., 2009). As early as 1917, *E. coli* Nissle was used for therapeutic purposes, and Mutaflo It was packaged in a pharmaceutical capsule called r. The therapeutic effect of E. coli Nissle is... It is generally accepted that safety has been proven to a satisfactory degree (Ukena et al.) , 2007).

[0069] Those skilled in the art will see that the genetic modifications disclosed herein are suitable for other species, strains, and subtypes of bacteria. It is important to understand that genes from one or more different species can be combined. Furthermore, genes from one or more different species can be derived from each other. It is acceptable for it to be introduced; for example, the PAL gene derived from Rhodosporidia torroides is derived from E. coli. It can be expressed in (Sarkissian et al., 1999), in prokaryotes and eukaryotes. Phenylalanine ammonia lyase is known to share sequence homology (X iang and Moore, 2005.

[0070] Unmodified Escherichia coli Nissle and the genetically modified bacteria of the present invention are, for example, also found in the digestive tract. Alternatively, protective factors in the serum (Sonnenborn et al., 2009), or administered Destruction will occur in the next few hours or days when the kill switch is activated. This is possible. Thus, genetically modified bacteria may require continuous administration. In some embodiments, residence time is calculated for human subjects. The residence time can be calculated for the genetically modified bacteria of the present invention (see, for example, Figure 68). (Referring to Teru).

[0071] In some embodiments, the genetically engineered bacteria of the present invention have a gene encoding PAL. The PAL gene is operably linked to a promoter that can be directly or indirectly induced. In some embodiments, bacteria contain non-natural PAL genes. Morphologically, the bacteria contain further copies of the native PAL gene. In some embodiments, The promoter is not naturally associated with the PAL gene. In some embodiments, A promoter is any one or more promoters disclosed herein.

[0072] In some embodiments, the genetically engineered bacteria of the present invention have a gene encoding PAH. The PAH gene is operably linked to a promoter that can be directly or indirectly induced. In some embodiments, bacteria contain non-natural PAH genes. In the application form, the bacteria contain further copies of the native PAH gene. In some embodiments In some embodiments, the promoter is not naturally associated with the PAH gene. A promoter is any one or more promoters disclosed herein.

[0073] In some embodiments, the genetically engineered bacteria of the present invention have a gene encoding LAAD. The LAAD gene contains offspring and can be activated by a promoter that can be directly or indirectly induced. They are linked. In some embodiments, bacteria contain a non-natural LAAD gene. In that embodiment, the bacteria contain further copies of the native LAAD gene. In the application morphology, the promoter is not naturally associated with the LAAD gene. Several applications In form, the promoter is any one or more promoters disclosed herein.

[0074] In some embodiments, genetically engineered bacteria are phenylalanine transporters The bacterium further comprises a gene encoding (PheP). In certain embodiments, the bacterium is phenyl It contains further copies of the natural gene encoding the alanine transporter, and phenyl Ranin transporter genes act on promoters that can be directly or indirectly induced. They are linked together. In an alternative embodiment, bacteria are transpo The gene encoding the phenylalanine transporter is directly Alternatively, it is operably connected to an indirectly induced promoter. Both embodiments are, This falls under the category of "unnatural" phenylalanine transporters. Several implementations Morphologically, the promoter is not naturally associated with the pheP gene. Several implementations Morphologically, the same promoter is used for PheP and PAL and / or PAH and / or It controls the expression of L-AAD. In some embodiments, it controls the expression of PheP. The promoter controls the expression of PAL and / or PAH and / or L-AAD. It is different from the promoter. In some embodiments, the promoter controls the expression of PheP. A promoter is any one or more promoters disclosed herein.

[0075] In some embodiments, it operates on PAL, PAH, LAAD, and / or pheP. Potentially linked promoters are directly or indirectly induced by extrinsic environmental conditions. In some embodiments, the promoter is exogenous and specific to the mammalian digestive tract. It is directly or indirectly induced by environmental conditions. In some embodiments, promotion The ter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of mammals. In some embodiments, the promoter is an exogenous environment specific to the mammalian large intestine. It is induced directly or indirectly depending on the conditions. In some embodiments, the promoter This is in low-oxygen or anaerobic conditions and / or low-oxygen conditions such as the environment of the digestive tract of mammals. Therefore, it is induced directly or indirectly. In some embodiments, the promoter is a pyol The presence of molecules or metabolites specific to the digestive tract of mammals, such as propionates, It is directly or indirectly induced. In some embodiments, the promoter is tetra It is induced directly or indirectly by exposure to cyclins. In some embodiments, The promoter is induced directly or indirectly by exposure to arabinose. In some embodiments, the promoter is directly or indirectly activated by exposure to IPTG. It is induced to rhamnose or something already known to those skilled in the art. In some embodiments, the promoter is rhamnose or something already known to those skilled in the art Directly or indirectly through exposure to other chemical and / or nutritional inducers of knowledge It is induced. In some embodiments, the promoter is directly induced by the extrinsic ambient temperature. It is controlled directly or indirectly. In some embodiments, the promoter is also IPTG This is induced directly or indirectly by exposure to other lacI-binding compounds. In that embodiment, the promoter is directly or indirectly stimulated by exposure to rhamnose. It is induced. In some embodiments, the promoter is directly and This is indirectly induced. In some embodiments, the promoter is activated by a decrease in temperature. It is induced directly or indirectly. In some embodiments, the promoter is the present invention It is directly or indirectly induced by molecules administered simultaneously with genetically modified bacteria. Such molecules are tetracycline or IPTG or arabinose or already known to those skilled in the art. It may be another chemical and / or nutritional inducer of knowledge.

[0076] In some embodiments, the promoter is located in a culture vessel (e.g., a flask or fermenter). or directly or indirectly induced by the conditions in other suitable culture vessels, within The strain is grown or maintained before in vivo administration. This is provided during the culture of the strain. Non-specific examples of eel conditions include low oxygen, anaerobic, microaerophilic, or aerobic conditions, and other constant conditions. Oxygen levels (as exemplified below), presence of arabinose, IPTG, rhamno S or other chemical and / or nutritional compounds described herein or known in the art. This includes the presence of growth-inducing substances. In some embodiments, the conditions in the culture vessel are specific to acidity. At the elementary level, for example, oxygen 1%~10%, oxygen 10%~20%, oxygen 20%~30%, oxygen 30%~40%, 40~50% oxygen, 60~70% oxygen, 70~80% oxygen, 80~ Set to 90%, 90-100% oxygen, and other oxygen levels as described herein. At that point in time, the promoter is directly or indirectly guided.

[0077] Reduction of hyperphenylalaninemia The genetically modified bacteria of the present invention encode phenylalanine metabolic enzyme (PME). Contains genes. In some embodiments, genetically engineered bacteria contain phenylalanine. It contains a gene encoding the phenoglycan enzyme (PME) and reduces hyperphenylalaninemia. It is possible.

[0078] Examples of phenylalanine metabolic enzymes include, but are not limited to, phenylalanine hydroxyl AA (PAH), phenylalanine ammonia lyase (PAL), aminotransferase L-amino acid deaminase (L-AAD), and phenylalanine dehydrogenase It contains phenylalanine hydroxylase, phenylalanine dehydrogenase. The reaction with -ase or aminotransferase requires a cofactor, but L-AAD and PAL does not require any additional cofactors. It is not bound by theory, but cofactors The fact that it does not require phenyl by enzymes encoded by genetically modified bacteria Alanine degradation depends on substrate availability and is not limited by cofactor availability. It means that.

[0079] In some embodiments, the manipulated bacteria are one or more phenylalanine hydroxy It includes a gene sequence encoding an enzyme (PAH) polypeptide. In some embodiments, it includes a gene sequence encoding an enzyme (PAH) polypeptide. The manipulated bacteria contain one or more phenylalanine ammonia lyase (PAL) polypeptides. Contains the gene sequence encoding thiosulfate. Phenylalanine ammonia lyase (PAL;E C4.3.1.24) L-phenylalanine is converted to ammonia and trans-cinnamon. It is an enzyme that catalyzes the reaction that converts to acid. Phenylalanine ammonia lyase is L- It is specific to Phe and to a lesser degree specific to L-tyrosine. The catalyzed reaction involves L-phenyl to produce trans-cinnamic acid and ammonia. This is a naturally occurring, non-oxidative deamination of alanine. Unlike mammalian enzymes (PAHs) PAL is a monomer and does not require a cofactor (MacDonald et al., Bioche m Cell Biol 2007;85:273~82. A modern v (Iew of phenylalanine ammonia lyase). In this context, it is because microorganisms use L-phenylalanine (LP) as their sole source of carbon and nitrogen. In one embodiment, the present invention has a catabolic role that enables the use of he(g) Genetically modified bacteria contain the PAL gene. PAL produces phenylalanine at a non-toxic level. It can be converted to trans-cinnamic acid and ammonia. The acid (TCA) can be further converted into its metabolites, benzoic acid and hippuric acid (S arkissian et al., J Mass Spectrom. June 2007; 42(6) :811~7 pages;Quantitation of phenylalanine an d its trans-cinnamic,benzoic and hippuri c acid metabolites in biological fluids (in a single GC-MS analysis). PAL enzyme activity is THB supplementation. Factor activity is not required.

[0080] In some embodiments, PAL is, but is not limited to, Achromobacter xylosophyton. Sidance, Pseudomonas aeruginosa, Photorhabdus luminescence, Anabaena • Derived from bacterial species, including *Variabilis* and *Agrobacterium tumefaciens*. Encoded by the PAL gene. In some embodiments, the bacterial species is Photolabda It is luminescence. In some embodiments, the bacterial species is Anabaena variabilis. In some embodiments, PAL is derived from eukaryotic species, such as yeast species and plant species. Encoded by the PAL gene. Multiple different PAL proteins are involved in this field. It is publicly known that when the PAL gene is expressed in genetically modified bacteria, under the same conditions... PA converts more phenylalanine than unmodified bacteria of the same bacterial subtype. Therefore, PA Genetically modified bacteria containing L have been associated with hyperphenylalaninemia, including PKU. It can be used to treat the condition by metabolizing phenylalanine in the body into non-toxic molecules. In some embodiments, the genetically modified bacteria are Anabaena variabilis PAL It expresses ("PAL1"). In some embodiments, the genetically engineered bacteria are fo It expresses Trabdus luminescence PAL ("PAL3"). Indefinitely the target PAL sequence Typical examples are shown in Table 2.

[0081] In some embodiments, the manipulated bacteria contain one or more LAAD polypeptides. It includes a gene sequence encoding a certain gene. In some embodiments, the manipulated bacteria consist of one or more bacteria. Gene sequences encoding the PAL polypeptide and one or more LAAD polypeptides It contains. LAAD, along with the production of ammonia and hydrogen peroxide via imino acid intermediates, Stereospecific oxidation, i.e., deamination of L-amino acids to α-keto acids, which consumes oxygen. It catalyzes L-AAD, a type of venom found in snake venom and many bacteria (Bifulco et al., 2013). Specifically, it is found in the cell membranes of bacteria belonging to the genera Proteus, Providencia, and Morganella. It is released. L-AAD (EC 1.4.3.2) is a flavin enzyme that has a dimeric structure. Each subunit is a non-covalent flavin adenine dinucleotide (FAD) cofactor. It contains two types of L-AA. Contains D (Duerre and Chakrabarty 1975). One is widespread. It has substrate specificity and is a keto acid of aliphatic and aromatic L-amino acids, typically L-phenyl Catalytic oxidation to rualanine (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 a coli). Other types primarily act on basic L-amino acids (GenBan k:EU669819.1). LAADs derived from bacteria, fungi, and plants are used as nitrogen sources. It is involved in the utilization of L-amino acids (i.e., ammonia produced by enzymatic activity). It appears that most eukaryotic and prokaryotic L-amino acid deaminases are membrane-bound. Except for the Proteus species LAAD, which is a combined form, it is secreted extracellularly. (Proteus mira) In bilis, L-AAD is found in cells that face outward to the periplasmic interstitial space where enzyme activity exists. 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 modified bacteria of the present invention contain the LAAD gene. LAAD is Phenylalanine can be converted to non-toxic levels of phenylpyrubate, and that phenyl Nylpirubate can also be further broken down into phenyllactate by, for example, liver enzymes. Phenylpirubate cannot cross the blood-brain barrier, and LAAD can cause other potential problems. It reduces phenylalanine levels in the brain without allowing the accumulation of toxic metabolites. It is possible. In some embodiments, LAAD may include, but is not limited to, the genus Proteus. LAAD genes derived from bacterial species, including bacteria of the genera Providencia and Morganella. Encoded by: In some embodiments, the bacterial species is Proteus mirabilis. In some embodiments, the bacterial species is Proteus vulgaris. Morphologically, genetically modified bacteria are Proteus mirabilis LAAD enzyme GenBank It expresses :U35383.1. Non-specific examples of the target LAAD sequence are shown in Table 2. In some embodiments, the LAAD enzyme is derived from snake venom. According to the present invention, gene manipulation When the resulting bacteria express the LAAD gene, they exhibit the same unmodified characteristics as the same bacterial subtype under the same conditions. It converts more phenylalanine than bacteria. Therefore, genetically modified organisms including LAAD The bacteria, in order to treat conditions associated with hyperphenylalaninemia, including PKU, are present in the body. It can be used to metabolize phenylalanine into non-toxic molecules.

