WO2023165521A1 - 一种用于治疗高苯丙氨酸血症的工程微生物及其用途 - Google Patents

一种用于治疗高苯丙氨酸血症的工程微生物及其用途 Download PDF

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WO2023165521A1
WO2023165521A1 PCT/CN2023/079034 CN2023079034W WO2023165521A1 WO 2023165521 A1 WO2023165521 A1 WO 2023165521A1 CN 2023079034 W CN2023079034 W CN 2023079034W WO 2023165521 A1 WO2023165521 A1 WO 2023165521A1
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expression cassette
gene encoding
promoter
engineered microorganism
microorganism according
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English (en)
French (fr)
Inventor
向斌
孙婉菊
刘翠翠
朱丽
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Commbio Therapeutics Co Ltd
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Commbio Therapeutics Co Ltd
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Priority to EP23762919.1A priority Critical patent/EP4446407A4/en
Priority to US18/725,311 priority patent/US20250082692A1/en
Publication of WO2023165521A1 publication Critical patent/WO2023165521A1/zh
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Definitions

  • the present disclosure relates to the field of genetic engineering, in particular to an engineered microorganism and an engineered microbial composition, a composition comprising the engineered microorganism or an engineered microbial composition, and using the engineered microorganism or engineered microbial composition to relieve and/or treat A method for a disease and/or disorder associated with hyperphenylalaninemia, wherein the engineered microorganism or engineered microorganism composition is used in the preparation of a medicament for alleviating or treating a disease and/or disorder associated with hyperphenylalaninemia Or use in health products.
  • Hyperphenylalaninemia hyperphenylalaninemia
  • HPA hyperphenylalaninemia
  • HPA hyperphenylalaninemia
  • Phe phenylalanine hydroxylase
  • Phe phenylalanine hydroxylase
  • phenylketonuria is caused by the mutation of phenylalanine hydroxylase (PAH) in the phenylalanine metabolic pathway, so that phenylalanine cannot be converted into tyrosine, resulting in phenylalanine Accumulate with ⁇ -ketoglutaric acid in blood and tissues, and excrete in large quantities in urine.
  • the content of phenylalanine in the blood of healthy people is about 100 ⁇ M. It is generally believed that when the content of phenylalanine in human blood is above 360 ⁇ M to 600 ⁇ M, there will be symptoms of phenylketonuria. In China, the incidence of phenylketonuria in newborns is about 1/11000. If a child with phenylketonuria is not diagnosed and treated in time, it will seriously affect the intellectual development of the child, causing symptoms such as epilepsy and movement disorders. And accompanied by melanin synthesis disorder.
  • the current treatment for this disease is mainly the control of food.
  • Patients with phenylketonuria can maintain serum phenylalanine levels in the range of 120-360 ⁇ M through dietary intervention or treatment, which may help alleviate the cognitive impairment associated with the disease.
  • this treatment often affects the growth of newborns or adolescents, as well as the development of fetuses in pregnant women; Diet therapy requires long-term, uninterrupted treatment, which causes a great financial burden on patients and families.
  • approved drugs for the treatment of phenylketonuria that have appeared in recent years include sapropterin hydrochloride and phenylalanine ammonia-lyase.
  • sapropterin hydrochloride cannot be used as a single treatment and needs to be used in combination with a controlled diet; phenylalanine ammonia lyase is recently approved for the treatment of adults with phenylketonuria, relying on systemic injection of phenylalanine Acid ammonia lyase (PAL), however, this therapy is often accompanied by severe allergic reactions and immune-mediated adverse reactions.
  • PAL phenylalanine Acid ammonia lyase
  • PAH phenylalanine hydroxylase gene
  • the present disclosure attempts to use engineered microorganisms as carriers for delivering phenylalanine-related degrading enzymes, using genetic engineering technology
  • engineered microorganisms that can degrade phenylalanine to produce non-toxic intermediate metabolites, and can effectively reduce the level of phenylalanine in animals (including humans).
  • an engineered microorganism introduced with an exogenous gene can be provided, wherein the exogenous gene includes: one or more genes encoding the ability to convert phenylalanine into phenylpyruvate one or more genes encoding enzymes capable of converting phenylpyruvate to phenylacetaldehyde; one or more genes encoding enzymes capable of converting phenylacetaldehyde into phenylethyl alcohol; and a one or more codes capable of converting phenylpropanoid A gene for a protein that transports amino acids into the engineered microorganism. .
  • an engineered microorganism composition comprising more than one engineered microorganism, each of the more than one engineered microorganism independently comprising any of the following genes one or more kinds:
  • the engineered microbial composition comprises a gene encoding an enzyme capable of converting phenylalanine into phenylpyruvate, a gene encoding an enzyme capable of converting phenylpyruvate into phenylacetaldehyde, and a gene encoding an enzyme capable of converting phenylacetaldehyde into phenylacetaldehyde.
  • a composition which comprises the engineered microorganism or engineered microorganism composition according to the present disclosure and a pharmaceutically, nutritionally or physiologically acceptable carrier.
  • a kit may be provided, which includes the engineered microorganism according to the present disclosure or the engineered microorganism composition according to the present disclosure or the composition according to the present disclosure.
  • a method for alleviating and/or treating diseases and/or conditions related to hyperphenylalaninemia comprising administering to a patient in need thereof according to The engineered microorganism of the present disclosure or the engineered microorganism composition according to the present disclosure or the composition according to the present disclosure or the kit according to the present disclosure.
  • the engineered microorganism according to the present disclosure or the engineered microorganism composition according to the present disclosure or the composition according to the present disclosure can be provided for relieving and/or treating those with high benzene Use in medicine or health care products for diseases and/or diseases related to alaninemia.
  • Figure 1 depicts a map of plasmid PCBT003.
  • Figure 2 depicts a map of plasmid PCBT001.
  • Figure 3 depicts the engineering strains EcN, CBT2001, CBT2002 and In vitro test results of CBT2003 degradation of phenylalanine.
  • Fig. 4 depicts the in vitro detection results of engineering strains EcN, CBT2001, CBT2002 and CBT2003 degrading phenylalanine under anaerobic conditions.
  • Figure 5 depicts the in vitro detection results of engineering strains CBT209 and CBT210 degrading phenylalanine.
  • Figure 6 depicts the in vitro detection results of engineering strain CBT201 degrading phenylalanine.
  • Figure 7 depicts the in vitro detection results of engineering strain CBT202 degrading phenylalanine.
  • Figure 8 depicts the in vitro detection results of engineering strains CBT205 and CBT206 degrading phenylalanine.
  • Figure 9 depicts the in vitro detection results of engineering strains CBT207 and CBT208 degrading phenylalanine.
  • Figure 10 depicts the in vitro detection results of engineering strains CBT201, CBT205, CBT207 and CBT211 degrading phenylalanine.
  • Figure 11 depicts the detection results of engineering strains CBT-201, CBT-212, CBT-213, CBT-205 and CBT-207 in vitro degradation of phenylalanine.
  • Figure 12 depicts the results of in vivo degradation of phenylalanine by engineering strains CBT-201, CBT-201-AA, CBT-205, negative control group EcN and active reference group SYNB1934.
  • Microorganism refers to a microscopic, submicroscopic or ultramicroscopic sized organism or microorganism usually consisting of single cells. Examples of microorganisms include, but are not limited to, bacteria, viruses, parasites, fungi, certain algae, and protozoa.
  • An "engineered microorganism” as used in this disclosure refers to a microorganism that has been genetically modified to exhibit desired properties or characteristics. Furthermore, in this disclosure, the term “engineered microorganism” is used interchangeably with “genetically engineered microorganism” or “genetically modified microorganism”.
  • Phenylalanine as used in this disclosure is used to refer to The amino acid of COOH. Phenylalanine is a precursor to tyrosine, dopamine, norepinephrine, and epinephrine. L-Phenylalanine is an essential amino acid and is mainly found in the form of phenylalanine in dietary proteins; the stereoisomer D-phenylalanine is found in lower amounts in dietary proteins; DL-phenylalanine is A combination of both forms. Phenylalanine may refer to one or more of L-phenylalanine, D-phenylalanine and DL-phenylalanine.
  • phenylpyruvate in this disclosure are also intended to cover phenylpyruvate, unless the context specifically dictates or is clearly contradicted.
  • Phenylpyruvate decarboxylase as used in the present disclosure has the same meaning as “phenylpyruvate decarboxylase” unless the context specifically states or is clearly contradicted.
  • aromatic amino acids transaminase refers to a phenylalanine metabolizing enzyme that converts or processes phenylalanine into phenylpyruvate.
  • L-amino acid deaminase refers to a phenylalanine metabolizing enzyme that converts or processes phenylalanine into phenylpyruvate.
  • Phenylalanine dehydrogenase in the present disclosure refers to a phenylalanine metabolizing enzyme that converts or processes phenylalanine into phenylpyruvate.
  • phenylalanine metabolizing enzyme refers to an enzyme capable of degrading or converting phenylalanine. Any phenylalanine metabolizing enzyme known in the art can be encoded by engineered microorganisms. Phenylalanine metabolizing enzymes include, but are not limited to, aromatic amino acid transaminase, phenylalanine dehydrogenase, L-amino acid deaminase, and the like.