[0083] In some embodiments, genetically modified bacteria produce wild-type enzymes similar to those found in nature. To code. In some embodiments, genetically engineered bacteria are genetically engineered to have a mutation in the wild-type sequence. It encodes an enzyme containing mutations. In some embodiments, the mutations increase the stability of the enzyme. To cause. In some embodiments, the mutation increases the catalytic activity of the enzyme. In the embodiment, the genetically modified bacteria possess one or more of the proteins listed in Table 2. It contains a gene that codes for the sequence number. In some embodiments, the genetically engineered bacteria have a sequence number. Gene sequences encoding one or more polypeptides containing any of sequences 1-8. Includes. In some embodiments, genetically engineered bacteria have sequences of sequence numbers 1-8. The difference is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, and 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% It contains a gene sequence encoding a polypeptide with 98% or 99% identity. In some embodiments, the genetically engineered bacteria include mutations, one of those in Table 2 or It encodes multiple enzymes. In some embodiments, genetically modified bacteria are wild-type P It contains a gene encoding AH. In some embodiments, the genetically engineered bacteria multiply Encodes mutant PAHs with added stability and / or activity. Several implementations Morphologically, genetically modified bacteria contain the gene that encodes wild-type PAL. In this embodiment, the genetically modified bacteria have increased stability and / or activity. It encodes a mutated PAL. In some embodiments, the genetically engineered bacteria are wild Contains a gene encoding type LAAD. In some embodiments, genetically engineered bacteria This encodes a mutant LAAD with increased stability and / or activity, which is desired. Methods for screening enzymes with specific properties are known in the art and are described herein. It will be listed.

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

[0085] PMEs, for example, the PAL, LAAD, or PAH gene(s), are genetically modified. It may be present on plasmids or chromosomes in bacteria. In some embodiments, PME A gene sequence(s) is expressed under the control of one or more constitutive promoters. In some embodiments, the PME gene is subjected to exogenous environmental conditions as described herein. It is expressed under the control of a promoter that is directly or indirectly induced by the substance. In that embodiment, the PME gene is a molecule or metabolite specific to the mammalian digestive tract. The control of promoters, which is directly or indirectly induced by extrinsic environmental conditions such as the presence of a promoter. It is expressed under certain conditions. In one embodiment, the PME gene is expressed in hypoxic, microaerophilic, or anaerobic conditions. It is expressed under the control of a promoter that is directly or indirectly induced depending on the conditions, and PME Gene expression, such as that of the PAL gene, is affected by hypoxic or anaerobic conditions, such as those found in the digestive tract environment of mammals. It is activated under specific environmental conditions.

[0086] In some embodiments, genetically engineered bacteria are given one or more PAL polypeptides. It includes a gene sequence that codes for a column. In some embodiments, the manipulated bacteria are one or more The gene sequence includes the above PAL polypeptide sequence, and the gene sequence is mammalian. It is directly or indirectly induced by hypoxic or anaerobic conditions, such as those found in the digestive tract. In some embodiments, the manipulated bacteria encode one or more LAAD polypeptides. It contains a gene sequence that does so. In some embodiments, the manipulated bacteria have one or more LAAs. The gene sequence includes a gene sequence encoding a D polypeptide, and the gene sequence is found in the stomach, duodenum, and Oxygen is supplied in the proximal intestines, including but not limited to the ileum. It is directly or indirectly induced by conditions such as oxygen, hypoxia, or microaerophilic conditions. In this embodiment, the manipulated bacteria encode one or more PME polypeptide sequences. The gene sequence includes a gene sequence that is naturally present in the digestive tract of mammals due to environmental factors. This is induced directly or indirectly. In other embodiments, genetically modified bacteria are induced Environmental factors that do not naturally exist in the digestive tract of mammals, such as arabinose or IPTG... Therefore, it encodes one or more PME gene sequences that are directly or indirectly induced. In this embodiment, genetically modified bacteria are naturally present in the digestive tract of mammals under inflammatory conditions. One or more PME gene sequences that are directly or indirectly induced by environmental factors are coded In some embodiments, the manipulated bacteria produce one or more PAL polypeptides. Genetic sequences encoding one or more L-AAD polypeptides This includes the gene sequence being controlled by the same promoter or different copies of the same promoter. The promoter is subject to any of the environmental conditions described herein and specified herein. Directly or indirectly induced by any of the promoters or other exogenous environmental conditions described above. In some embodiments, the manipulated bacteria are led to one or more PAL polypeptides. Gene sequences encoding one or more L-AAD polypeptides and gene sequences encoding one or more L-AAD polypeptides The sequence includes a column, and the gene sequence is under the control of a different promoter, and the promoter is Any of the environmental conditions described herein and any of the promoters described herein It is directly or indirectly induced by any extrinsic environmental conditions. In some embodiments, The manipulated bacteria have one or more gene sequences encoding PAL polypeptides and one The gene sequence includes the above L-AAD polypeptide encoding, and the gene sequence is constitutive It is under the control of a promoter. In some embodiments, the manipulated bacteria are controlled by one or more The gene sequence encoding the PAL polypeptide and one or more L-AAD polypeptides The PAL gene sequence includes an encoding gene sequence, and the PAL gene sequence is under the control of a constitutive promoter. Furthermore, the LAAD gene sequence is under the control of an inducible promoter. Several embodiments The manipulated bacteria then have a gene sequence that encodes one or more PAL polypeptides and The LAAD gene comprises one or more gene sequences encoding L-AAD polypeptides. The sequence is under the control of a constitutive promoter, and the PAL gene sequence in question is controlled by an inductive promoter. It is under the control of one or more Phetra. In any of these embodiments, the bacteria are under the control of one or more Phetra. The gene sequence encoding the propeller polypeptide may further include the gene The sequence can be under the control of a constitutive promoter or an inductive promoter, Pa The promoters that control the l and / or LAAD gene sequences are the same as or different from the promoters that control them. It is possible to be a worker.

[0087] In other embodiments, the manipulated bacteria are directly administered in bacterial cell culture before in vivo administration. Encoding one or more PME gene sequences that are directly or indirectly induced; i.e. , one or more PME gene sequences are expressed under the control of an inducible promoter, and the inducible promoter The motor controls bacterial growth in flasks, fermenters, or other culture vessels. Specific molecules or metabolites, temperature, oxygen levels, or other parameters supplied to nutrients Responds to the data. In some embodiments, the manipulated bacteria are in bacterial cell culture. One or more PME gene sequences that are directly or indirectly induced before vivo administration Encodes; one or more PME gene sequences are expressed under hypoxic or anaerobic conditions. In some embodiments, the manipulated bacteria were used before in vivo administration in bacterial cell culture. Encoding one or more PME gene sequences that are directly or indirectly induced; one The above PME gene sequences are expressed under aerobic conditions. In some embodiments, the manipulated The bacteria are induced directly or indirectly before in vivo administration during bacterial cell culture. Encodes one or more PME gene sequences; one or more PME gene sequences are microscopic It is expressed under aerobic conditions. In some embodiments, the manipulated bacteria are expressed during bacterial cell culture. One or more PME genes that are directly or indirectly induced before in vivo administration. Encoding a child sequence; one or more PME gene sequences are expressed in the presence of arabinose. In some embodiments, the manipulated bacteria are administered in vivo during bacterial cell culture. Encodes one or more PME gene sequences that are previously directly or indirectly induced; One or more PME gene sequences are expressed in the presence of IPTG.

[0088] Bacteria possess evolved transcription factors that can detect oxygen levels. The oxygen transport pathway can be triggered by different oxygen levels and occurs with different dynamics. Level-dependent promoters are nuclei to which one or more oxygen level-sensing transcription factors can bind. It is an acid sequence, and 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 more specific terms, the PAL gene is associated with low-oxygen environments such as the digestive tract of mammals. Alternatively, it is under the control of an oxygen-level-dependent promoter that is activated in an anaerobic environment.

[0089] In certain embodiments, genetically modified bacteria regulate fumarate and nitrate reductase. One or more PME gene sequences(s) expressed under the control of a factor (FNR) promoter, For example, PAL is included. In E. coli, FNR controls the transition from aerobic to anaerobic metabolism. It is a major transcriptional activator (Unden et al., 1997). Anaerobic and / or Under hypoxic conditions, FNR activates hundreds of genes involved in adaptation to anaerobic growth. It dimerizes into an active DNA-binding protein. Under aerobic conditions, FNR is activated by oxygen. Dimerization is inhibited, and the substance is inactive. In some embodiments, multiple different FNR nucleic acids are used. The sequence is inserted into the genetically modified bacteria. In an alternative embodiment, the genetically modified bacteria This refers to alternative oxygen level-dependent promoters, such as the ANR promoter (Ray et al., 19 (1997), PM is expressed under the control of the DNR promoter (Trunk et al., 2010). E, for example, PAL, is included. In some embodiments, phenylalanine metabolism occurs in the gastrointestinal tract, etc. It is particularly activated in low-oxygen or anaerobic environments.

[0090] In Pseudomonas aeruginosa, arginine deiminase and nitrate reduction Anaerobic regulatory (ANR) transcription factors are "physiologically induced under oxygen-restricted or anaerobic conditions." "Required for the expression of scientific function" (Winteler et al., 1996; Sawers 1 991). Pseudomonas aeruginosa ANR is homologous to Escherichia coli FNR, and " The census FNR site (TTGAT----ATCAA) is determined by ANR and FNR. "Efficiently recognized" (Winteler et al., 1996). Similar to FNR, anaerobic conditions In this state, ANR activates numerous genes responsible for adaptation to anaerobic growth. In this state, ANR is inactive. Pseudomonas fluorescein (Pseud Omonas fluorescens, Pseudomonas putida (Pseudomo Pseudomonas syringae ringae), and Pseudomonas mendocina All of the docina have functional analogues of ANR (Zimmermann et al., 19 (1991). Promoters regulated by ANR, e.g., arcDABC operon The promoter is publicly known in the field (for example, Hasegawa et al., 1998). (See reference).

[0091] The FNR family also includes "Anaerobic nitrate respiration of Pseudomonas aeruginosa" Hasegawa et al. (1998) Transcriptional regulators required in conjunction with ANR It also includes catabolic nitrate respiratory regulators (DNRs) (Arai et al., 1995). Regarding genes, the FNR binding motif is "probably recognized only by DNR." Hasegawa et al., 1998). Controlled by extrinsic environmental conditions and corresponding regulatory regions. Any suitable transcription regulator can be used. Non-limiting examples include ArcA / B. This includes ResD / E, NreA / B / C, and AirSR, and others in the relevant field. It is publicly known.

[0092] FNR promoter sequences are publicly known in the art, and any appropriate FNR promoter The ter sequence can be used in the genetically modified bacteria of the present invention. Any suitable FNR The motor may be combined with any suitable PAL. Non-limited FNR motor The sequences are provided in Table 3, and non-restrictive PAL sequences are also provided herein. In that embodiment, the genetically modified bacteria of the present invention are sequence numbers 9, 10, and 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 nirB promoter fused to the ribosome binding site (SEQ ID NO: 15), strong ribosome The ydfZ promoter (SEQ ID NO: 16) is fused to the binding site, and small R is anaerobically induced. The NA gene is fnrS (fnrS1 promoter, SEQ ID NO: 9 or fnrS2 promoter). (SEQ ID NO: 17), nirB promoter fused to the CRP binding site (SEQ ID NO: 18) ), and one or more of the following: fnrS (SEQ ID NO: 19) fused to the CRP binding site. Includes.