  • Phenylalanine metabolites in the present disclosure refer to metabolites produced due to the degradation of phenylalanine. Metabolites can be directly catalyzed by phenylalanine production by enzymes that use phenylalanine as a substrate, or metabolites can be produced indirectly by different enzymes acting downstream of the metabolic pathway of phenylalanine metabolites. Phenylalanine metabolites can be produced by engineered microorganisms encoding phenylalanine metabolizing enzymes.
  • phenylpyruvate decarboxylase refers to a phenylpyruvate metabolizing enzyme that converts or processes phenylpyruvate into phenylacetaldehyde.
  • ⁇ -keto acid decarboxylase refers to a phenylpyruvate metabolizing enzyme that converts or processes phenylpyruvate into phenylacetaldehyde.
  • aldehyde reductase (Aldehyde dehydrogenase) refers to the Phenylacetaldehyde metabolizing enzyme that converts or processes aldehydes to phenylethyl alcohol.
  • Glutamate dehydrogenase (Glutamate dehydrogenase)
  • glutamic acid salt
  • Phenylalanine transporter as used in this disclosure is used to refer to a membrane transporter capable of transporting phenylalanine into cells.
  • the phenylalanine transporter gene encodes a high-affinity phenylalanine-specific phenylalanine transporter responsible for phenylalanine transport.
  • the phenylalanine transporter can be encoded by a phenylalanine transporter gene derived from bacteria including, but not limited to, Acinetobacter calcoaceticus, Salmonella enteritidis, and Escherichia coli .
  • phenylalanine transporters include the general amino acid transporter encoded by the arop gene, which is capable of transporting three aromatic amino acids, including phenylalanine, with high affinity and is thought to be responsible, together with the phenylalanine transporter Most of the phenylalanine input.
  • low levels of phenylalanine transport activity have been traced to the activity of the LIV-I/LS system, which is composed of two periplasmic binding proteins, LIV-binding protein (LIV-I system) and LS -binding protein (LS system), and a branched-chain amino acid transporter composed of membrane component LIVHMGF.
  • an enzyme capable of converting phenylalanine to phenylpyruvate an enzyme capable of converting phenylpyruvate into phenylacetaldehyde, an enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol, Phenylalanine transporter and glutamate dehydrogenase or their coding genes also include functional equivalents of the above enzymes, proteins or their coding genes; the "functional equivalents" refer to amino acid sequences or polynucleotides Any relevant variant that differs in sequence or chemical structure but retains at least in part one or more biological functions of a naturally occurring gene/enzyme.
  • the "expression cassette" in the present disclosure refers to a nucleic acid molecule capable of directing the expression of at least one polynucleotide/encoding gene of interest in an appropriate host cell. At least in some embodiments, the nucleic acid molecule also contains an operable Linked to the promoter and/or other regulatory elements of the polynucleotide/encoding gene of interest.
  • “Operably linked” in the present disclosure means that the nucleic acid sequence is linked to the regulatory region sequence in a manner that allows expression of the nucleic acid sequence (eg, acting in cis).
  • Regulatory area can refer to Nucleic acid that directs transcription of a gene of interest, such as a promoter, optionally including enhancers, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated region, transcription initiation site, termination sequence, polyadenylation sequence and/or intron.
  • the "promoter” in this disclosure refers to a polynucleotide sequence that can control the transcription of a coding sequence.
  • the promoter sequence includes specific sequences sufficient for RNA polymerase to recognize, bind and initiate transcription.
  • the promoter sequence may include, optionally, sequences that regulate the recognition, binding, and transcription initiation activity of RNA polymerase in the engineered microorganisms provided in the present disclosure.
  • a promoter can affect the transcription of a gene located on the same nucleic acid molecule as itself or a gene located on a different nucleic acid molecule than itself.
  • inducible promoter refers to a coding sequence that can be initiated in one or more cell types under its control by external stimuli, such as chemicals, light, hormones, stress, anaerobic conditions, or pathogens or a regulated promoter that increases the level of transcription of a gene.
  • inducible promoters and variants are well known in the art and include, but are not limited to, PLteto1, galP1, PLlacO1, Pfnrs, PBAD, and Pvan.
  • the "directly inducible promoter” in the present disclosure refers to a regulatory region operably linked to a gene encoding a protein or polypeptide, and the protein or polypeptide is expressed or repressed in the presence of an inducer of the regulatory region.
  • an “indirectly inducible promoter” as used in this disclosure refers to a regulatory system comprising two or more regulatory regions, e.g., a first regulatory region operably linked to a gene encoding a first molecule, e.g., a transcriptional A modulator capable of modulating a second regulatory region operably linked to the gene encoding the effector molecule.
  • the second regulatory region can be activated or repressed, thereby activating or repressing the expression of the effector molecule.
  • Both directly and indirectly inducible promoters are encompassed by "inducible promoter".
  • a “constitutive promoter” in the present disclosure refers to a promoter capable of promoting continuous transcription of a coding sequence or gene under its control and/or operably linked thereto.
  • Constitutive promoters and variants of Escherichia coli Nissle1917 are well known in the art, including but not limited to BBa_J23119, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23109, BBa_J23110, BBa_J23114, BBa_J23117, USP45_promoter, OmpA_promoter, BBa_J23100, BBa_J23104, BBa_J23105, BBa_I14018, BBa_J45992, BBa_J23118, BBa_J23116, BBa_J23115, BBa_J23113, BBa_J2 3112, BBa__
  • a “foreign promoter” as used herein refers to a promoter operably associated with a coding region, wherein the promoter is not a promoter naturally associated with the coding region in the genome of an organism.
  • a promoter in the genome that is naturally associated with or linked to a coding region is referred to as the "endogenous promoter" for that coding region.
  • exogenous environmental conditions refer to the environment in which the promoters described herein are directly or indirectly induced, refer to the environment or environmental conditions outside the engineered microorganisms, and are related to the in vitro culture conditions of the microorganisms.
  • Exogenous environmental conditions may also refer to conditions during the growth, production and manufacture of engineered microorganisms. Such conditions include aerobic culture conditions, anaerobic culture conditions, hypoxic culture conditions and other conditions at a set oxygen concentration. Such conditions also include the presence in the medium of chemical and/or nutritional inducers, such as tetracycline, arabinose, IPTG, rhamnose, and the like. Such conditions also include the ambient temperature of the engineered microorganism in the mammal.
  • “Gut” as used in this disclosure refers to the organs, glands, tracts and systems responsible for the transfer and digestion of food, the absorption of nutrients, and the excretion of waste.
  • the gut comprises the gastrointestinal tract (GI), which begins at the mouth and ends at the anus, and additionally contains the esophagus, stomach, small intestine, and large intestine.
  • the intestine also includes accessory organs and glands such as the spleen, liver, gallbladder, and pancreas.
  • the upper gastrointestinal tract consists of the esophagus, stomach, and duodenum of the small intestine.
  • the lower gastrointestinal tract includes the rest of the small intestine, namely the jejunum and ileum, and all of the large intestine, namely the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, such as the gastrointestinal tract.
  • oxygen level-dependent promoter or “oxygen level-dependent regulatory region” mentioned in the present disclosure refers to a nucleic acid sequence that one or more oxygen level-sensitive transcription factors can bind, wherein the corresponding transcriptional binding and/or activating factors Activates downstream gene expression.
  • Non-pathogenic bacteria refers to bacteria that cannot cause disease or harmful responses in the host.
  • Non-pathogenic bacteria may be commensal bacteria.
  • examples of non-pathogenic bacteria include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus (Enterococcus), Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces and Staphylococcus, Bacillus coagulans, Bacillus subtilis ( Bacillus subtilis), Bacteroides fragilis, Bacteroides subtili, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lac
  • Probiotic as used in this disclosure is used to refer to live, non-pathogenic microorganisms, such as bacteria, which are capable of conferring a health benefit on a host organism containing the microorganism in appropriate amounts.
  • the host organism may be a mammal, in particular a human.
  • auxotrophy-associated gene refers to a gene required for the survival of a host cell (eg, a microorganism such as a bacterium). Auxotrophy-associated genes may be required for the production of nutrients necessary for the microorganism to survive or grow, or may be required for the detection of signals in the environment that regulate the activity of transcription factors, where the absence of a signal Failure will result in cell death.
  • the terms “alleviation” and “treatment” and their synonyms refer to the amelioration of a disease, disorder and/or condition.
  • "Relief” and “treatment” can be an improvement in at least one measurable physical parameter, which is not necessarily identifiable by the patient.
  • "Alleviation” and “treatment” can also be physical (eg, stabilization of identifiable symptoms), physiological (eg, stabilization of physical parameters), or both, inhibiting the development of a disease, disorder and/or condition.
  • “Alleviating” and “treating” can also mean slowing down or reversing the progression of a disease, disorder and/or condition.
  • prevention As used herein, the term “treatment” is intended to encompass “prevention”. As used herein, “prevention” and synonyms thereof refer to delaying the onset of or reducing the risk of acquiring a particular disease, disorder and/or condition or symptoms associated with such disease, disorder and/or condition.
  • composition in the present disclosure refers to the preparation of the engineered microorganism or engineered microorganism composition described in the present disclosure and other components such as pharmaceutically acceptable carriers and/or excipients.