[0093] In some embodiments, genetically engineered bacteria are represented by sequence numbers 9, 10, 11, 12. DNA sequences 13, 14, 15, 16, 17, 18, 19, or 20 or their functions Fragments and 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 approximately 99% homologous. [Table 3] [Table 4-1] [Table 4-2]

[0094] In other embodiments, one or more PMEs, for example, PAL, are transcription activators, for example, CRP. It is expressed under the control of an oxygen-level-dependent promoter fused to the binding site for C RP (Cyclic AMP receptor protein or catabolite activating protein, i.e., CAP) ) is not very beneficial when rapidly metabolizable carbohydrates such as glucose are present. By suppressing genes responsible for carbon source uptake, metabolism, and assimilation in bacteria, It plays a major regulatory role (Wu et al., 2015). This preference for glucose is, This is called glucose inhibition and carbon catabolite inhibition (Deutscher, 2 (Gorke and Stulke, 2008). In some embodiments, P ME, for example, PAL expression, is linked to an oxygen-level-dependent promoter fused to the CRP binding site. Therefore, it is controlled. In some embodiments, PAL expression is fused to the CRP binding site. It is controlled by the FNR promoter. In these embodiments, the cyclic AMP is glycated. It binds to CRP when the nucleotide is not present in the environment. This binding alters the conformation of CRP. A change is triggered, allowing CRP to bind strongly to its binding site. Then, CRP binding to the FNR promoter via direct protein-protein interaction By recruiting polymerase, the transcription of PME genes, such as the PAL gene, is activated. In the presence of glucose, cyclic AMP does not bind to CRP, but to PME, for example, PAL. Genetic transcription is suppressed. In some embodiments, the binding site for the transcription activator A fused oxygen level-dependent promoter (e.g., an FNR promoter) is, for example, G By adding glucose to the growth medium in vitro, a sufficient amount of glucose can be obtained. If present, ensure that PME, for example PAL, is not expressed under anaerobic conditions. It is used for [purpose].

[0095] In another embodiment, one or more PMEs, such as LAAD, are expressed in the presence of glucose. Inducible suppression occurs when a transcription activator, such as CRP, is fused to the binding site. It is expressed under the control of a capable promoter.

[0096] In some embodiments, LAAD is under the control of the FNR promoter. In this embodiment, LAAD is under the control of a promoter that is not an FNR promoter. AAD requires oxygen to catalyze the breakdown of phenylalanine into phenylpilbert. Therefore, under conditions where oxygen is supplied, or under hypoxic conditions where LAAD is active. In the lower region, it is desirable to induce LAAD expression.

[0097] In some embodiments, one or more PMEs, for example, PAL and / or LAAD , is expressed under the control of a promoter that is inducible in response to specific molecules or metabolites in an environment (e.g., the mammalian gastrointestinal tract). For example, short-chain fatty acid propionate is a major microbial fermentation metabolite localized in the gastrointestinal tract (Hosseini et al., 2011). In one embodiment, the expression of one or more PME genes is under the control of a promoter that is inducible by propionate. In a more specific embodiment, the PME gene expression is under the control of one or more promoters that are inducible by propionate and are activated by the presence of propionate in the mammalian gastrointestinal tract. In a healthy and / or diseased state, any molecule or metabolite found in the mammalian gastrointestinal tract can be used to induce PME gene expression. Non-limiting examples include propionate, bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma-glutamyltransferase, hepatitis antigen and antibody, alpha-fetoprotein, anti-mitochondria, smooth muscle, and anti-nuclear antibody, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese. In an alternative embodiment, the expression of PME, e.g., PAL and / or LAAD genes, is under the control of a P promoter that is activated in the presence of arabinose. In one embodiment, the LAAD expression is under the control of a P promoter. In one embodiment, the expression of LAAD occurs under aerobic or microaerobic conditions. In one embodiment, the PAL expression is under the control of a P promoter. In one embodiment, are included. In an alternative embodiment, the expression of PME, e.g., PAL and / or LAAD genes, is under the control of a P promoter that is activated in the presence of arabinose. In one embodiment, the LAAD expression is under the control of a P promoter. In one embodiment, the expression of LAAD occurs under aerobic or microaerobic conditions. In one embodiment, the PAL expression is under the control of a P promoter. In one embodiment, are included. In an alternative embodiment, the expression of PME, e.g., PAL and / or LAAD genes, is under the control of a P promoter that is activated in the presence of arabinose. In one embodiment, the LAAD expression is under the control of a P araBAD promoter. In one embodiment, the LAAD expression is under the control of a P promoter. In one embodiment, the LAAD expression is under the control of a P araBAD promoter. In one embodiment, the expression of LAAD occurs under aerobic or microaerobic conditions. In one embodiment, the PAL expression is under the control of a P promoter. In one embodiment, the PAL expression is under the control of a P araBAD promoter. In one embodiment, PAL expression occurs under aerobic or microaerobic conditions. In one embodiment, PAL expression is aerobic. LADD expression occurs under aerobic or microaerobic conditions, while 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 the promoter.

[0098] In some embodiments, one or more PME genes (e.g., PAL and / or The LAAD gene is induced by exposure to chemical and / or nutrient inducers. It is expressed under the control of a promoter. In some embodiments, one or more PMEs The gene (e.g., PAL and / or LAAD gene) is affected by exposure to tetracycline. It is expressed under the control of a promoter induced by. In some embodiments, one The above PME genes (e.g., PAL and / or LAAD genes) are arabinose It is expressed under the control of a promoter induced by exposure to. In some embodiments, In this case, one or more PME genes (e.g., PAL and / or LAAD genes) Under promoter control induced by exposure to IPTG or other LaciI inducers It is expressed in some embodiments. In some embodiments, one or more PME genes (e.g., PAL and The promoter of the LAAD gene is induced by exposure to rhamnose. It is expressed under control. In some embodiments, one or more PME genes (e.g., PA) The L and / or LAAD genes are induced by exposure to tetracycline. It is expressed under the control of a morphogenetic motor. For example, the PAL and LAAD genes are expressed, and each gene has a different promoter, for example For example, under the control of any of the promoters discussed in this paragraph and elsewhere in this specification It is expressed.

[0099] In some embodiments, one or more PME genes, e.g., PAL and / or LA The AD gene is induced by a temperature change from an unacceptable temperature to an acceptable temperature. It is expressed under control. In some embodiments, gene expression is known in the art. Methods, e.g., optimization of ribosome binding sites, manipulation of transcription regulators, and / or mR Further optimization is achieved through increased NA stability. For fine-tuning and optimization of RBS. Ioinformatics tools are well known in this field.

[0100] In any of the embodiments described above (and elsewhere in this specification) However, the manipulated bacteria have one or more gene sequences encoding Phe transporters Furthermore, it may include, and the gene sequence may be controlled by any of the promoters described herein. It is possible to be under His authority.

[0101] In some embodiments, genetically engineered bacteria express PAL in host cells. The host cells may be able to do this in vitro, for example, in culture medium, and / or in vivo, for example. For example, to survive and / or multiply in the digestive tract, a stable organism possessing the PAL gene is maintained. Contains a plasmid or chromosome. In some embodiments, genetically modified chromosomes. The bacteria contain two or more different PAL genes. In some embodiments, genetically engineered Bacteria contain multiple copies of the same PAL gene. In some embodiments, the PAL gene It is present on the plasmid and can be operably linked to a promoter that can be directly or indirectly induced. In some embodiments, the PAL gene is located on a plasmid and is hypoxic. Alternatively, it is operablely linked to a promoter induced under anaerobic conditions. Several implementations Morphologically, the PAL gene is located on the chromosome and is directly or indirectly induced by promotion. It is operably linked to the ter. In some embodiments, the PAL gene is located on the chromosome. It is operably linked to a promoter that is induced under hypoxic or anaerobic conditions. In some embodiments, the PAL gene is located on a plasmid and is exposed to tetracycline. It is operably connected to a dew-induced promoter. In some embodiments, The PAL gene resides on a plasmid and is induced by exposure to arabinose. It is operably connected to a motor. In some embodiments, the PAL gene is a plasmid. It is present above and is induced by exposure to IPTG or another LacI inducer. It is operably linked to the plasmid. In some embodiments, the PAL gene is placed on the plasmid. It is present and activatably linked to a promoter induced by exposure to rhamnose. In some embodiments, the PAL gene is located on a plasmid and is directed towards tetracycline. It is operably coupled to a promoter induced by exposure. In some embodiments, The PAL gene is located on the plasmid and reacts to temperature changes from non-permissible to permissible temperatures. It is operablely coupled to the promoter to be induced. In some embodiments, the PAL gene The offspring resides on the chromosome and is activated by a promoter induced by exposure to arabinose. It is linked to the function. In some embodiments, the PAL gene is located on the chromosome and IPTG Alternatively, it can be operably linked to a promoter induced by exposure to another LacI inducer. In some embodiments, the PAL gene is located on the chromosome and is exposed to rhamnose. It is operably coupled to a dew-induced promoter. In some embodiments, The PAL gene is located on the chromosome and is induced by exposure to tetracycline. It is operably linked to the motor. In some embodiments, the PAL gene is located on the chromosome. It is present and activatable in promoters induced by temperature changes from non-permissible temperatures to permissible temperatures. It is connected to.

[0102] In some embodiments, genetically engineered bacteria express LAAD in host cells. It may be done, and the host cells in vitro, for example in culture medium, and / or in vivo. For example, a stable organism possessing the LAAD gene that can survive and / or proliferate in the gastrointestinal tract. Includes plasmids or chromosomes maintained in some embodiments. The bacteria contain two or more different LAAD genes. In some embodiments, genetic engineering The bacteria contain multiple copies of the same LAAD gene. In some embodiments, LA The AD gene resides on a plasmid and can be directly or indirectly induced by a promoter. It is movably linked. In some embodiments, the LAAD gene is located on a plasmid. And, for example, a promoter that can be induced by arabinose or tetracycline. It is operablely linked. In some embodiments, the LAAD gene is located on the chromosome. , operably linked to a promoter that can be directly or indirectly induced. In this embodiment, the LAAD gene is located on a chromosome, for example, arabinose or tetracycline. It is operably coupled to a promoter induced by Klin. Several embodiments So, the LAAD gene is located on a plasmid and is induced by exposure to tetracycline. It is operablely linked to the promoter. In some embodiments, the LAAD gene The offspring resides on the plasmid and is produced by a promoter induced by exposure to arabinose. It is movably linked. In some embodiments, the LAAD gene is located on a plasmid. Furthermore, the promoter is induced by exposure to IPTG or other LacI inducers. It is movably linked. In some embodiments, the LAAD gene is located on a plasmid. It is activatably linked to a promoter induced by exposure to rhamnose. In some embodiments, the LAAD gene is located on a plasmid and is rated from an unacceptable temperature to an acceptable temperature. It is operably linked to a promoter that is induced by a temperature change to several degrees. In this embodiment, the LAAD gene is located on a plasmid and is activatable by a constitutive promoter. It is linked to. In some embodiments, the LAAD gene is located on a plasmid, and It is operably linked to a promoter induced by exposure to tracycline. In some embodiments, the LAAD gene is located on a chromosome and reacts to exposure to arabinose. It is operably linked to a promoter that is induced by this. In some embodiments, LA The AD gene is located on the chromosome and is activated by exposure to IPTG or other LacI inducers. It is operablely connected to the induced promoter. In some embodiments, LAAD The gene is located on the chromosome and is activated by a promoter induced by exposure to rhamnose. They are linked as possible. In some embodiments, the LAAD gene is located on the chromosome, and non It is operably connected to a promoter that is induced by a temperature change from one allowable temperature to another. In some embodiments, the LAAD gene is located on the chromosome and is constitutively promoted. It is operably connected to the tar.

[0103] This bacterial gene containing PME genes (e.g., PAL, PAH, and / or LAAD) In any of these embodiments, the bacteria encode one or more Phe transporters. It may further include a gene sequence that is a plastic Phe transporter gene sequence. It can be present on a plasmid or chromosome. The plasmid or chromosome is PME The presence of a gene can be identical or different. Phe transporter The gene sequence may be under the control of the same or different promoter as the PMR gene sequence.

[0104] In some embodiments, genetically engineered bacteria are oxygen-level-dependent transcription factors. For example, FNR, ANR, or DNR, and the corresponding promoters derived from different bacterial species. —Includes. Unnatural oxygen level-dependent transcription regulators and promoters under the same conditions. Compared to natural transcription regulators and promoters in bacteria, hypoxic or anaerobic rings At the boundary, a gene operably linked to the promoter, for example, PAL or P Increases AH transcription. In certain embodiments, unnatural oxygen level-dependent transcription factors. This is an FNR protein derived from Neisseria gonorrhoeae. (See, for example, Isabella et al., 2011). In some embodiments, The corresponding wild-type transcription regulator remains intact and retains its wild-type activity. In alternative embodiments, the corresponding wild-type transcription factor reduces wild-type activity, or It is deleted or mutated to be removed.