  • the "pharmaceutically acceptable carrier” in the present disclosure refers to a carrier or diluent or adjuvant used for the preparation or administration of engineered microorganisms or engineered microorganism compositions, which itself is not a necessary active ingredient, and there is no Excessive toxicity.
  • Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art and include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and topical Active agents include, for example, polysorbate 20.
  • physiologically acceptable carrier in the present disclosure refers to a carrier or diluent or adjuvant that does not cause significant irritation to the organism and does not eliminate the biological activity and characteristics of the administered engineered microorganism or engineered microorganism composition .
  • the "diseases and/or conditions associated with hyperphenylalaninemia” in this disclosure include, but are not limited to: phenylketonuria (such as classic or typical (classical or typical) phenylketonuria and atypical (atypical) phenylketonuria), mild (mild) hyperphenylalaninemia, non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, Dihydropterin reductase deficiency, tetrahydropterin synthase deficiency, Segawa's disease and liver disease.
  • phenylketonuria such as classic or typical (classical or typical) phenylketonuria and atypical (atypical) phenylketonuria
  • mild (mild) hyperphenylalaninemia such as classic or typical (classical or typical) phenylketonuria and atypical (atypical)
  • “Hyperphenylalaninemia” or “excess phenylalanine” as used in the present disclosure refers to an increased or abnormally high concentration of phenylalanine in the body.
  • the hyperphenylalaninemia Diagnostic signs are blood phenylalanine levels of at least 2 mg/dl, at least 4 mg/dl, at least 6 mg/dl, at least 8 mg/dl, at least 10 mg/dl, at least 12 mg/dl, at least 14 mg/dl, at least 16 mg/dl, at least 18mg/dl, at least 20mg/dl or at least 25mg/dl.
  • “Hyperphenylalaninemia” in this disclosure includes, but is not limited to, phenylketonuria (such as classic or typical (classical or typical) phenylketonuria and non-classic (atypical) phenylketonuria), Permanent mild (mild) hyperphenylalaninemia, non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropterin reductase deficiency , tetrahydropterin synthase deficiency, Segawa's disease and liver disease.
  • phenylketonuria such as classic or typical (classical or typical) phenylketonuria and non-classic (atypical) phenylketonuria
  • Permanent mild (mild) hyperphenylalaninemia non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency,
  • Diseases and/or disorders related to phenylalanine metabolism disorders are synonymous with “diseases and/or disorders associated with hyperphenylalaninemia", including: phenylketonuria (such as classic Or typical (classical or typical) phenylketonuria and non-classic (atypical) phenylketonuria), permanent mild (mild) hyperphenylalaninemia, non-phenylketonuric hyperphenylalanine Acidemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropterin reductase deficiency, tetrahydropterin synthase deficiency, Segawa's disease, and liver disease.
  • phenylketonuria such as classic Or typical (classical or typical) phenylketonuria and non-classic (atypical) phenylketonuria
  • permanent mild (mild) hyperphenylalaninemia non-phenylketonuric hyperpheny
  • engineered microorganisms described in the present disclosure are capable of reducing excess phenylalanine.
  • engineered microorganisms are bacteria or yeast.
  • engineered microorganisms are non-pathogenic bacteria.
  • engineered microorganisms are commensal bacteria.
  • the engineered microorganism is a fungus.
  • the engineered microorganisms are probiotics.
  • engineered microorganisms are naturally pathogenic bacteria that have been modified or mutated to reduce or eliminate pathogenicity.
  • engineered microorganisms are Gram-negative bacteria.
  • engineered microorganisms are Gram-positive bacteria.
  • engineered microorganisms are selected from the group including but not limited to Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus ) and Lactococcus.
  • the engineered microorganism is Escherichia coli. In some embodiments, the engineered microorganism is Escherichia coli Nissle1917 (E. coli Nissle), a Enterobacteriaceae that "has evolved into one of the best characterized probiotics" Gram-negative bacteria (Enterobacteriaceae). The strain is characterized in that it is completely harmless and has a GRAS (generally recognized as safe) status. Genome sequencing confirmed that E. coli Nissle lacks prominent virulence factors (eg, E. coli alpha-hemolysin, P-fimbrial adhesin). In addition, it has been shown that E.
  • E. coli Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and is not uropathogenic.
  • E. coli Nissle was packaged as a drug capsule (called Mutaflor) for therapeutic use. Since then, E. coli Nissle has been used in vivo to treat ulcerative colitis in humans, to treat human inflammatory bowel disease, Crohn's disease, and pouchitis in vivo, and to inhibit intestinal invasion in vitro Salmonella, Legionella, Yersinia and Shigella.
  • the general consensus is that the safety and utility of E. coli Nissle as a therapeutic vector has been convincingly demonstrated.
  • the engineered microorganism is auxotrophic.
  • the auxotrophs are uracil, thymine, leucine, histidine, tryptophan, lysine, methionine, adenine, non-naturally occurring amino acids, di Any one or combination of aminoacrylic acid auxotrophs.
  • the auxotroph is a diaminoacrylic acid auxotroph and/or a thymidine auxotroph.
  • auxotrophs are replenished when engineered microorganisms are present in the gut of a mammal.
  • the engineered microorganism comprises an inactivation or deletion of at least one auxotrophy-associated gene.
  • the auxotroph-related genes are selected from the group consisting of: thyA, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, uraA, dapF, flhD, metB, metC, proAB, yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, th
  • the modified flagellar structure of the engineered microorganism has a functional defect or loss, so that the residence time of the engineered microorganism in the intestinal tract is increased, which will be more conducive to the metabolism of phenylalanine in vivo .
  • the modification of the flagellar structure is achieved by mutation or deletion of part or all of the genes related to flagellar synthesis.
  • the Escherichia coli is used as the microbial organism, and the genes related to flagella synthesis are bscA and/or fliC genes.
  • an engineered microorganism introduced with exogenous genes can be provided, wherein the exogenous genes include: one or more encoding enzymes capable of converting phenylalanine into phenylpyruvate gene; one or more genes encoding an enzyme capable of converting phenylpyruvate to phenylacetaldehyde; one or more genes encoding an enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol; and one or more Various genes encoding proteins capable of transporting phenylalanine into the engineered microorganism.
  • the engineered microorganism is capable of metabolizing phenylalanine in the gut of a human and/or mammal.
  • the exogenous gene is integrated in the genome or located on an expression plasmid.
  • the gene encoding an enzyme capable of converting phenylalanine into phenylpyruvate is selected from the group consisting of genes encoding transaminases, genes encoding dehydrogenases, genes encoding deaminases, and One or more of functional equivalents which retain at least part of the activity of the relevant enzyme.
  • the gene encoding dehydrogenase is a gene encoding phenylalanine dehydrogenase; in other embodiments, the gene capable of converting phenylalanine into phenylpyruvate Enzymes include phenylalanine dehydrogenase and transaminases (eg, aromatic amino acid transaminase).
  • phenylalanine dehydrogenases eg, aromatic amino acid transaminase.
  • the gene encoding phenylalanine dehydrogenase is derived from viruses, fungi and/or bacteria. In some embodiments of the present disclosure, the gene encoding phenylalanine dehydrogenase is derived from Sarcina ureae and/or Bacillus, for example, Sarcina ureae SCRC-R04 (Sporosarcina ureae SCRC-R04) , Lysinibacillus sphaericus SCRC-R79a, Bacillus badius, and/or Bacillus sp. SLBN-3.
  • the gene encoding phenylalanine dehydrogenase is selected from PheDH (Phenylalanine Dehydrogenase) of Bacillus sp. SLBN-3.
  • the phenylalanine dehydrogenation The enzyme has the amino acid sequence shown in SEQ ID NO:6; or the gene encoding the phenylalanine dehydrogenase has the nucleotide sequence shown in SEQ ID NO:17.
  • the gene encoding a transaminase is a gene encoding an aromatic amino acid transaminase.
  • the gene encoding aromatic amino acid transaminase is derived from viruses, fungi and/or bacteria.
  • the gene encoding aromatic amino acid transaminase is derived from Escherichia coli and/or yeast, such as Escherichia coli BL21 (DE3) (Escherichia coli BL21 (DE3)) and/or Saccharomyces cerevisiae S288C (Saccharomyces cerevisiae S288C).
  • the gene encoding aromatic amino acid transaminase is selected from TyrB (Tyrosine aminotransferase) of Escherichia coli BL21 (DE3) or ARO8 (Bifunctional 2-aminoadipate transaminase/aromatic-amino-acid) of Saccharomyces cerevisiae S288C :2-oxoglutarate transaminase).
  • the aromatic amino acid transaminase has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or the gene encoding the aromatic amino acid transaminase has a sequence such as SEQ ID NO: 15 or the nucleotide sequence shown in SEQ ID NO:90.
  • the engineered microorganism further comprises one or more of the gene encoding glutamate dehydrogenase and its functional equivalent, and the functional equivalent retains at least part of the relevant enzyme activity, especially when transaminases (eg, aromatic amino acid transaminases) are included in the engineered microorganism.
  • Glutamate dehydrogenase can couple the transamination reaction of phenylalanine with the reduction reaction of phenylacetaldehyde, realize the regeneration of cofactors, and provide more ⁇ -ketoglutarate ( ⁇ -ketoglutaric acid), which further increases the metabolic rate of phenylalanine.
  • the gene encoding glutamate dehydrogenase is derived from viruses, fungi and/or bacteria. In some embodiments of the present disclosure, the gene encoding glutamate dehydrogenase is derived from yeast and/or Clostridium difficile, such as Saccharomyces cerevisiae S288C and/or Clostridioides difficile. In some embodiments of the present disclosure, the gene encoding glutamate dehydrogenase is selected from S288C of Saccharomyces cerevisiae or GDH2 (Glutamate dehydrogenase (NAD+)) of Clostridium difficile.
  • the glutamate dehydrogenase has the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12; or the gene encoding the glutamate dehydrogenase has the amino acid sequence as shown in Nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:97 List.
  • the gene encoding deaminase is a gene encoding L-amino acid deaminase. In some embodiments of the present disclosure, the gene encoding L-amino acid deaminase is derived from viruses, fungi and/or bacteria. In some embodiments of the present disclosure, the gene encoding L-amino acid deaminase is derived from Escherichia coli, yeast, and/or Proteus mirabilis, such as Escherichia coli BL21 (DE3), Saccharomyces cerevisiae S288C, and/or mirabilis Proteus mirabilis HI4320 (Proteus mirabilis HI4320).
  • the gene encoding L-amino acid deaminase is selected from LAAD (L-amino acid deaminase) of Escherichia coli BL21 (DE3), Saccharomyces cerevisiae S288C, or Proteus mirabilis HI4320.
  • LAAD L-amino acid deaminase
  • the L-amino acid deaminase has the amino acid sequence shown in SEQ ID NO: 89
  • the gene encoding the L-amino acid deaminase has the amino acid sequence shown in SEQ ID NO: 91 The nucleotide sequence shown.
  • the gene encoding an enzyme capable of converting phenylalanine into phenylpyruvate includes at least one of the following groups: (1) a gene encoding phenylalanine dehydrogenase; (2) gene encoding aromatic amino acid transaminase; (3) gene encoding aromatic amino acid transaminase and gene encoding phenylalanine dehydrogenase; (4) gene encoding aromatic amino acid transaminase and encoding L-amino acid deamination (5) gene encoding phenylalanine dehydrogenase and gene encoding L-amino acid deaminase; or (6) gene encoding aromatic amino acid transaminase, gene encoding phenylalanine dehydrogenase and the gene encoding L-amino acid deaminase.
  • the gene encoding an enzyme capable of converting phenylpyruvate into phenylacetaldehyde is selected from a gene encoding phenylpyruvate decarboxylase, a gene encoding ⁇ -keto acid decarboxylase, and a function thereof One or more of equivalents which retain at least part of the activity of the relevant enzyme.
  • the gene encoding phenylpyruvate decarboxylase is derived from viruses, fungi and/or bacteria.
  • the gene encoding phenylpyruvate decarboxylase is derived from yeast, such as Saccharomyces cerevisiae S288C. In some embodiments of the present disclosure, the gene encoding phenylpyruvate decarboxylase is ARO10 (Phenylpyruvate decarboxylase) of Saccharomyces cerevisiae S288C. In some embodiments of the present disclosure, the phenylpyruvate decarboxylase has the amino acid sequence shown in SEQ ID NO: 7; or encodes the phenylpyruvate decarboxylase The gene of carboxylase has the nucleotide sequence shown in SEQ ID NO:18.
  • the gene encoding ⁇ -ketoacid decarboxylase is derived from viruses, fungi and/or bacteria. In some embodiments of the present disclosure, the gene encoding ⁇ -ketoacid decarboxylase is derived from yeast and/or Proteus mirabilis, such as Saccharomyces cerevisiae S288C and/or Proteus mirabilis JN458 (Proteus mirabilis JN458).
  • the gene encoding ⁇ -ketoacid decarboxylase is selected from at least one of KDC (Alpha-keto-acid decarboxylase) of Saccharomyces cerevisiae S288C or Proteus mirabilis JN458.
  • KDC Alpha-keto-acid decarboxylase
  • the ⁇ -ketoacid decarboxylase has the sequence shown in SEQ ID NO:8; or the gene encoding the ⁇ -ketoacid decarboxylase has the sequence shown in SEQ ID NO:95 the nucleotide sequence.
  • the gene encoding an enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol is selected from one or more of genes encoding aldehyde reductase and functional equivalents thereof, the function Equivalents retain at least some of the activity of the relevant enzyme.
  • the gene encoding aldehyde reductase is derived from viruses, fungi and/or bacteria.
  • the gene encoding aldehyde reductase is derived from Escherichia coli and/or Lactobacillus brevis, such as Escherichia coli str.K-12substr.MG1655 and/or Lactobacillus brevis.
  • the gene encoding aldehyde reductase is selected from YahK (NADPH-dependent aldehyde reductase) of Escherichia coli str.K-12substr.MG1655 or ADH (Alcohol dehydrogenase) of Lactobacillus brevis.
  • the aldehyde reductase has an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10; or the gene encoding the aldehyde reductase has a sequence such as SEQ ID NO: 19 or The nucleotide sequence shown in SEQ ID NO:96.
  • the gene encoding a protein capable of transporting phenylalanine into the engineered microorganism is selected from one of genes encoding phenylalanine transporters and functional equivalents thereof or more, said functional equivalent retains at least part of the activity of the relevant enzyme.
  • the gene encoding the phenylalanine transporter is derived from viruses, fungi and/or bacteria.
  • the phenylalanine transporter is encoded by a phenylalanine transporter gene derived from bacteria including, but not limited to, Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Salmonella enteritidis and Escherichia coli.
  • the gene encoding the phenylalanine transporter is derived from Escherichia coli.
  • the phenylalanine transporter is encoded by the PHEP gene of bacterial origin.
  • the phenylalanine transporter is encoded by the AROP gene of bacterial origin.
  • the phenylalanine transporter is encoded by the LIV-binding protein and LS-binding protein and LIVHMGF genes of bacterial origin.
  • the engineered microorganism comprises at least one type of phenylalanine transporter selected from PHEP, AROP and LIV-I/LS.
  • the gene encoding the phenylalanine transporter is PheP (Phenylalanine:H(+)symporter) of Escherichia coli.
  • the phenylalanine transporter has the amino acid sequence shown in SEQ ID NO: 13; or the gene encoding the phenylalanine transporter has the amino acid sequence shown in SEQ ID NO: 21 The nucleotide sequence shown.
  • the gene encoding an enzyme capable of converting phenylalanine into phenylpyruvate the gene encoding an enzyme capable of converting phenylpyruvate into phenylacetaldehyde, the gene encoding an enzyme capable of any one, two, three, Four, five, six, more or all are optionally operably linked to one or more same or different promoters.
  • the gene encoding aromatic amino acid transaminase and the gene encoding glutamate dehydrogenase share the same promoter.
  • the gene encoding phenylpyruvate decarboxylase and the gene encoding aldehyde reductase share the same promoter. In some embodiments of the present disclosure, the gene encoding phenylalanine dehydrogenase and the gene encoding phenylalanine transporter share the same promoter. In some embodiments of the present disclosure, the gene encoding L-amino acid deaminase is operably linked to a promoter alone. In some embodiments of the present disclosure, the gene encoding the phenylalanine transporter is operably linked to a promoter alone.
  • the promoter is an endogenous promoter or an exogenous promoter. In some embodiments of the present disclosure, the promoter is an inducible promoter or a constitutive promoter. In some embodiments of the present disclosure, the promoter is a directly or indirectly inducible promoter. in some implementations In, the promoter is a pH-dependent promoter. In some embodiments, the promoter is an oxygen level dependent promoter. In some embodiments of the present disclosure, the promoter is directly or indirectly induced by exogenous environmental conditions. In some embodiments of the present disclosure, exogenous environmental conditions refer to exogenous environmental conditions in the gut of a mammal. In some embodiments of the present disclosure, mammalian gut refers to human gut.
  • the exogenous environmental condition refers to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental condition refers to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental condition is the small intestine of a mammal. In some embodiments, exogenous environmental conditions refer to hypoxic, microaerophilic or anaerobic conditions, such as the environment of a mammalian gut. In some embodiments, an exogenous environmental condition refers to a molecule or metabolite present in the gut of a mammal in a healthy or diseased state. In some embodiments, the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule.
  • the exogenous environmental condition is a low pH environment.
  • the promoter is selected from at least one of PfnrS, FDHF, Ptet, Pbba, Ptrc, Pvan, or PBAD.
  • the sequence of the PfnrS promoter is shown in SEQ ID NO.22.
  • the sequence of the FDHF promoter is shown in SEQ ID NO.24.
  • the sequence of the Ptet promoter is shown in SEQ ID NO.100.
  • the sequence of the Pbba promoter is shown in SEQ ID NO.98.
  • the sequence of the Ptrc promoter is shown in SEQ ID NO.99.
  • the Pvan promoter is a promoter induced by vanillic acid, and its sequence is shown in SEQ ID NO:101.