[0105] In some embodiments, genetically engineered bacteria exhibit wild-type oxygen-level-dependent transcriptional regulation. Nodal factors, such as FNR, ANR, or DNR, and wild-type bacteria of the same subtype. Includes the corresponding promoter which has been mutated compared to the promoter. The promoter, compared to the wild-type promoter under the same conditions, exhibits a lower oxygen or anaerobic environment. At the boundary, the binding to wild-type transcription regulators is improved, and the promoter, for example PA Increases transcription of genes operably linked to L or PAH. Several implementations In this state, genetically modified bacteria have a wild-type oxygen level-dependent promoter, for example, F NR, ANR, or DNR promoters, and wild-type transcriptional modulo derived from bacteria of the same subtype. Includes the corresponding transcription regulator that has been mutated against the nodal factor. Mutant transcription regulator The factor, compared to wild-type transcription regulators under the same conditions, exhibits improved performance in hypoxic or anaerobic environments. This improves binding to the wild-type promoter, for example, PAL or P It increases the transcription of genes operably linked to AH. In certain embodiments, mutations Different oxygen-level-dependent transcription factors enhance dimerization and FNR activity. It is an FNR protein that includes acid substitution (see, for example, Moore et al., 2006). ).

[0106] In some embodiments, the genetically engineered bacteria of the present invention regulate oxygen level-sensing transcription. This includes multiple copies of endogenous genes that encode factors, such as the FNR gene. In this embodiment, the gene encoding the oxygen level sensing transcription regulator is located on a plasmid. In some embodiments, the gene encoding the oxygen level sensing transcription regulator and P The gene encoding AL resides on a different plasmid. In some embodiments, acid The gene encoding the elementary level detection transcription regulator and the gene encoding PAL are the same It is present on rasmid. In some embodiments, it encodes an oxygen level sensing transcription regulator. The genes that perform this function are located on the chromosome. In some embodiments, oxygen level sensing transcription regulators The genes that code for the offspring and the genes that code for PAL are located on different chromosomes. In some embodiments, the gene encoding the oxygen level sensing transcription regulator and PAL The genes that encode these genes reside on the same chromosome. In some cases, this improves expression stability. Therefore, expressing an oxygen level sensing transcription regulator under the control of an inducible promoter is possible. It may be beneficial. In some embodiments, the expression of transcription factors is phenylalanine-based. The expression of the gene encoding the enzyme is controlled by a different promoter. It is controlled. In some embodiments, the expression of transcription regulators is controlled by phenylalanine metabolic yeast. It is controlled by the same promoter that controls the primary expression. In some embodiments, Regulatory factors and phenylalanine metabolic enzymes are transcribed in a wide variety of ways from the promoter region.

[0107] In some embodiments, the genetically engineered bacteria of the present invention are the same subtype under the same conditions. Compared to unmodified bacteria, blood phenylalanine levels were at least approximately 1.5 times higher, and at least approximately 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, less At least 7 times, at least 8 times, at least 9 times, at least 10 times, and at least Also about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, or less At the very least, to reduce it by approximately 50 times, extrinsic environmental conditions such as the hypoxic environment in the digestive tract of mammals It produces PAL under certain conditions. In some embodiments, the genetically modified bacteria of the present invention PAL is produced under exogenous environmental conditions such as a hypoxic environment in the digestive tract of mammals, and under the same conditions Compared to unmodified bacteria of the same subtype, the amount of hippuric acid in the urine was at least about 1.5 times higher, at least Approximately twice, at least three times, at least four times, at least five times, at least six times, At least approximately 7 times, at least approximately 8 times, at least approximately 9 times, at least approximately 10 times, less Approximately 15 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, or It increases by at least approximately 50 times. Certain unmodified bacteria reach detectable levels of phenylalanine. They would not have the ability to process nin into hippuric acid. Using genetically modified versions of these bacteria In the embodiment used, PAL-mediated processing of phenylalanine was performed under exogenous environmental conditions. It can be detected.

[0108] In some embodiments, the genetically engineered bacteria of the present invention are in vitro bacteria It produces PAL under exogenous environmental conditions such as culture conditions, and under the same conditions, it produces the same subtype of unmodified PAL. Compared to bacteria, the trans-cinnamic acid in the culture medium is at least about 1.5 times, and at least about 2 times. double, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, less At least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least Approximately 15 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, or less It increases by at least 50 times. Phenylalanine is used by methods known in the field, 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 field to evaluate it. Cinnamic acid Raw phenylalanine is directly correlated with phenylalanine degradation, and in some embodiments, cinnamic acid is strain It can be used as an alternative biomarker for activity (Figure 16B). Cinnamic acid is used in liver yeast It can be further broken down into hippuric acid by the element, and both can be measured as described in Examples 24-26. It is possible. As shown herein, in vivo TCA rapidly converts hippurin It is converted into hippuric acid, which then accumulates in the urine. Therefore, the amount of hippuric acid in the blood and especially in the urine is significant. Uric acid is a far superior biomarker for phenylalanine degradation in vivo. It is possible. In some embodiments, PAL expression is performed by methods known in the art, for example. It is measured by measuring phenylalanine levels in the blood. Hippuric acid is used in the examples described herein. and can be measured by methods known in the field.

[0109] In some embodiments, the genetically engineered bacteria of the present invention are the same subtype under the same conditions. Compared to unmodified bacteria, blood phenylalanine levels were at least approximately 1.5 times higher and at least approximately 2 times higher. double, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 6 times, less At least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least Approximately 15 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, or less At best, it produces LAAD to reduce the level by approximately 50 times. Certain non-recombinant bacteria can be detected. They lack phenylalanine processing at a certain level. These genetically modified bacteria are used. In the embodiment used, LAAD-mediated processing of phenylalanine is performed under exogenous environmental conditions. It can be detected below. Phenylalanine can be detected by methods known in the art, for example, by blood sampling. It can be measured by mass spectrometry. Pyruvate is a degradation product produced by LAAD. And phenylpyrubate was analyzed using mass spectrometry as described in Examples 24-26 It can be measured and used as further readout information for LAAD activity.

[0110] In some embodiments, the genetically engineered bacteria of the present invention are used in vivo or i Under exogenous environmental conditions such as in vitro bacterial culture conditions, two or more PMEs, for example, P It produces AL, PAH, and / or LAAD, and under the same conditions as unmodified bacteria of the same subtype. Compared to that, 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, less At least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least At least 30 times, at least 40 times, or at least 50 times, the amount of phenylalanine in the blood. These embodiments reduce nin and / or increase trans-cinnamic acid in the culture medium. In either case, the bacteria encode one or more Phe transporter polypeptides. It may further include the gene sequence that does this.

[0111] In some embodiments, one or more PMEs, for example, PAL, LAAD, and / or Alternatively, PAH is expressed on a low-copy plasmid. In some embodiments, low-copy Plasmids may be useful for increasing expression stability. In some embodiments, Low-copy plasmids may be useful in reducing expression leaks under non-inducible conditions. In some embodiments, one or more PMEs, for example, PAL, LAAD, and / or P AH is expressed on high-copy plasmids. In some embodiments, high-copy plasmids Mid increases PME expression, such as PAL, LAAD, and / or PAH. It may be useful for increasing phenylalanine metabolism, thereby increasing phenylalanine levels. It reduces blood smegma. In some embodiments, PME expressed on high-copy plasmids For example, genetically modified bacteria including PAL, LAAD, and / or PAH are different In the absence of further copies of species pheP and natural pheP, on low-copy plasmids Genes that express the same PME, e.g., PAL, LAAD, and / or PAH. Compared to manipulated bacteria, either the bacteria do not increase phenylalanine metabolism or phenylalanine metabolism. It does not reduce nin levels. The same PME gene(s) on high and low copy plasmids. For example, genetically modified PAL, LAAD, and / or PAH genes(s) Bacteria were generated. For example, PAL1 on high-copy plasmids and low-copy plasmids. Either PAL3 or PAL3 is produced, and each is metabolized to produce phenylalanine at a similar level. It was reduced to (Figure 15). Therefore, in some embodiments, phenylalanine The rate-limiting step in metabolism is the availability of phenylalanine (see, for example, Figure 16). In these embodiments, the transport of phenylalanine to cells is increased, thereby increasing the phenylalanine transport. Improving rualanine metabolism may be beneficial. In combination with pheP, low copy PAL Even plasmids can be almost completely removed from test samples (see, for example, Figure See 16A). Furthermore, in some embodiments that incorporate pheP, high Transformed bacteria with improved PAL expression stability while maintaining phenylalanine metabolism. To reduce the negative selective pressure mentioned above, use a low-copy PAL expression plasmid in combination. This may be even more beneficial. In an alternative embodiment, phenylalanine transporter It is used in combination with high-copy plasmids.

[0112] In some embodiments, the transporter increases phenylalanine degradation. This is not possible. For example, in Proteus mirabilis LAAD, enzyme catalysis occurs in the surrounding material. It is localized to the cell membrane. Phenylanine is transported through the outer membrane without the need for transporters. It can be easily crossed. Therefore, the implementation of genetically modified bacteria expressing LAAD Morphologically, transporters do not necessarily require phenylalanine metabolism, or This does not need to be improved.

[0113] In some embodiments, PME(s), e.g., PAL, LAAD, and / or Alternatively, the PAH gene is expressed on the chromosome. In some embodiments, expression from the chromosome This may be useful in increasing the stability of PME expression. In some embodiments, P ME genes, such as PAL, LAAD, and / or PAH genes, are genetically modified. It is incorporated into the bacterial chromosome at one or more integration sites in the bacteria. In some embodiments, PME genes, such as PAL, LAAD, and / or PA, are used. The H gene is located at the following insertion sites in *E. coli* Nissle: malE / K, insB / I araC / BAD, lacZ, agal / rsml, thyA, and malP / T It is inserted into the bacterial genome in one or more of these locations. Any suitable insertion site is used. It may be inserted in a different location (see, for example, Figure 66). The insertion site may be within the genome, for example, (nutritionally required). (To create a strain) genes necessary for survival and / or proliferation, such as thyA Within the genome, within the active regions of the genome such as near genome replication sites, and / or arabinose opioid To reduce the risk of unintended transcription, such as between AraB and AraC of Ron, It may be located anywhere between the branch promoters. In some embodiments, PME A gene, for example, more than one copy of PAL, PAH, and / or LAAD, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies However, in one or more integration sites in genetically modified bacteria, the bacterial chromosome It is incorporated. More than one copy of the PME gene is more than one copy of the same PME gene. It may be a copy or more than one copy of a different PME gene.

[0114] Exemplary constructs are shown in 4–13 below. Table 4 shows Phe for chromosome insertion. Sequence of an exemplary construct containing genes encoding P and FNR promoter sequences (sequence) Number 21) is shown, the pheP sequence is underlined, and the FNR promoter sequence is in bold. Table 5 shows the results of coding the PAL1 and FNR promoter sequences on high-copy plasmids. The sequence of an exemplary construct containing the gene (SEQ ID NO: 22) is shown, and the PAL1 sequence is as follows: Lines are drawn, and the FNR promoter sequence is in bold. Table 6 shows the results on high-copy plasmids. The arrangement of exemplary constructs including genes encoding the PAL3 and FNR promoter sequences The column (sequence number 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 distribution on high-copy plasmids. The sequence of an exemplary construct containing the gene encoding the sequence (SEQ ID NO: 24) is shown, and PAL1 Columns are underlined, and Tet promoter sequences are in bold. Table 8 shows high copy plastics. Exemplary structures including genes encoding PAL3 and Tet promoter sequences on Smid The sequence of structures (sequence number 25) is shown, with the PAL3 sequence underlined, and Tet Promo The tracer sequence is shown in bold. Table 9 shows PAL1 and FNR promo on low copy plasmids. The sequence of an exemplary construct containing the gene encoding the vector sequence (SEQ ID NO: 26) is shown, P The AL1 sequence is underlined, and the FNR promoter sequence is in bold. Table 10 shows low Contains genes encoding PAL3 and FNR promoter sequences on a copy plasmid. An example construct sequence (sequence number 27) is shown, with the PAL3 sequence underlined, F NR promoter sequences are shown in bold. Table 11 shows PAL1 and on low-copy plasmids. Sequence of an exemplary construct containing the gene encoding the Tet promoter sequence (SEQ ID NO: 28) The diagram shows the PAL1 sequence, with the PAL1 sequence underlined and the Tet promoter sequence in bold. Table 12 encodes the PAL3 and Tet promoter sequences on low-copy plasmids. The sequence of an example construct containing the gene (SEQ ID NO: 29) is shown, with the PAL3 sequence underlined. Table 13 shows the genes encoding pheP. The offspring, the gene encoding TetR, and the Tet promoter for insertion into the chromosome An example construct sequence (sequence number 30) including columns is shown, with the pheP sequence underlined. The TetR sequence is enclosed in a square, 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 bacteria are one of the cells in SEQ ID NOs. 21-30. It contains a gene sequence that includes one or more of the following sequences. In some embodiments, the gene The manipulated bacteria have at least 75% of one of the sequences from sequence numbers 21-30. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, A legacy comprising one or more sequences having 96%, 97%, 98%, or 99% identity. It contains a genetic sequence.