  • the sequence of the PBAD promoter is shown in SEQ ID NO:102. It is known that the activity of the vanillic acid promoter can be inhibited by the repressor expression cassette VanRAM (its coding sequence is shown in SEQ ID NO: 111), and the above inhibition can be relieved by adding vanillic acid, so that the expression of the vanillic acid promoter Promoter activity. Through this mechanism, the induction and regulation of the promoter can be achieved.
  • the vanillic acid promoter When there is no VanRAM in the engineered microorganism, the vanillic acid promoter is in a continuous activation state. At this time, in order to achieve inducible expression, additional VanRAM needs to be added. Therefore, in some embodiments of the present disclosure, when the promoter is the Pvan promoter, it is also possible to simultaneously transfer into the genome of the engineered microorganism a vanilla gene capable of expressing one or more copies. Expression cassette for the oxalate repressor VanRAM.
  • the gene encoding an enzyme capable of converting phenylalanine into phenylpyruvate the gene encoding an enzyme capable of converting phenylpyruvate into phenylacetaldehyde
  • the gene encoding an enzyme capable of The gene for an enzyme that converts phenylacetaldehyde to phenylethyl alcohol, the gene encoding glutamate dehydrogenase, and/or the gene encoding a phenylalanine transporter are located in one or more expression cassettes.
  • the expression cassette exists in the form of single copy, double copy, triple copy, quadruple copy or more copies.
  • the expression cassette is present on a plasmid. In some embodiments of the present disclosure, the expression cassette is stably integrated in one, two, or more identical or different genomic loci of the engineered microorganism. In some embodiments of the present disclosure, the genomic site is selected from yicS site, malPT site, malE site, exo site, rhtB/C site, agaI/rsml site, araBD site, yghx site at least one of site, ldhA site, araAB site, lacZ site, kefB site, maeB site, nth/tppB site, and/or tkrA site.
  • the engineered microorganism of the present disclosure comprises a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first promoter, and the aromatic amino acid transaminase-encoding gene operably linked thereto Gene and the gene encoding glutamate dehydrogenase; the second expression cassette comprises a second promoter, and the gene encoding phenylpyruvate decarboxylase and the gene encoding aldehyde reductase operably linked thereto; the The first or second promoters are the same or different.
  • the engineered microorganism further comprises a third expression cassette comprising the gene encoding the phenylalanine transporter operably linked to a third promoter, the third The promoter may be the same as the first promoter and/or the second promoter, or different from both the first and the second promoter.
  • the first expression cassette exists in the form of single copy or double copy.
  • the second expression cassette exists in the form of single copy, double copy, triple copy or quadruple copy.
  • the third expression cassette is present in a single copy.
  • the first expression cassette is integrated at the yghx site and/or the araAB site.
  • the second expression cassette is integrated at the ldhA site, the lacZ site, the yghx site, the yjcS site and/or the agaI/rsml site.
  • the third expression cassette is integrated at the kefB site.
  • the engineered microorganisms of the present disclosure comprise a fourth expression cassette and a second expression cassette, wherein the fourth expression cassette comprises a fourth promoter, and the encoding phenylalanine deactivator operably linked thereto.
  • the fourth expression cassette comprises a fourth promoter, and the encoding phenylalanine deactivator operably linked thereto.
  • the engineered microorganism further comprises a first expression cassette comprising a first promoter, the gene encoding aromatic amino acid transaminase and the glutamic acid encoding gene operably linked thereto.
  • a dehydrogenase gene; the first promoter may be the same as the fourth promoter and/or the second promoter, or different from both the fourth promoter and the second promoter.
  • the engineered microorganism further comprises a fifth expression cassette, the fifth expression cassette comprises a fifth promoter, the gene encoding L-amino acid deaminase operably linked thereto; the fifth The promoter is the same or different from the fourth promoter, the second promoter and/or the first promoter.
  • the expression cassette exists in the form of single copy, double copy, triple copy, quadruple copy or more copies.
  • the fourth expression cassette exists in the form of single copy or double copy.
  • the second expression cassette exists in the form of single copy, double copy, triple copy or quadruple copy.
  • the first expression cassette exists in the form of single copy or double copy.
  • the fifth expression cassette is present in a single copy.
  • the fourth expression cassette is integrated at the kefB site, ldhA site, nth/tppB site, maeB site and/or tkrA site.
  • the second expression cassette is integrated at the ldhA site, yghx site, lacZ site, yjcS site and/or agal/rsml site.
  • the first expression cassette is integrated at the yghX site and/or the araAB site.
  • the fifth expression cassette is integrated at the rhtB/C site.
  • all or part of the first promoter, the second promoter, the third promoter, and the fourth promoter are selected from PfnrS (sequence such as any of SEQ ID NO: 22-23 item) and/or Pvan (sequence shown in SEQ ID NO:101) At least one of them, the fifth promoter is PBAD (sequence shown in SEQ ID NO: 102).
  • the engineered microorganism of the present disclosure includes a single copy of the fourth expression cassette, which is integrated at the kefB site; a double copy of the second expression cassette, and the second expression cassette is respectively Integrated at the ldhA and lacZ sites; double copies of the first expression cassette integrated at the yghX and araAB sites, respectively; and a single copy of the fifth expression cassette integrated at the rhtB/ C site; wherein, the promoter used in the fourth, second and first expression cassettes is the PfnrS promoter (sequence shown in SEQ ID NO: 22), and the promoter used in the fifth expression cassette is PBAD Promoter (sequence is shown in SEQ ID NO:101).
  • the engineered microorganisms of the present disclosure include a single copy of the fourth expression cassette, which is integrated at the nth/tppB site; three copies of the second expression cassette, the second expression cassette Cassettes are integrated at the yjcS, ldhA and agaI/rsml sites, respectively; double copies of the first expression cassette, which are respectively integrated at the yghX and araAB sites; and a single copy of the fifth expression cassette, the fifth The expression cassette is integrated at the rhtB/C site; wherein, the promoter used in the fourth, second and first expression cassettes is the Pvan promoter (sequence shown in SEQ ID NO: 101), and in the fifth expression cassette The promoter used is the PBAD promoter (sequence shown in SEQ ID NO: 102).
  • the engineered microorganisms of the present disclosure include double copies of the fourth expression cassette, which are respectively integrated at the nth/tppB and tkrA sites; three copies of the second expression cassette, the The second expression cassette is integrated at the yjcS, ldhA and agaI/rsml sites respectively; the double copy of the first expression cassette is respectively integrated at the yghX and araAB sites; and the fifth expression cassette of a single copy, so The fifth expression cassette is integrated at the rhtB/C site; wherein, the promoter used in the fourth, second and first expression cassettes is the Pvan promoter (sequence shown in SEQ ID NO: 101), the fifth The promoter used in the expression cassette is the PBAD promoter (sequence shown in SEQ ID NO: 102).
  • the engineered microorganism of the present disclosure includes a single copy of the fourth expression cassette, which is integrated at the maeB site; three copies of the second expression cassette, and the second expression cassette is respectively Integrated at the yjcS, lacZ and agaI/rsml sites; double copy of the first expression cassette integrated at the yghX and araAB sites respectively point; and a single copy of the fifth expression cassette, which is integrated at the rhtB/C site; wherein, the single copy of the fourth expression cassette uses the PfnrS promoter, and the three copies of the second expression cassette use respectively PfnrS promoter (sequence as shown in any one of SEQ ID NO:22), PfnrS promoter (sequence as shown in SEQ ID NO:22) and Pvan promoter (sequence as shown in SEQ ID NO:101), double The first expression cassette of copy uses PfnrS promoter (sequence as shown in SEQ ID NO:22
  • the engineered microorganism of the present disclosure includes double copies of the fourth expression cassette, which is integrated at the maeB and kefB sites; three copies of the second expression cassette, the second expression cassette Cassettes are integrated at the yjcS, ldhA and agaI/rsml sites, respectively; double copies of the first expression cassette, which are respectively integrated at the yghX and araAB sites; and a single copy of the fifth expression cassette, the fifth The expression cassette is integrated at the rhtB/C site; wherein, the fourth expression cassette of the double copy uses the PfnrS promoter, and the second expression cassette of the three copies uses the Pvan promoter, the Pvan promoter and the PfnrS promoter respectively (sequence such as SEQ shown in ID NO:22), the first expression cassette of double copy uses Pvan promotor (sequence as shown in SEQ ID NO:101), the promotor that the 5th expression cassette uses is PBAD promotor (s
  • the engineered microorganism of the present disclosure includes a single copy of the fourth expression cassette, which is integrated at the kefB site; four copies of the second expression cassette, and the second expression cassette is respectively Integrated at the ldhA and lacZ sites; double copies of the first expression cassette integrated at the yghX and araAB sites, respectively; and a single copy of the fifth expression cassette integrated at the rhtB/ C site; wherein, the promoter used in the fourth, second and first expression cassettes is the PfnrS promoter (sequence shown in SEQ ID NO: 22), and the promoter used in the fifth expression cassette is PBAD Promoter (sequence is shown in SEQ ID NO:102).
  • the engineered microorganisms of the present disclosure include double copies of the fourth expression cassette, which are integrated at the ldhA and maeB sites; double copies of the second expression cassette, the second expression cassette Cassettes are respectively integrated at the yghX and lacZ sites; and a single copy of the fifth expression cassette, which is integrated at the rhtB/C site; wherein,
  • the promoter used in the fourth and second expression cassettes is the PfnrS promoter (sequence shown in SEQ ID NO: 22), and the promoter used in the fifth expression cassette is the PBAD promoter (sequence shown in SEQ ID NO: 102).