[0125] Phenylanine transport PAL1 and PAL3, respectively, exhibit high efficacy in genetically modified E. coli Nissle. It was expressed on P-plasmids and low-copy plasmids. Surprisingly, each construct The substance metabolizes and reduces phenylalanine to a similar level (Figure 15), and phenylalanine The rate-limiting step in phenylalanine metabolism was the availability of phenylalanine (Figure 16). Therefore, In some embodiments, the transport of phenylalanine to cells is increased, thereby increasing the phenylalanine transport. Improving rualanine metabolism is beneficial. Unexpectedly, with low-copy PAL plasmids... Even when expressed in combination with pheP, it is possible to almost completely remove Phe from the test sample. This can be achieved (Figure 16A). Furthermore, it is possible to maintain high phenylalanine metabolism while stabilizing PAL expression. To improve quality and reduce negative selective pressure on transformed bacteria, use in combination with pheP. Using a low-copy PAL expression plasmid may be even more beneficial. Alternative implementation Morphologically, phenylalanine transporters are used in combination with high-copy plasmids. ru.

[0126] Genetically modified bacteria have the gene that codes for the phenylalanine transporter. It also contains. Phenylalanine transporters improve the transport of phenylalanine into cells. To achieve this, the genetically modified bacteria of the present invention may be expressed or modified obtain.

[0127] PheP is a membrane transport protein that can transport phenylalanine to bacterial cells (for example) See Pi et al., 1991. In some embodiments, the genetically modified organisms of the present invention The naturally occurring pheP gene in bacteria is not modified. In some embodiments, The genetically modified bacteria of the invention contain multiple copies of the natural pheP gene. In the embodiment, the genetically engineered bacteria of the present invention have multiple copies of the non-natural pheP gene. Includes. In some embodiments, the genetically modified bacteria of the present invention have their natural promo Promoters, induceable promoters, promoters stronger than natural promoters, for example GlnRS promoter or P(Bla) promoter, or constitutive promoter It includes the pheP gene controlled by. In some embodiments, the pheP gene Expression is the expression of genes encoding phenylalanine metabolic enzymes and / or transcription regulators. It is controlled by a promoter that controls the current state and a different promoter. Several implementations In this state, pheP gene expression is related to phenylalanine metabolic enzymes and / or transcriptional regulators. It is controlled by the same promoter that controls the expression of the offspring. In some embodiments, p heP genes and phenylalanine metabolic enzymes and / or transcriptional regulators are promo It is transferred in various ways from the ter region. In some embodiments, PheP, phenylalanine The expression of each gene encoding metabolic enzymes and transcription regulators is influenced by different promoters. —is controlled by. In some embodiments, PheP, a phenylalanine metabolizing enzyme. The expression of genes encoding transcription factors is controlled by the same promoter. ru.

[0128] In some embodiments, the native pheP gene in genetically modified bacteria is modified. Furthermore, one or more additional copies of the natural pheP gene control PAL expression. The expression of the same inducible promoter, such as the FNR promoter or PAL, is controlled. Control of a promoter that is different from the controlled promoter, or control of a constitutive promoter. It is inserted into the genome below. In alternative embodiments, the native pheP gene is not modified. Copies of the non-native pheP gene from different bacterial species control the same induction of PAL expression. A promoter that can be used, such as the FNR promoter, or a promoter that controls PAL expression. Generators under the control of a different inducible promoter, or a constitutive promoter It is inserted into the body.

[0129] In some embodiments, the native pheP gene in genetically modified bacteria is modified. Furthermore, one or more additional copies of the natural pheP gene are present on the plasmid, PA The expression of L can be controlled by the same inducible promoter, such as the FNR promoter, or A promoter that controls PME expression and a different inducible promoter, or a constitutive promoter It exists in bacteria under the control of a morphogenetic. In an alternative embodiment, the native pheP gene is modified. Unadorned, non-natural copies of the pheP gene from different bacterial species are present on the plasmid, P The same inducible promoter that controls AL expression, for example, the FNR promoter, or This involves a promoter that controls PAL expression and a different inducible promoter, or a constitutive promoter. It exists in bacteria under the control of a promoter.

[0130] In some embodiments, the native pheP gene is mutagenic, and phenylalanine A mutant showing increased transport was selected, and the mutagenesized pheP gene was isolated. It is then inserted into genetically modified bacteria (e.g., Pi et al., 1996; Pi et al., 1996) (See Year 8). The phenylalanine transporter modifications described herein are It may be present on rasmids or on chromosomes.

[0131] In some embodiments, the genetically engineered bacterium is E. coli Nissle, and E. coli The natural pheP gene in Nissle is not modified, and one or more further genes The Nissle pheP gene in the naturally occurring E. coli pheP controls the expression of PAL, as does the same induction. Possible promoters, such as the FNR promoter, or a promoter that controls PAL expression. E. coli under the control of a motor-inducible promoter or a constitutive promoter different from the motor. It is inserted into the Nissle genome. In an alternative embodiment, in E. coli Nissle... The natural pheP gene is unmodified, while copies of the non-natural pheP gene from different bacteria are present. However, the same inducible promoter that controls PAL expression, for example, the FNR promoter, Alternatively, a promoter that can induce PAL expression but is different from the promoter that controls PAL expression, Under the control of a constitutive promoter, it is inserted into the *E. coli* Nissle genome. In this embodiment, the genetically modified bacterium is E. coli Nissle, and E. coli Nissle The natural pheP gene in this case is not modified, and one or more further copies of the natural gene The E. coli Nissle pheP gene controls the expression of PAL on the plasmid. Control the expression of reducible promoters, such as the FNR promoter or PAL. A promoter that is different from the promoter, or a constitutive promoter under its control. It is present in bacteria. In an alternative embodiment, the native pheP gene in *E. coli* Nissle. It is not modified, and copies of the non-natural pheP gene from different bacteria are present on the plasmid. If the same inducible promoter controls PAL expression, for example, the FNR promoter, or a promoter that controls PAL expression and a different induceable promoter or configuration It exists in bacteria under the control of a specific promoter.

[0132] In other embodiments, one or more genes encoding Phe transporters may be present. (The group) may be located on a plasmid or chromosome, and its gene expression is as specified herein. It can be controlled by any promoter that is disclosed, and that promoter is PM The promoter that regulates the E gene(s) must be the same as or different from the promoter. but can.

[0133] E. coli has five different transport systems for accumulating aromatic amino acids (AroP, Mtr, It has been reported that it possesses PheP, TnaB, and TyrP. aroP gene The common amino acid permease encoded by contains three amino acids, including phenylalanine. It transports aromatic amino acids with high affinity and, together with PheP, significantly increases phenylalanine uptake. It is thought to play a role in this. Furthermore, the accumulation of low levels of phenylalanine is thought to be related to aromatic amino acids. Acid transporter-deficient E. coli strain (ΔaroP ΔpheP Δmtr Δtna Δ Observed in tyrP), the activity of the LIV-I / LS system was traced, and its LIV-I The / LS system consists of two peripheral-binding proteins, LIV-binding protein (LIV-I system). ) and LS-binding protein (LS system), as well as membrane components and branching consisting of LivHMGF It is a chain amino acid transporter (Koyanagi et al., and the references within it; I dentification of the LIV-I / LS System as the Third Phenylalanine Transporter in E Scherichia coli K-12).

[0134] In some embodiments, genetically engineered bacteria contain the aroP gene. In this embodiment, the genetically modified bacterium is E. coli Nissle, and E. coli Nissle The natural aroP gene in this case is not modified, and the natural E. coli Nissle aroP gene One or more further copies of the offspring express PME on the plasmid or chromosome. Control the same inducible promoter, such as the FNR promoter or araB promoter. AD promoter, promoter that controls PME expression, and different inducible promoters - or present in bacteria under the control of a constitutive promoter. In an alternative embodiment, *Escherichia coli*. The native aroP gene in Nissle is not modified, and the non-native aroP gene from different bacteria is not modified. Copies of the roP gene, either on a plasmid or in a chromosome, control the expression of the same PME. Inducible promoters, such as the FNR promoter or the AraBAD promoter. , or a promoter that controls PME expression and is different from an inducible promoter, or It exists in bacteria under the control of a constitutive promoter.

[0135] In other embodiments, genetically engineered bacteria are the same or different inducible or constituent Under the control of the promoter, it includes AroP and PheP.

[0136] In some embodiments, the pheP gene is expressed on the chromosome. In this state, chromosomal expression may be useful in increasing the stability of pheP expression. In some embodiments, the pheP gene is one or multiple in genetically engineered bacteria. It is incorporated into the bacterial chromosome at several integration sites. In some embodiments, The eP gene is found at the following insertion sites in *E. coli* Nissle: malE / K, insB / I, araC / BAD, lacZ, agal / rsml, thyA, and malP / T It is inserted into the bacterial genome in one or more of the following locations. Any suitable insertion site is used. It may be used (see, for example, Figure 66). The insertion site is within the genome, for example, ( (In order to create a strain that requires nutrients) genes necessary for the survival and / or propagation of thyA, etc. Within genes, within the active regions of the genome such as near genome replication sites, and / or arabinose To reduce the risk of unintended transcription, such as between AraB and AraC in the operon. It can be located anywhere between the branching promoters.

[0137] In some embodiments, genetically engineered bacteria are transported by one or more Phe transporters —codes the Phe transporter, and the Phe transporter directly or indirectly before in vivo administration. Pre-inducible (for example, during the production of the strain before in vivo administration, in flasks, fermenters) or induction in response to specific molecules or metabolites supplied to the culture medium in other culture vessels (Expressed under the control of the sex promoter.)

[0138] In other embodiments, genetically engineered bacteria possess one or more Phe transporters. The Phe transporter is then induced directly or indirectly upon in vivo administration. (For example, response conditions in an exogenous in vivo environment (e.g., the gastrointestinal tract)) Alternatively, it may be expressed under the control of an inducible promoter for a specific molecule or metabolite. In some embodiments, the promoter is a molecule or hypoxic state specific to the gastrointestinal tract. It is induced by. In some embodiments, the bacterial strain is chemical and / or nutrient-based. It is administered in combination with an inducer.

[0139] In some embodiments, genetically engineered bacteria have multiple mechanisms of action and / or one Or it may include multiple nutritional requirements. In certain embodiments, bacteria have different combinations of chromosomes. Inclusion sites (for example, malE / K, yicS / nepI, malP / T, agaI / rs) Oxygen level-dependent promoters (e.g., P) inserted in mI and cea) f nrS -PAL3) controls five copies of PAL, and different incorporations on the chromosome. An oxygen level-dependent promoter (e.g., lacZ) is inserted at a site (e.g., P fnrS Under the control of -pheP, one of the phenylalanine transporter genes They are genetically engineered to contain pea. In a more specific embodiment, the bacteria are genetically engineered to have kanamycin resistance. Genetically engineered to include the gene and further thyA nutritional requirement, the thyA gene It is deleted and / or replaced with an unrelated gene.

[0140] Oxygen level-independent inducible promoter In some embodiments, genetically engineered bacteria are induced via an arabinose induction system. Contains one or more possible gene sequences. The gene for arabinose metabolism is PAraBAD pro It is organized into one operon controlled by a motor, AraBAD. The D (or Para) promoter adequately meets the criteria for an inducible expression system. PAraB AD likely involves the dual suppression of AraC, which acts as both an inducer and a repressor. For its role, it provides more precise control of payload gene expression than many other systems. Furthermore, ParaBAD-based expression levels fine-tune the expression levels of the payload. To regulate it, it can be adjusted over a wide range of L-arabinose concentrations. However, Cell populations exposed to subsaturated L-arabinose concentrations consist of two types: induced and uninduced cells. They are divided into two subgroups, and which one belongs to depends on the L-arabinose transporter. Determined by differences between individual cells 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 (Vol. 78, No. 16, pp. 5831-5838). Alternatively, induced emissions from ParaBad. Currently, this is controlled by optimizing the ribosome binding site (RBS) as described herein. It can be fine-tuned. Exemplary construct (control of arabinose-inducible promoter) The construct for PAL expression shown below is Figure 62C.

[0141] In one embodiment, one or more PMEs (e.g., PAL and / or LAAD), and and / or Phe transporters (e.g., PheP), and / or transcription regulators Expression of (for example, FNRS24Y) is controlled by one or more arabinose-inducible promoters. This is driven directly or indirectly. In one embodiment, PAL expression is driven by arabinose It is driven directly or indirectly by an inductive promoter. In one embodiment, Phe P expression is driven directly or indirectly by an arabinose-inducible promoter. In one embodiment, LAAD expression is directly induced by an arabinose-inducible promoter. Or it is driven indirectly. In one embodiment, the expression of FNRS24Y is arabinose-induced. It is driven directly or indirectly by a conductive promoter.

[0142] In another embodiment, one or more arabinose-inducible promoters are used, along with one or more bis- Drives the expression of stronic messages. Arabinose-induced biscyst A radio message is sent from 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 It may include transcription regulators (e.g., FNRS24Y). Such bicistronic compounds Non-restrictive examples of the message include PAL and PheP, PAL in both directions. and LAAD, PAL and FNRS24Y, PheP and LAAD, PheP and This includes FNRS24Y, LAAD and FNRS24Y. Bi-Cistronic Mechanism Sage also has two transcripts of the same gene, for example, PAL-PAL and LAAD-LA AD, PheP-PheP and / or FNRSY24S-FNRSY24S are included. Non-exclusive examples of bicistronic messages are described herein, for example, arrays. Para-FNRS24Y-LAAD is included in number 73.

[0143] In one embodiment, one or more arabinose-inducible promoters are tricistronic promoters It drives the expression of message. Tricistronic message induced by arabinose. The page 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 transcription regulators ( For example, it can include FNRS24Y. Such a tricistronic message For example, (1) transcription of three messages from the same gene; (2) one of the first genes (3) The message of the first gene, and the two messages of the second gene; (3) the three messages of three different genes It may include a transcription of one or more messages. One or more PMEs (e.g., PAL and / (or LAAD) and / or any combination of PheP, tricistronic me It can be included in the message. An unrestricted example of a tricistronic message is: As detailed in the manual, for example, PAL-PAL-PheP contained in sequence number 95 (bold) It is included.

[0144] In one embodiment, one or more arabinose-inducible promoters are used in a multi-cistrone Drives the expression of the message. Multi-cistronic induced by arabinose. The message is from one or more PMEs (e.g., PAL or LAAD) and / or one The above Phe transporters (e.g., PheP) and / or one or more transcriptional regulators It can include factors (e.g., FNRS24Y). Such multi-cistronic A message is, for example, (1) the transcription of several messages from the same gene; 2) the One or more messages from gene 1, and one or more messages from gene 2; also (3) transcription of one or more messages from one or more different genes (DN Arranged on A like beads on a string. ). One or more PMEs (e.g., PAL or LAAD) and / or one or more Ph e-transporters (e.g., PheP) and / or one or more regulatory factors (e.g., Any combination of FNRS24Y can be included in a multi-cistronic message.

[0145] In some embodiments, the arabinose-inducible promoter is one or more PMEs (e.g., For example, PAL and / or LAAD) and / or Phe transporters (for example) in vivo analysis of PheP and / or transcription regulators (e.g., FNRS24Y) It is useful for expression or is induced during in vivo expression. In some embodiments, , one or more PMEs and / or Phe transporters (e.g., PheP) and / or the expression of a transcription regulator (e.g., FNRS24Y) induces one or more arabinose induction It is driven directly or indirectly in vivo by a conductive promoter. In some embodiments, the promoter is co-administered with the genetically modified bacteria of the present invention. It is directly or indirectly induced by the donated 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 The expression of the factor (e.g., FNRS24Y) is observed in vitro before in vivo administration. During the growth, preparation, or manufacture of the strain, one or more arabinose-inducible promoters are used. It is driven directly or indirectly. In some embodiments, arabinose-inducible pro The motor grows during cultivation, for example, in a flask, fermenter, or another suitable culture vessel. It is induced inside. In some embodiments, before administration, the expression is induced to the bacterial payload. Molecules added to bacterial cultures for preloading, such as arabinose, The promoter is induced directly or indirectly. In some embodiments, arabino The culture induced by is grown aerobically. In some embodiments, Arabic The cultures induced by North are grown anaerobically.

[0147] In one embodiment, the arabinose-inducible promoter is a second promoter (for example, In conjunction with a second constitutive or inducible promoter, one or more PMEs (e.g., PA L and / or LAAD) and / or Phe transporter (e.g., PheP ) and / or the expression of a construct containing a transcription regulator (e.g., FNRS24Y) In some embodiments, two promoters are located proximal to the construct, and their expression is controlled. The arabinose-inducible promoter drives expression under the first set of exogenous conditions. The second promoter drives expression under the second set of exogenous conditions. In a non-limiting example, The first and second conditions are two consecutive culture conditions (i.e., flask, fermenter or During the preparation of cultures in other suitable culture vessels, for example, arabinose and IPT G) is possible. In another non-limiting example, the first induction condition may also be the culture condition (for example) The second induction condition may be an in vivo condition (including the presence of arabinose). Such in vivo conditions include hypoxia, microaerophilic or anaerobic conditions, and gastrointestinal conditions. This includes the presence of metabolites and / or metabolites administered in combination with bacterial strains. In some embodiments, one or more arabinose promoters express the same gene sequence. In conjunction with the FNR promoter that drives it, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporter (e.g., PheP) and / Alternatively, it drives the expression of transcription factors (e.g., FNRS24Y).

[0148] In some embodiments, the arabinose-inducible promoter is as described herein. Low-copy plasmids or high-copy plasmids or Biosafety System Plus From Mid, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcription regulators (e.g., F It drives the expression of NRS24Y). In some embodiments, arabinose-inducible promo The filter extracts one or more PMEs (e.g., PAL) from structures incorporated into bacterial chromosomes. (and / or LAAD) and / or Phe transporter (e.g., PheP) It drives the expression of a transcription factor (e.g., FNRS24Y). (Example insertion) The parts 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 The factor (e.g., FNRS24Y) is knocked into the arabinose operon, and natural arabinose Driven by a North-inducible promoter. In some embodiments, FNRS24 Y is knocked into the arabinose operon and, by the natural arabinose-inducible promoter... It is driven by arabinose. In some embodiments, FNRS24Y-LAAD is arabinose It is knocked into Perone and driven by a natural arabinose-inducible promoter.

[0150] In some embodiments, genetically engineered bacteria are associated with any of the sequences in 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 includes one or more gene sequences having 99% identity. In some embodiments, The arabinose-inducible construct further contains the gene encoding AraC, and AraC is, One or more PMEs (e.g., PAL or LAAD), and / or one or more Phe Transporters (e.g., PheP), and / or one or more regulatory factors (e.g., It is transcribed in various ways from the same promoter as FNRS24Y). In some embodiments, Genetically modified bacteria have at least 80%, 81%, of any of the sequences in sequence number 66. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Having 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity It contains one or more gene sequences. In some embodiments, the genetically engineered bacteria A polypeptide encoded by any of the sequences in sequence number 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 contains one or more gene sequences that encode polypeptides of the same identity.

[0151] In some embodiments, genetically engineered bacteria can be induced by a rhamnose induction system. It contains one or more gene sequences that can perform the rhaBAD gene, which is the rhaBAD promoter. —It is organized into a single operon controlled by. The rhaP BAD promoter is It is regulated by two activators, RhaS and RhaR, and the corresponding gene is Rha It belongs to a single transcription unit that branches off in the opposite direction from BAD and is transcribed. (Presence of L-rhamnose) Below, RhaR binds to the rhaP RS promoter, generating RhaR and RhaS. It activates RhaS, then together with L-rhamnose, rhaP BAD and rh It binds to the aP T promoter and activates the transcription of structural genes. AraC is supplied, In contrast to the arabinose system, which is transcribed by branching in the gene sequence, the rhamnose expression system Furthermore, even on multicopy plasmids, the transcription from chromosomes is activated to such an extent that it can be activated. Since the expression level is sufficient, there is no need to express more of the regulatory protein. Therefore Only the rhaP BAD promoter is cloned upstream of the gene that should be expressed. Complete induction of rhaBAD transcription is also a key regulator of catabolism inhibition, CRP -Requires binding of the cAMP complex. Alternatively, inducible expression from rhaBAD is As described in the specification, the ribosome binding site (RBS) can be controlled or refined by optimization. It can be controlled. An example structure is shown in Figure 62B (Control of a rhamnose-inducible promoter). The construct for PAL expression is shown below.

[0152] In one embodiment, one or more PMEs (e.g., PAL and / or LAAD), and and / or Phe transporters (e.g., PheP), and / or transcription regulators Expression of (for example, FNRS24Y) is mediated by one or more rhamnose-inducible promoters. It is driven directly or indirectly. In one embodiment, PAL expression is rhamnose-induced. Driven directly or indirectly by a sex promoter. In one embodiment, PheP Expression is driven directly or indirectly by a rhamnose-inducible promoter. In the application morphology, LAAD expression is directly or indirectly induced by the rhamnose-inducible promoter. It is driven indirectly. In one embodiment, the expression of FNRS24Y is rhamnose-inducible promo It is driven directly or indirectly by a motor. In non-limited examples, PAL expression is For example, by constructs containing rhamnose-inducible PAL, such as those included in Sequence ID No. 106. It is driven by.

[0153] In another embodiment, one or more rhamnose-inducible promoters are used, along with one or more biscis Drives the expression of tronic messages. Bi-cistrone induced by rhamnose The message is from one or more PMEs (e.g., PAL or LAAD) and / or One or more Phe transporters and / or one or more transcription regulators (e.g., F It may include NRS24Y), and the arabinose derivative is the same as described above.

[0154] In one embodiment, one or more rhamnose-inducible promoters are tricistronic methotrex Driving sage expression: Rhamnose-induced tricistronic messages This refers to one or more PMEs (e.g., PAL and / or LAAD) and / or Phe Transporters (e.g., PheP) and / or transcription regulators (e.g., FNRS) It may include 24Y). Tricistronic messages induced by rhamnose are One or more PMEs (e.g., PAL or LAAD) and / or one or more Pheto Contains a lanceporter and / or one or more transcription regulators (e.g., FNRS24Y) It appears that the arabinose induction is the same as described above.

[0155] In one embodiment, one or more rhamnose-inducible promoters are multi-cistronic Drives message expression. Multicistronic messaging induced by rhamnose. Sage contains one or more PMEs (e.g., PAL or LAAD) and / or one or more The Phe transporter (e.g., PheP) and / or one or more regulatory factors (e.g., For example, it may include FNRS24Y). Multi-cistrone induced by rhamnose. The message is from one or more PMEs (e.g., PAL or LAAD) and / or 1 One or more Phe transporters and / or one or more transcription regulators (e.g., FN It may include RS24Y), and the arabinose derivative is the same as described above.

[0156] In some embodiments, the rhamnose-inducible promoter is one or more PME(PA) L or LAAD) and / or one or more Phe transporters (e.g., Phe P) and / or one or more regulatory factors (e.g., FNRS24Y) in vivo It is currently useful or is induced during in vivo expression. In some embodiments, One or more PMEs (e.g., PAL or LAAD) and / or one or more Pheto Lance porters (e.g., PheP) and / or one or more regulators (e.g., FN) RS24Y) is directly induced in vivo by one or more rhamnose-inducible promoters. Driven indirectly or indirectly. In some embodiments, the promoter is a legacy of the present invention. Molecules administered co-administered with genetically modified bacteria, such as rhamnose, either directly or indirectly. It is indirectly induced.

[0157] In some embodiments, one or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or one or more The above regulatory factors (e.g., FNRS24Y) are used in vitro before in vivo administration. During the propagation, preparation, or manufacture of the strain, one or more rhamnose-inducible promoters are used. It is driven directly or indirectly. In some embodiments, rhamnose-inducible promo The culturer is growing during cultivation, for example, in a flask, fermenter, or another suitable culture vessel. It is induced. In some embodiments, before administration, the expression is induced to make the bacteria in the payload Molecules added to bacterial cultures for preloading, such as rhamnose, promote The motor is induced directly or indirectly. In some embodiments, by rhamnose The induced culture is grown aerobically. In some embodiments, induction is performed by rhamnose. The cultured material is grown anaerobically.