  • the engineered microorganism of the present disclosure comprises a single copy of the fourth expression cassette, which is integrated at the ldhA site; and a single copy of the second expression cassette, the second expression cassette Integrated at the yghX site; wherein, the promoter used in the fourth and second expression cassettes is the PfnrS promoter (sequence shown in SEQ ID NO: 22).
  • the engineered microorganism of the present disclosure comprises a single copy of the first expression cassette, which is integrated at the yghX site; a single copy of the second expression cassette, which is integrated at the ldhA site; and a single copy of the third expression cassette, which is integrated at the kefB site; wherein, the promoter used in the first, second and third expression cassettes is the PfnrS promoter ( The sequence is shown in SEQ ID NO:22).
  • the engineered microorganism of the present disclosure includes a single copy of the first expression cassette, which is integrated at the yghX site; and a single copy of the second expression cassette, the second expression cassette Integrated at the ldhA site; wherein, the promoter used in the first and second expression cassettes is the PfnrS promoter (sequence shown in SEQ ID NO: 22).
  • the engineered microorganisms of the present disclosure include double copies of the first expression cassette, which are respectively integrated at the yghX and araAB sites; and double copies of the second expression cassette, the second expression cassette
  • the two expression cassettes are integrated at the ldhA and lacZ sites; wherein, the promoter used in the first and second expression cassettes is the PfnrS promoter (sequence shown in SEQ ID NO: 22).
  • the engineered microorganisms of the present disclosure include double copies of the first expression cassette, which are respectively integrated at the yghX and araAB sites; double copies of the second expression cassette, the second The expression cassettes are respectively integrated at the ldhA and lacZ sites; and a single copy of the third expression cassette, which is integrated at the kefB site; wherein the promoters used in the first, second and third expression cassettes
  • the promoter is the PfnrS promoter (sequence shown in SEQ ID NO: 22).
  • an engineered microorganism composition comprising more than one engineered microorganism, each of the more than one engineered microorganism independently comprising any of the following genes One or more of: a gene encoding an enzyme capable of converting phenylalanine to phenylpyruvate; a gene encoding an enzyme capable of converting phenylpyruvate into phenylacetaldehyde; a gene encoding an enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol The gene of enzyme; the gene of encoding the protein that can transport phenylalanine to described engineering microorganism; A gene for an enzyme converting phenylpyruvate into phenylacetaldehyde, a gene encoding an enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol, and a gene encoding a protein capable of transporting phenylalanine into the
  • each of the more than one engineered microorganisms independently comprises any one or more of the following genes: the gene encoding the enzyme capable of converting phenylpyruvate to phenylacetaldehyde; the gene encoding the enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol; the gene encoding glutamate dehydrogenase; the gene encoding the enzyme capable of converting phenylacetaldehyde Alanine is transported to the gene of the protein in the engineered microorganism; wherein the engineered microorganism composition comprises the gene encoding the enzyme that can convert phenylalanine into phenylpyruvate, the gene encoding the enzyme that can convert phenylpyruvate into phenylethyl The gene of the enzyme of aldehyde, the gene encoding the enzyme capable of converting phenylacetaldehyde into phenylethyl alcohol, the gene encoding the enzyme capable of converting
  • a composition which comprises the engineered microorganism or engineered microorganism composition described in the present disclosure and a pharmaceutically, nutritionally or physiologically acceptable carrier.
  • the composition is a pharmaceutical composition, which can be used for treating, alleviating, managing, improving and/or preventing diseases and/or conditions related to hyperphenylalaninemia.
  • the engineered microorganism can be present in the composition in an amount ranging from about 104 to about 1013 colony forming units (CFU).
  • CFU colony forming units
  • the effective amount of engineered microorganisms can be about 10 5 CFU to about 10 13 CFU, preferably about 10 6 CFU to about 10 13 CFU, preferably about 10 7 CFU to about 10 12 CFU, more preferably about 10 8 CFU to about 10 12 CFU in amounts.
  • project Microorganisms can be living cells or can be dead cells.
  • the composition is an edible composition. In some embodiments of the present disclosure, the composition is a probiotic composition. In some embodiments of the present disclosure, the composition is a food supplement. In some embodiments, a composition disclosed herein may be formulated for oral administration and may be a nutritional or nourishing composition, such as a food, food supplement, feed or feed supplement, such as a dairy product, such as a fermented dairy product , such as yogurt or yogurt drinks. In this case, the composition may comprise a nutritionally acceptable carrier, which may be a suitable food base.
  • compositions disclosed herein can also be formulated as medicaments in capsules, pills, liquid solutions, eg, as encapsulated lyophilized bacteria, and the like.
  • the composition is a probiotic composition.
  • compositions disclosed herein can be formulated to be effective for a given individual in a single administration or in multiple administrations.
  • a single administration is substantially effective to reduce the monitored symptoms of the targeted disease condition in the mammalian subject administered the composition.
  • the composition is formulated such that a single oral dose contains at least about 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 CFU of engineered microorganisms (eg bacterial entities and/or fungal entities).
  • the composition comprises 1 ⁇ 10 8 -1 ⁇ 10 12 CFU of the engineered microorganism of the present invention.
  • the composition contains at least about 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, by mass, 90% or greater than 90% of the microorganisms of the present disclosure.
  • the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 mg or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 grams of the engineering microorganisms provided by the application.
  • Physiologically acceptable carriers for compositions disclosed herein can include, for example, physiologically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, Buffers, Antioxidants, Suspending/Dispersing Agents, Chelating Agents (sequestering/chelating agent), diluent, adjuvant, excipient or non-toxic auxiliary substance, other components known in the art, or various combinations thereof.
  • the compositions described in the present disclosure are liquid formulations, solid formulations, semi-solid formulations.
  • the liquid formulation is selected from the group consisting of solution products or suspension products.
  • aqueous vehicles may include, for example, Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles may include Comprising, for example, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; the antimicrobial agent may be at a bacteriostatic or fungistatic concentration, and/or may be added to the composition in a multi-dose container, the Container contains phenol or cresol, amalgam, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride.
  • Isotonic agents can contain, for example, sodium chloride or dextrose; buffers, such as phosphate or citrate buffers; antioxidants, such as sodium bisulfate; suspending and dispersing agents, such as carboxymethylcellulose Sodium, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; chelating agents may include, for example, ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Suitable excipients may comprise, for example, water, saline, dextrose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may comprise, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins. reagents.
  • the compositions provided herein can be pharmaceutical compositions.
  • the compositions provided herein can be food supplements.
  • the compositions provided herein may comprise, in addition to the genetically modified engineered microorganisms provided herein, a pharmaceutically, nutritionally/alimentarily or physiologically acceptable carrier.
  • the preferred form will depend on the intended mode of administration and (therapeutic) application.
  • the carrier can be any material suitable for delivering the genetically modified engineered microorganisms provided herein to the gastrointestinal tract of a mammal, such as a human, preferably near or within the intestinal mucosal barrier, more preferably the colonic mucosal barrier, of a mammal.
  • Compatibility Physiologically acceptable non-toxic substances.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of powder, powder, tablet, sugar-coated agent, capsule, granule, suspension, solution, syrup, Drops, sublingual tablet, or a combination thereof.
  • composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder.
  • Oral formulations can contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions of the present disclosure may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active ingredients into compositions for pharmaceutical use .
  • the pharmaceutical composition is subjected to tabletting, lyophilization, direct compression, conventional mixing, dissolution, granulation, milling, emulsification, encapsulation, entrapment or spray drying to form tablets, granules, nanoparticles , nanocapsules, microcapsules, microtablets, pills, or powders, which may be enteric-coated or uncoated. Proper formulation depends upon its route of administration.
  • the engineered microorganisms described in the present disclosure can be formulated in any suitable dosage form (e.g., liquid for oral administration, capsule, sachet, hard capsule, soft capsule, tablet, enteric-coated tablet, suspension powder) , granules or matrix sustained release formulations) and pharmaceutical compositions of any suitable type of administration (eg, oral, immediate release, pulsed release, delayed release or sustained release).
  • suitable dosage form e.g., liquid for oral administration, capsule, sachet, hard capsule, soft capsule, tablet, enteric-coated tablet, suspension powder
  • granules or matrix sustained release formulations e.g, oral, immediate release, pulsed release, delayed release or sustained release.
  • the composition can be administered one or more times daily, weekly or monthly.
  • Engineered microorganisms can be formulated to contain one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surfactants, neutral or cationic lipids, lipoplexes, liposomes, Pharmaceutical compositions of penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or agents.
  • the engineered microorganisms described in the present disclosure can be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.
  • Pharmaceutical compositions for oral use can be manufactured using a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. .
  • Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulosic compositions such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl Cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).
  • PVP polyvinylpyrrolidone
  • PEG polyethylene glycol
  • a disintegrant such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate is added.
  • Tablets or capsules can be prepared by conventional methods together with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized cornstarch, polyvinylpyrrolidone, hydroxypropyl Methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, dextrose, sorbitol, starch, gums, kaolin, and tragacanth); fillers (for example, lactose, microcrystalline cellulose, or dibasic calcium phosphate) ; Lubricants (for example, calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silicon dioxide); disintegrants (for example, starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silicon dioxide powder); or wetting agents (for example, sodium lauryl sulfate).
  • binding agents e.g., pregelatinized corn
  • Coating shells may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydrides, other biodegradable polymers, alginate-polylysine-alginate alginate) (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroxymethyl acrylate-methyl methacrylate (HEMA- MMA), multilayer HEMA-MMA-MAA, polyacrylonitrile vinyl chloride (PAN-PVC), acrylonitrile/sodium methylsulfonate (AN-69), polyethylene Glycol/Pentamethylcyclopentasiloxane/Polydimethylsiloxane (PEG/PD5/PDMS), Poly N,N-Dimethacrylamide (PDMAAm), Silica Encapsulant, Sulfate Fiber Sodium alginate/polymethylene-co-guanidine (CS/A
  • APA alginate-polylys
  • Liquid preparations for oral administration may take the form of solutions, syrups, suspensions or dry products for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional methods together with pharmaceutically acceptable agents: such as suspending agents (for example, sorbitol syrups, cellulose derivatives or hydrogenated edible fats); emulsifying agents; (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethanol, or fractionated vegetable oils); and preservatives (e.g., Methylparaben or Propylparaben or Sorbic Acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Formulations for oral administration may be suitably formulated for slow, controlled or sustained release of the engineered microorganisms described in this disclosure.
  • engineered microorganisms described in the present disclosure can be administered orally, for example, with an inert diluent or an assimilable edible carrier.
  • Compounds may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the subject's diet.
  • the compounds can be mixed with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers and the like.
  • To administer a compound of the present disclosure by means other than parenteral administration it may be necessary to coat or co-administer the compound with a material to prevent its inactivation.
  • the composition is formulated for enteral administration, intrajejunal administration, duodenal administration via nanoparticles, nanocapsules, microcapsules, or microtablets (enteric-coated or uncoated). intraileal administration, gastric bypass administration or intracolonic administration.
  • the pharmaceutical compositions of the present disclosure may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.
  • the present disclosure provides pharmaceutically acceptable compositions in a single dosage form.
  • Single dosage forms can be in liquid or solid form.
  • a single dosage form may be administered directly to the patient without modification, or may be diluted or reconstituted prior to administration.
  • a single dosage form may be administered as a bolus injection, eg, a single injection, a single oral dose, including oral doses comprising multiple tablets, capsules, pills, and the like.
  • a single dosage form may be administered over a period of time, eg, by infusion.
  • a single dosage form of a pharmaceutical composition of the present disclosure may be prepared by dispensing the pharmaceutical composition into smaller aliquots, into a single dose container, into a single dose liquid form or a single dose solid form, such as a tablet , granules, nanoparticles, nanocapsules, microcapsules, microtablets, pellets or powders, which may be enteric-coated or uncoated.
  • Single doses in solid form can be administered to the patient by adding a liquid (usually usually sterile water or saline solution) for reconstitution.
  • Dosage regimens can be adjusted to provide a therapeutic response. For example, a single bolus injection can be administered at once, several divided doses can be administered over a predetermined period of time or the dose can be decreased or increased as indicated by the therapeutic circumstances.
  • the dosage specification is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time according to the individual needs and the professional judgment of the treating clinician.
  • compositions can be delivered in a controlled release or sustained release system.
  • a pump can be used to achieve controlled or sustained release.
  • polymeric materials may be used to achieve controlled or sustained release of the therapies of the present disclosure.
  • polymers for sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), ), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide ( PLA), poly(lactide-co-glycolide) (PLGA) and polyorthoesters.
  • Polymers for use in sustained release formulations can be inert, free of leachable impurities, stable on storage, sterile and biodegradable.
  • controlled-release or sustained-release systems can be placed near the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to those skilled in the art may be used.
  • Dosage can depend on several factors, including severity and responsiveness of the disease, route of administration, duration of treatment (days to months to years), and time to amelioration of the disease.
  • the present disclosure provides a kit comprising the engineered microorganism described in the present disclosure or the composition of engineered microorganism described in the present disclosure or the composition described in the present disclosure.
  • the kit may further comprise one or more of various conventional pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as described for the It will be apparent to those skilled in the art. Instructions may also be included in the kit, either as an insert or as a label, indicating the amounts of the components to be administered, directions for administration and/or directions for mixing the components.
  • the engineered microorganisms of the present disclosure comprise a gene encoding an enzyme capable of converting phenylalanine into phenylpyruvate, a gene encoding an enzyme capable of converting phenylpyruvate into phenylacetaldehyde, a gene encoding an enzyme capable of converting phenylacetaldehyde into phenylacetaldehyde,
  • One or more of the genes of the enzymes of ethanol, the genes encoding glutamate dehydrogenase, and/or the genes encoding phenylalanine transporters which can relieve and/or treat hyperphenylalanineemia related diseases and/or conditions.
  • the engineered microorganisms constructed in the present disclosure can effectively degrade phenylalanine and/or phenylpyruvate, and experiments have proved that the engineered microorganisms can effectively degrade phenylalanine and phenylalanine in the body of Pah R408 W mice with phenylketonuria Metabolites, showing good application prospects.
  • the present disclosure provides a method for alleviating and/or treating a disease and/or condition associated with hyperphenylalaninemia, the method comprising administering an engineered microorganism according to the present disclosure to a patient in need thereof Or the engineered microbial composition according to the present disclosure or the composition according to the present disclosure or the kit according to the present disclosure.
  • the engineered microorganisms described in the present disclosure or the engineered microorganism compositions described in the present disclosure can be provided for alleviating and/or treating diseases related to hyperphenylalaninemia and /or the application in the medicine of disease or health product.
  • the diseases and/or conditions associated with hyperphenylalaninemia include but are not limited to: phenylketonuria (such as classic or typical (classical or typical) phenylketonuria and atypical (atypical) phenylketonuria), permanent mild (mild) hyperphenylalaninemia, non-phenylketonuric hyperphenylalaninemia, phenylalanine hydroxylase deficiency, cofactor deficiency, dihydropterin reductase deficiency, tetrahydropterin synthase deficiency, Segawa's disease and liver disease.
  • phenylketonuria such as classic or typical (classical or typical) phenylketonuria and atypical (atypical) phenylketonuria
  • permanent mild (mild) hyperphenylalaninemia such as classic or typical (classical or typical) phenylketonuria and atypical (atypical) phenylketonuri
  • Table 1 Specific sequence information of enzymes, promoters and expression cassettes used in the examples
  • Table 2 The sgRNA sequence information corresponding to the insertion site in gene editing
  • N20NGG 20bp sequence connecting the NGG PAM sequence on the two strands of the target integration site sequence and blast the EcN genome.
  • the 300-500bp sequences upstream and downstream of the sgRNA were selected as the left homology arm (LHA) and right homology arm (RHA).
  • the sgRNA sequence was added to the 5' end of the gRNA backbone reverse primer, amplified by PCR and digested with restriction endonucleases PstI and SpeI, and the digested PCR product fragment was combined with the same digested plasmid pCBT003 (SEQ ID NO: 84, the plasmid map is shown in Figure 1) were ligated to form the pCBT003_sgRNA plasmid.
  • the target gene to be integrated into the EcN genome is synthesized by GeneScript on a cloning plasmid (eg, pUC57).
  • the target gene was amplified using the synthesized plasmid as a template, and the LHA and RHA of the selected integration site were amplified using the EcN genome as a template.
  • the PCR primers used to amplify these fragments have 15-20bp of homologous sequences to each other, so they can be joined by overlapping PCR to obtain LHA-ordered
  • the PCR product of the marker gene-RHA was used as the donor gene fragment.
  • the pCBT003_sgRNA plasmid expressing the sgRNA of the integration site obtained from 1.2 and the PCR product containing LHA-target gene-RHA obtained from 1.3 were used MultiS one-step cloning kit for connection, transformation into Top10 competent cells, screening with ampicillin resistance plate (100 ⁇ g/mL), picking transformants for colony PCR verification and sequencing, and selecting the correctly sequenced PCBT003-target gene- sgRNA recombinant plasmid.
  • the PCBT001 plasmid (plasmid expressing Cas9 protein, SEQ ID NO.85, plasmid map shown in Figure 2) was electrotransformed into E. ) on the LB solid plate to obtain EcN/PCBT001 transformants.
  • EcN/PCBT001 A single colony of EcN/PCBT001 was picked and cultured in LB liquid medium containing spectinomycin and streptomycin (50 ⁇ g/mL) overnight at 30°C and 220 rpm. The next day, the bacterial solution was inoculated into 30mL LB liquid medium according to the inoculum amount (V/V) of 1:100. When the bacterial concentration OD600 was 0.2, IPTG with a final concentration of 1mM was added for induction, and the culture continued for 2-3h to reach the OD value. 0.6-0.8 to prepare electroporation competent cells.