[0158] In one embodiment, the rhamnose-inducible promoter is a second promoter (for example, the second In conjunction with two constitutive or inducible promoters, 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 regulators (e.g., FNRS24Y) Driven. In some embodiments, two promoters are located near the structure, The construct expression is driven by the rhamnose-inducible promoter, which is expressed under the first set of exogenous conditions. The second promoter drives expression under the 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 cultures in a fermenter or other suitable culture vessel, for example, rhamnose It could be (and arabinose). In another non-limiting example, the first induction condition is the culture condition, For example, it may include the presence of rhamnose, and the second induction condition is in vivo. This could be the case. Such in vivo conditions include low oxygen, microaerophilic, or anaerobic conditions. The presence of gastrointestinal metabolites, and / or metabolites administered in combination with bacterial strains Included. In some embodiments, one or more rhamnose promoters are the same gene In conjunction with an FNR promoter that drives sequence expression, one or more PMEs and / or Phe transporters (e.g., PheP) and / or transcription regulators (e.g., F It drives the expression of NRS24Y.

[0159] In some embodiments, the rhamnose-inducible promoter is a low-copy promoter as described herein. - Plasmid, high-copy plasmid, or biosafety system plasmid Furthermore, one or more PMEs (e.g., PAL or LAAD) and / or one or more Ph e-transporters (e.g., PheP) and / or one or more regulatory factors (e.g., It drives the expression of FNRS24Y). In some embodiments, rhamnose-inducible promo The filter extracts one or more PMEs (e.g., PAL) from structures incorporated into bacterial chromosomes. (or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or drive the expression of one or more regulators (e.g., FNRS24Y). Typical insertion sites are described herein.

[0160] In some embodiments, genetically engineered bacteria are one of the sequences in sequence number 107. and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 It contains one or more gene sequences that have % or 99% identity.

[0161] In some embodiments, genetically engineered bacteria are isopropyl β-D-1-thioga Other transcriptions induced from lactopyranoside (IPTG) induction systems or Lac promoters. It contains one or more gene sequences that can be induced via a compound. IPTG is a lac operon Allolactose is a lactose metabolite that activates transcription. It is a molecular mimic of ) allolactose. In contrast to allolactose, the sulfur atoms in IPTG are not hydrolyzed. It forms a decomposing chemical bond, preventing the decomposition of IPTG and maintaining a constant concentration. IPTG is lac The tetramerized repressor is bonded to the repressor from the lac operator in an allosteric manner. It releases (lacI), thereby enabling gene transcription in the lac operon. IPTG is not metabolized by E. coli, so its concentration remains constant, and Lac Promoter-controlled expression rates are strictly controlled both in vivo and in vitro. It is controlled. Other transport routes are also involved, so IPTG intake affects lactose permease Independent of action. Inducible expression from PLac is performed as described herein, as ribosomes. The junction site (RBS) can be controlled or fine-tuned by optimization. Instead of IPTG, other compounds that inactivate LacI can be used.

[0162] In one embodiment, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcription regulators (e.g., For example, the expression of FNRS24Y) is directly induced by one or more IPTG-inducible promoters. It is driven directly or indirectly. In one embodiment, PAL expression is driven by IPTG-induced promo It is driven directly or indirectly by a motor. In one embodiment, PheP expression is It is driven directly or indirectly by an IPTG-inducible promoter. In one embodiment, LAAD expression is driven directly or indirectly by the IPTG-inducible promoter. In one embodiment, FNRS24Y expression is induced by an IPTG-inducible promoter. Driven directly or indirectly. Includes PAL under the control of an IPTG-inducible promoter. Non-exclusive examples of structures (for example, those included in Sequence ID No. 74) (see, for example, Figure 60) ).

[0163] In another embodiment, one or more IPTG-inducible promoters are provided by one or more biscythians. Drives the expression of bicistronic messages. IPTG-induced bicistronic The message is from one or more PMEs (e.g., PAL or LAAD) and / or one The above Phe transporters (e.g., PheP) and / or one or more transcriptional regulators It may include factors (e.g., FNRS24Y) and, regarding arabinose induction, as described above. It is the same as the one. In one embodiment, one or more IPTG-inducible promoters are used. Drives the expression of stronic messages. IPTG-induced tricistrone The message is from one or more PMEs (e.g., PAL or LAAD) and / or One or more Phe transporters (e.g., PheP) and / or one or more transcriptions It may include regulatory factors (e.g., FNRS24Y) and the above regarding arabinose induction. It is the same as the above. In one embodiment, one or more IPTG-inducible promoters are used Drives the expression of multi-situ cistronic messages. IPTG-induced multi-situ A stronic message is one or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or one or more It can include the above transcription regulators (e.g., FNRS24Y) and is involved in arabinose induction. This is the same as described above.

[0164] In some embodiments, the IPTG-inducible promoter includes one or more PMEs (e.g., , PAL and / or LAAD) and / or Phe transporter (e.g., P in vivo expression of heP) and / or transcription regulators (e.g., FNRS24Y) It is useful for, or is induced during in vivo expression. In some embodiments, 1 One or more PMEs and / or Phe transporters (e.g., PheP) and / or Alternatively, the expression of a transcription regulator (e.g., FNRS24Y) is associated with one or more IPTG-induced prostate problems. Driven directly or indirectly in vivo by a motor. Several implementations Morphologically, the promoter is a molecule that is administered simultaneously with the genetically modified bacteria of the present invention, for example If so, it is directly or indirectly induced by the 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 The expression of the factor (e.g., FNRS24Y) is observed in vitro before in vivo administration. During the propagation, preparation, or manufacture of the strain, one or more IPTG-inducible promoters directly It is driven directly or indirectly. In some embodiments, the IPTG-inducible promoter is During cultivation, for example, during growth in a flask, fermenter or another suitable culture vessel, In some embodiments, the bacteria are preloaded with the payload by inducing expression before administration. Molecules added to bacterial cultures to control the promoter, such as IPTG, allow the promoter to function. It is induced directly or indirectly. In some embodiments, it is induced by IPTG. The culture is grown aerobically. In some embodiments, induction by IPTG The cultured material is grown anaerobically.

[0166] In one embodiment, the IPTG-inducible promoter is a second promoter (for example, a second In conjunction with a constitutive or inducible promoter, one or more PMEs (e.g., PAL) (and / or LAAD) and / or Phe transporter (e.g., PheP) It drives the expression of constructs containing and / or transcription regulators (e.g., FNRS24Y). In some embodiments, two promoters are located near the structure, and the structure The IPTG-inducible promoter drives expression under the first set of exogenous conditions. The second promoter drives expression under the external conditions of the second set. Non-restrictive example So, the first and second conditions are two consecutive culture conditions (i.e., flask, fermenter). Or during the preparation of cultures in other suitable culture vessels, for example, arabinose and It could be IPTG). In another non-limiting example, the first induction condition is the culture condition (e.g., (including the presence of IPTG), and the second induction condition was in vivo conditions. Such in vivo conditions may include hypoxic, microaerophilic, or anaerobic conditions. The presence of gastrointestinal metabolites, and / or metabolites administered in combination with bacterial strains. In some embodiments, one or more IPTG-inducible promoters are linked to the same gene. In conjunction with an FNR promoter that drives sequence expression, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporter (e.g., PheP) and / or drive the expression of transcription regulators (e.g., FNRS24Y).

[0167] In some embodiments, the IPTG-inducible promoter is a low copy as described herein. From plasmids, high-copy plasmids, or biosafety system plasmids , one or more PMEs (e.g., PAL and / or LAAD) and / or Pheto Lansporters (e.g., PheP) and / or transcription regulators (e.g., FNRS2) It drives the expression of 4Y). In some embodiments, the IPTG-inducible promoter is fine From a construct integrated into the microbial chromosome, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporter (e.g., PheP) and / or It drives the expression of transcription regulators (e.g., FNRS24Y). The exemplary insertion site is shown in this specification. It will be written in the book.

[0168] In some embodiments, genetically engineered bacteria are associated with any of the sequences in 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% or comprising one or more gene sequences having 99% identity. In some embodiments, The IPTG-inducible construct further includes the gene encoding lacI, and the lacI gene is One or more PMEs (e.g., PAL or LAAD) and / or one or more Pheto Lance porters (e.g., PheP) and / or one or more regulators (e.g., FN) It is transcribed in many ways from the same promoter as R24Y). In some embodiments, the gene The manipulated bacteria had at least 80%, 81%, or 82% of any of the sequences in Sequence ID No. 75. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 1 having 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity It contains one or more gene sequences. In some embodiments, the genetically engineered bacteria have sequence numbers polypeptide encoded by any of sequence 75 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 It contains one or more gene sequences that encode polypeptides.

[0169] In some embodiments, genetically engineered bacteria are transmitted via a tetracycline induction system. Contains one or more inducible gene sequences. The first system (Tight control of gene expression in mammalian cells by tetracycline-responsi ve promoters., Gossen M & Bujard H. PNAS, 1 June 15, 992; Vol. 89(12): pp. 5547-51) is tetracycline o Known as: In the presence of tetracycline, from the tet-inducible promoter The expression of tetracycline regulatory transactivator (tTA) decreases. to the C-terminal domain of VP16 (virion protein 16) derived from herpes simplex virus It was created by fusion. In the absence of tetracycline, tTA's te The tR region binds to the tetO sequence within the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, and tTA It prevents binding to the tetO sequence. Next, the presence of tetracycline for induction rather than inhibition. A reverse Tet repressor (rTetR) based on the current technology has been developed. The activator rtTA (reverse tetracycline-regulating trans-activator) is rTe It was prepared by fusing tR and VP16. The tetracycline-one system is r It is also known as a tTA-dependent system.

[0170] In one embodiment, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcription regulators (e.g., For example, the expression of FNRS24Y is regulated by one or more tetracycline-inducible promoters. This is driven directly or indirectly. In one embodiment, the expression of PAL is tetracycline It is driven directly or indirectly by a phosphorus-inducible promoter. In one embodiment, P heP expression is directly or indirectly driven by a tetracycline-inducible promoter. In one embodiment, LAAD expression is controlled by a tetracycline-inducible promoter. Therefore, it is driven directly or indirectly. In one embodiment, the expression of FNRS24Y is driven by It is driven directly or indirectly by a tracyclin-inducible promoter.

[0171] In another embodiment, one or more tetracycline-inducible promoters are used, one or more It drives the expression of isisstronic messages. Tetracycline-induced Isistronic messages are sent to one or more PMEs (e.g., PAL or LAAD) and / or one or more Phe transporters (e.g., PheP) and / or 1 It may contain one or more transcription regulators (e.g., FNRS24Y) and arabinose induction The same applies to the above. In one embodiment, one or more tetracycline derivatives Sex promoters drive the expression of tricistronic messages. Tetracycline The tricistronic message 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 transcription regulators (e.g., FNRS24Y) The same applies to arabinose induction as described above. In one embodiment, one or more Tetracycline-inducible promoters drive the expression of multicistronic messages. Tetracycline-induced multicistronic messages are one or more The above PME (e.g., PAL or LAAD) and / or one or more Phe transformers Porter (e.g., PheP) and / or one or more transcription regulators (e.g., FNR) It may contain S24Y), and the arabinose derivation is the same as described above.

[0172] In some embodiments, the tetracycline-inducible promoter is one or more PMEs. (PAL and / or LAAD) and / or Phe transporters (e.g., P in vivo expression of heP) and / or transcription regulators (e.g., FNRS24Y) It is useful for, or is induced during in vivo expression. In some embodiments, 1 One or more PMEs and / or Phe transporters (e.g., PheP) and / or Alternatively, the expression of a transcription regulator (e.g., FNRS24Y) is associated with one or more tetracycline-induced reactions. Driven directly or indirectly in vivo by a conductive promoter. In one embodiment, the promoter is administered simultaneously with the genetically modified bacteria of the present invention. It is directly or indirectly induced 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 The expression of the factor (e.g., FNRS24Y) is observed in vitro before in vivo administration. During the growth, preparation, or manufacture of the strain, one or more tetracycline-inducible promoters Therefore, it is driven directly or indirectly. In some embodiments, tetracycline induction The conductive promoter is used during culture, for example, in a flask, fermenter, or another suitable culture vessel. It is induced during growth in [location]. In some embodiments, before administration, the expression is induced to [unclear] the bacteria. Molecules added to bacterial cultures for preloading with Irod, for example, tetracycline The promoter is induced directly or indirectly by the signal. In some embodiments, The cultures induced by tetracycline are grown aerobically. Several implementations Morphologically, tetracycline-induced cultures grow anaerobically.

[0174] In one embodiment, the tetracycline-inducible promoter is a second promoter (for example) If, in conjunction with a second constitutive or inductive promoter, one or more PMEs (e.g., PAL and / or LAAD) and / or Ph transporters (e.g., Ph The expression of constructs containing eP) and / or transcription regulators (e.g., FNRS24Y) is driven To move. In some embodiments, two promoters are located near the structure, and the structure The tetracycline-inducible promoter drives the expression of the structure under the first set of exogenous conditions. The first promoter drives expression, and the second promoter drives expression under the second set of exogenous conditions. In limited cases, the first and second conditions are two consecutive culture conditions (i.e., F During preparation of cultures in a fermenter or other suitable culture vessel, for example, teto This could be lacyclin and IPTG. In another non-limiting example, the first induction condition The first induction condition is under culture conditions (e.g., the presence of tetracycline), and the second induction condition is in v These could be in vivo conditions. Such in vivo conditions include hypoxia, microaerophilia, or Anaerobic conditions, the presence of gastrointestinal metabolites, and / or administration in combination with bacterial strains The product contains a reaction product. In some embodiments, one or more tetracycline promoters It works in conjunction with an FNR promoter that drives the expression of the same gene sequence to one or more PMEs. (For example, PAL and / or LAAD) and / or Phe transporters (e.g.) For example, it drives the expression of PheP and / or transcription regulators (e.g., FNRS24Y). do.

[0175] In some embodiments, the tetracycline-inducible promoter is as described herein. Low-copy plasmids or high-copy plasmids or Biosafety System Plus From Mid, one or more PMEs (e.g., PAL and / or LAAD) and / or Phe transporters (e.g., PheP) and / or transcription regulators (e.g., F It drives the expression of NRS24Y). In some embodiments, tetracycline-inducible p The chromotor is derived from one or more PMEs (e.g., PA) from a construct integrated into the bacterial chromosome. L and / or LAAD) and / or Phe transporter (e.g., PheP ) and / or drive the expression of transcription factors (e.g., FNRS24Y). Exemplary The insertion site is described herein.

[0176] In some embodiments, genetically engineered bacteria have the bold sequence (te) of sequence number 39. (The t promoter is in bold) and at least 80%, 81%, 82%, 83%, 84% %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% One or more gene pairs having 95%, 96%, 97%, 98%, or 99% identity Includes a column. In some embodiments, the tetracycline-inducible construct is coated with AraC. It further contains genes that do, and AraC contains one or more PMEs (e.g., PAL or LAA) D), and / or one or more Phe transporters (e.g., PheP), and / Alternatively, one or more regulators (e.g., FNR24Y) are transcribed in various ways from the same promoter. In some embodiments, genetically engineered bacteria are represented by the italicized form of SEQ ID NO: 39. At least 80% of the sequence (Tet repressor is italicized) and 8 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 Identical percentages of 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% It contains one or more gene sequences having sex. In some embodiments, genetically modified bacteria This is the italicized sequence of sequence number 39 (the Tet repressor is italicized). polypeptide encoded by either and at least 80%, 81%, 82%, 83% %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 Polypeptide having 94%, 95%, 96%, 97%, 98%, or 99% identity It contains one or more gene sequences that encode cide.

[0177] In some embodim...

Claims

1. a) One or more genes encoding phenylalanine ammonia lyase (PAL) It is a gene that is not naturally associated with the PAL gene and can be directly or indirectly induced. A gene that is operablely linked to a promoter, b) One or more genes encoding a phenylalanine transporter In nature, it is not associated with the phenylalanine transporter gene, either directly or indirectly. A gene that is operablely linked to a promoter that can be indirectly induced, c) One or more encoding variant fumarate and nitrate reductases (FNRs) The gene is one that is not naturally associated with the FNR gene and is directly or indirectly induced. Genes that are operablely linked to possible promoters and Genetically modified bacteria, including [specific organisms].

2. One or more genes encoding L-amino acid deaminase (LAAD) , a promo that is not naturally associated with the LAAD gene, and can be directly or indirectly induced The genetically engineered according to claim 1, further comprising a gene operably linked to the ter Bacteria.

3. A mutant FNR can activate the FNR promoter under oxygenation conditions, claim. A genetically modified bacterium as described in 1 or 2.

4. The genetically modified bacterium according to claim 3, wherein the mutant FNR is FNRS24Y.

5. A promoter and phenylalanine operably linked to the gene encoding PAL A promoter operably linked to the gene encoding the transporter, A genetically modified molecule according to any one of claims 1 to 4, which is a separate copy of the molecule. Bacteria.

6. The gene encoding PAL and the gene encoding the phenylalanine transporter Claims 1 to 1, wherein the gene is operably linked to the same copy of the same promoter. A genetically modified bacterium as described in any of section 4.

7. The gene encoding LAAD is operably linked to the gene encoding PAL. Operablely linked to genes encoding motors and phenylalanine transporters. Claims 2 to 6, which are operably connected to a promoter different from the promoter that was selected. A genetically modified bacterium as described in any one of the following items.

8. The promoter, phenylalanine, is operably linked to the gene encoding PAL. The promoter operably linked to the gene encoding the ranin transporter - and the promoter operably linked to the gene encoding LAAD , which are directly or indirectly induced by extrinsic environmental conditions, any one of claims 2 to 7 Genetically modified bacteria as described in the section.

9. The promoter and pheny are operably linked to the gene encoding PAL. The promo operably linked to the gene encoding the lualanine transporter The mites are directly or indirectly influenced by exogenous environmental conditions found in the digestive tract of mammals. A genetically modified bacterium according to any one of claims 1 to 8, which is induced.

10. A promoter and phenylalanine operably linked to the gene encoding PAL A promoter operably linked to a gene encoding a transporter triggers the mammalian movement Directly or indirectly induced by exogenous environmental conditions found in the small intestine of an animal, claim Genetically modified bacteria as described in 9.

11. A promoter and phenylalanine operably linked to the gene encoding PAL A promoter operably linked to the gene encoding the transporter triggers a hypoxic or is directly or indirectly induced under anaerobic conditions, any one of claims 1 to 10 Genetically modified bacteria as described above.

12. A promoter and phenylalanine operably linked to the gene encoding PAL A promoter operably linked to a gene encoding an FNR transporter responds to FNR A genetically modified bacterium according to claim 11, which is a responsive promoter.

13. The aforementioned gene encoding LAAD is naturally present in the digestive tract of mammals due to environmental factors The following claims 2 to 12 are under the control of a promoter that is directly or indirectly induced A genetically modified bacterium as described in any of the items.

14. The aforementioned gene encoding LAAD is not naturally present in the digestive tract of mammals due to environmental factors. Claims 2-12, under the control of a promoter directly or indirectly induced by A genetically modified bacterium as described in any one of the following items.

15. The gene encoding LAAD is arabinose, IPTG, tetracycline, and Directly due to the presence of chemical and / or nutrient derivatives selected from rhamnose The genetically modified organism according to claim 14 is under the control of a promoter that is indirectly induced. A bacterium that has been exposed to bacteria.

16. The gene encoding the mutant FNR is arabinose, IPTG, tetracycline, Directly due to the presence of chemical and / or nutrient derivatives selected from rhamnose Or, under the control of an indirectly induced promoter, as described in any one of claims 1 to 15. Genetically modified bacteria.

17. The gene encoding the phenylalanine transporter stains the bacteria. A genetically modified bacterium located on the body, according to any one of claims 1 to 16.

18. The gene encoding the phenylalanine transporter is found in the bacteria A genetically modified bacterium according to any one of claims 1 to 16, located on a smid.

19. The gene encoding PAL is located on a plasmid in the bacterium, claim A genetically modified bacterium described in any one of items 1 to 18.

20. Claims 1 to 1, wherein the gene encoding PAL is located on the chromosome in the bacterium. A genetically modified bacterium as described in any one of item 18.

21. The gene encoding the mutant FNR is located on the plasmid in the bacterium, A genetically modified bacterium according to any one of claims 1 to 20.

22. The gene encoding the mutant FNR is located on the chromosome in the bacterium, A genetically modified bacterium as described in any one of items 1 to 20.

23. The gene encoding LAAD is located on a plasmid in the bacterium, A genetically modified bacterium as described in any one of items 2 to 22.

24. Claim 2, the gene encoding LAAD is located on a chromosome in the bacterium. A genetically modified bacterium as described in any one of items 22.

25. The gene encoding mutant FNR and the gene encoding LAAD are both promoting the same gene. A genetically modified bacterium according to any one of claims 2 to 24, under the control of a developer.

26. The promoter is derived from arabinose, IPTG, tetracycline, and rhamnose. Directly or indirectly induced by the presence of selected chemical and / or nutrient inducers. The genetically modified bacterium described in claim 25, which is derived from the bacterium.

27. a) Two or more genes encoding phenylalanine ammonia lyase (PAL) There are progenitor genes that are not naturally associated with the PAL gene, and which can be directly or indirectly induced. Genes that are operablely linked to the motor, b) One or more genes encoding a phenylalanine transporter In nature, it is not associated with the phenylalanine transporter gene, either directly or indirectly. A gene that is operablely linked to a promoter that can be indirectly induced and Genetically modified bacteria, including [specific organisms].

28. The aforementioned chemical and / or nutrient derivatives include arabinose, IPTG, and tetracycline. A genetically modified bacterium according to claim 27, selected from phosphorus and rhamnose.

29. At least two genes encoding PAL are found in the exogenous digestive tract of mammals. Operablely connected to a directly or indirectly inducible promoter induced by environmental conditions. A genetically modified bacterium according to claim 27 or 28.

30. A promoter operably linked to at least two genes encoding PAL, In the small intestine of mammals, directly or indirectly induced by exogenous environmental conditions, The genetically modified bacterium according to claim 29.

31. A promoter operably linked to at least two genes encoding PAL, The hereditary gene according to claim 30, which is directly or indirectly induced under hypoxic or anaerobic conditions. Manipulated bacteria.

32. A promoter operably linked to at least two genes encoding PAL and Promo operably linked to the gene encoding the phenylalanine transporter The genetically modified bacterium according to claim 11, wherein the promoter is an FNR-responsive promoter.

33. The aforementioned PAL is Anabaena variabilis. (PAL1) or Photorhabdus luminescence A legacy according to any one of claims 1 to 32, derived from inescens) (PAL3). Genetically modified bacteria.

34. Any of claims 1 to 33, wherein the phenylalanine transporter is PheP. Genetically modified bacteria as described in item 1.

35. The bacterial is a probiotic bacterium, according to any one of claims 1 to 34. Genetically modified bacteria.

36. The aforementioned bacteria include Bacteroides, Bifidobacterium, Clostridium, and Ethyl. Selected from the group consisting of the genera Helicia, Lactobacillus, and Lactococcus. A genetically modified bacterium as described in item 35.

37. The genetically modified bacterium according to claim 36, wherein the bacterium is the E. coli strain Nissle. 。

38. The aforementioned bacteria provide the nutritional needs of genes that complement the bacteria when they are present in the digestive tract of mammals. A genetically modified bacterium according to any one of claims 1 to 37, which is the strain to seek.

39. The genetically modified bacterium according to claim 38, wherein the mammalian digestive tract is the human digestive tract.

40. The aforementioned bacteria are a nutrient requirement strain for enzymes in the diaminopimelic acid or thymidine biosynthesis pathway. The genetically modified bacterium according to claim 38 or 39.

41. The bacteria further possess genes that encode substances toxic to the bacteria. The gene is manipulated and directly transmitted to the mammalian digestive tract by environmental factors that do not naturally exist in the mammalian digestive tract. Any one of claims 1 to 40, under the control of a promoter that is directly or indirectly induced. Genetically modified bacteria as described in the section.

42. A drug comprising a bacterium according to any one of claims 1 to 41 and a pharmaceutically acceptable carrier. A scientifically acceptable composition.

43. The composition according to claim 42, formulated for oral administration.

44. To reduce hyperphenylalaninemia or disease associated with hyperphenylalaninemia A method for treating a disease, wherein the composition according to claim 42 or 43 is required A method including the step of administering to a subject.

45. The aforementioned disease is phenylketonuria, classical or typical phenylketonuria, atypical phenylketonuria. Nilketonuria, persistent mild hyperphenylalaninemia, nonphenylketonuria hyperphenylalaninemia Alaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydroptase Lysine reductase deficiency, tetrahydropterin synthase deficiency, Segawa disease, and liver The method according to claim 44, selected from the group consisting of diseases.

46. A method for measuring PAL activity in a subject, wherein a PAL-based drug is used as the subject. A method comprising administering a substance to the body and subsequently measuring the amount of hippuric acid produced in the urine of the subject. 。

47. A method for monitoring the therapeutic activity of a PAL-based drug in a subject, PA An L-based drug was administered to the subjects, and subsequently, the amount of hippuric acid produced in the subjects' urine was measured. A method that includes determining something.

48. A method for adjusting the dosage of a PAL-based drug administered to a subject, wherein the PAL-based drug administered to the subject Administration of AL-based drugs, measurement of hippuric acid produced in the urine of subjects, and in subjects Increasing or decreasing the dosage of a drug in order to increase or decrease PAL activity, A method that includes this.

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