  • the PCBT003-target gene-sgRNA plasmid was electrotransfected into EcN/PCBT001 competent cells (electroporation conditions: 2.5kV, 200 ⁇ , 25 ⁇ F), and coated with ampicillin (50 ⁇ g/mL), spectinomycin and streptomycin ( 50 ⁇ g/mL) on LB plates, cultured for 1 day, and picked single colonies growing on the resistant plates.
  • Embodiment 2 Construction scheme of auxotrophic strain
  • the thyA gene was knocked out by CRISPR/Cas9 technology to realize the deletion of the thyA gene in the EcN strain.
  • the specific construction scheme is as follows. According to the construction scheme in Example 1, first construct the recombinant plasmid PCBT003-thyA-HA-sgRNA (thyA The sequence of -HA-sgRNA is shown in SEQ ID NO:131), and then it is co-electrotransduced with the pCBT001 plasmid expressing Cas9 protein into a competent EcN strain, and after removing the redundant plasmid, an auxotrophic EcN_ ⁇ thyA strain is obtained.
  • the uppercase ununderlined part is the left homology arm
  • the uppercase underlined part is the right homology arm
  • the uppercase and boxed part is the sgRNA sequence.
  • the key gene fliC of the flagellar system and the adhesion-related gene (cellulose synthase gene) bscA were knocked out by using the CRISPR/Cas9 gene editing system.
  • the specific construction plan is to first prepare a competent EcN_ ⁇ thyA strain, and then construct the recombinant plasmid PCBT003-fliC-HA-sgRNA/PCBT003-bscA-HA containing the donor fragment and sgRNA targeting fliC or bscA -sgRNA (the sequence of fliC-HA-sgRNA shown in SEQ ID NO:132, the sequence of bscA-HA-sgRNA shown in SEQ ID NO:133), together with the pCBT001 plasmid expressing Cas9 protein, electroporated the EcN strain and removed excess After plasmid extraction, an auxotrophic EcN_ ⁇ thyA ⁇ bscA ⁇ fliC strain was obtained.
  • the uppercase ununderlined part is the left homology arm
  • the uppercase underlined part is the right homology arm
  • the uppercase and boxed part is the sgRNA sequence.
  • sequence of bscA-HA-sgRNA (SEQ ID NO: 133):
  • the uppercase ununderlined part is the left homology arm
  • the uppercase underlined part is the right homology arm
  • the uppercase and boxed part is the sgRNA sequence.
  • Embodiment 4 Construction of engineering strains that degrade phenylalanine
  • EcN as the chassis bacterium or the knockout flagella gene and/or auxotrophic strain constructed in Example 2 or 3 as the chassis bacterium, knocking in the relevant gene that can convert phenylalanine into a product without toxic side effects, Thereby obtaining the engineering strain capable of degrading phenylalanine.
  • sequences of the expression cassettes used in the construction process are as follows: vanRAM repressor (SEQ ID NO:103), Pfnrs-TyrB-gdh2 (SEQ ID NO:104), Pfnrs-Aro10-yahk (SEQ ID NO:105), Pfnrs -pheDH-pheP (SEQ ID NO:106), Pvan-TyrB-gdh2 (SEQ ID NO:107), Pvan-Aro10-yahk (SEQ ID NO:108), Pvan-pheDH-pheP (SEQ ID NO:109) , PBAD-LAAD (SEQ ID NO: 110).
  • the optional insertion site of the target gene expression cassette in EcN is selected from: yicS site, malPT site, maeB site, nth/tppB site, tkrA site, malE site, exo site, rhtB/C site One or more of agaI/rsml site, araBD site, yghX site, ldhA site, araAB site, lacZ site and/or kefB site.
  • the bacterial strain E.coli Nissle1917 (EcN) genome is transformed, comprising the following steps:
  • PCBT003-yghX-sgRNA PCBT003-yghX-sgRNA
  • yghX target site X sequence
  • the PCBT003-yghX-sgRNA plasmid obtained above was digested with restriction endonucleases PstI and XbaI, and the digested large fragment (plasmid backbone) was gel-recovered.
  • Utilize amplification primer yghX-LH-f (SEQ ID NO.30) and yghX-LH-r1 (SEQ ID NO.31) and yghX-RH-f1 (SEQ ID NO.32) and yghX-RH-r (SEQ ID NO.33) Amplify the upstream homology arm fragment and the downstream homology arm fragment of the yghX site from the genome template of strain E.coli Nissle1917, and purify the PCR product.
  • pUC-fnrs-TyrB-gdh2 plasmid (SEQ ID NO.86) (GenScript Synthetics) as template, fnrs-TyrB-f1 (SEQ ID NO.28) and gdh2-r1 (SEQ ID NO.29) as template Primers, PCR amplification to obtain the Pfnrs-TyrB-gdh2 fragment, about 5260bp; the enzyme-digested fragment (plasmid backbone) recovered from the gel, the upstream and downstream homology arm PCR products and the Pfnrs-TyrB-gdh2 fragment recovered by purification, used MultiS one-step cloning kit for ligation, transformed into Top10 competent cells, screened with ampicillin resistance plate (100 ⁇ g/mL), picked transformants for colony PCR verification and sequencing, and selected PCBT003-Pfnrs-TyrB with correct sequencing - gdh2-yghX-sgRNA
  • the PCBT001 plasmid (SEQ ID NO.85) was electrotransformed into E. coli EcN chemically competent cells, and screened on LB solid plates containing spectinomycin hydrochloride (50 ⁇ g/mL) to obtain EcN/PCBT001 transformants.
  • EcN/PCBT001 A single colony of EcN/PCBT001 was picked and cultured in LB liquid medium containing spectinomycin (50 ⁇ g/mL) overnight at 30° C. and 220 rpm. The next day, the bacterial solution was inoculated into 30mL LB liquid medium according to the inoculum amount (V/V) of 1:100. When the bacterial concentration OD600 was 0.2, IPTG with a final concentration of 1mM was added for induction, and the culture continued for 2-3h to reach the OD value. 0.6-0.8 to prepare electroporation competent cells.
  • the PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA plasmid was electrotransfected into EcN/PCBT001 competent cells (electroporation conditions: 2.5kV, 200 ⁇ , 25 ⁇ F), and coated with ampicillin (50 ⁇ g/mL) and spectinomycin (50 ⁇ g/mL) on LB plates, cultured for 1d.
  • Pick a single bacterium colony grown on the resistant plate utilize primers yghX-verify-f (SEQ ID NO.34) and yghX-verify-r (SEQ ID NO.35) to verify the integration of the gene by the method of colony PCR,
  • the positive fragment is about 6662bp;
  • PstI and speI were used to digest the PCBT003 plasmid, and the digested fragment (about 3123bp) recovered from the gel was ligated with the PCR product recovered from purification and transformed into E.coli Top10 competent cells. Screen on the ampicillin resistance plate (100 ⁇ g/mL) to obtain the PCBT003-ldhA-sgRNA plasmid.
  • PCBT003-ldhA-sgRNA plasmid was digested with restriction endonucleases PstI and XbaI, and the digested large fragment (plasmid backbone) was gel-recovered.
  • amplification primers ldhA-LH-f (SEQ ID NO.40) and ldhA-LH-r1 (SEQ ID NO.41) and ldhA-RH-f1 (SEQ ID NO.42) and ldhA-RH-r (SEQ ID NO.43) amplify the upstream homology arm fragment of ldhA site and The downstream homology arm fragments are about 500bp respectively.
  • the digested fragment (plasmid backbone) recovered from the gel, the upstream and downstream homology arm PCR product and the Pfnrs-ARO10-YahK fragment recovered by purification were used
  • the MultiS one-step cloning kit was used for ligation, transformed into Top10 competent cells, and screened with ampicillin resistance plates (100 ⁇ g/mL) to obtain PCBT003-Pfnrs-TyrB-gdh2-ldhA-sgRNA recombinant plasmids.
  • the engineering bacterium CBT2001 was finally obtained, which is a derivative of Escherichia coli Nissle1917, and a single copy of the aromatic amino acid transaminase gene TyrB, glutamate dehydrogenase gene gdh2, aldehyde reductase gene YahK and Phenylpyruvate decarboxylase gene ARO10.
  • the genotype of the engineered strain CBT2001 Nissle_ ⁇ yghx::Pfnrs-TyrB-gdh2_ ⁇ ldha::Pfnrs-ARO10-YahK.
  • araAB-sgRNA-f SEQ ID NO.46
  • araAB-sgRNA-r SEQ ID NO.47
  • PstI and speI were used to digest the PCBT003 plasmid, and the digested fragment (about 3123bp) recovered from the gel was ligated with the PCR product recovered from purification and transformed into E.coli Top10 competent cells. Screen on the ampicillin resistance plate (100 ⁇ g/mL) to obtain the PCBT003-araAB-sgRNA plasmid.
  • PCBT003-araAB-sgRNA plasmid was digested with restriction enzymes PstI and XbaI, and the large fragment (plasmid backbone) digested was gel-recovered.
  • araAB-LH-f SEQ ID NO.50
  • araAB-LH-r SEQ ID NO.51
  • araAB-RH-f SEQ ID NO.52
  • araAB-RH-r SEQ ID NO.53
  • pUC-fnrs-TyrB-gdh2 plasmid (SEQ ID NO.86) (GenScript Synthetics) as template, fnrs-TyrB-f2 (SEQ ID NO.48) and gdh2-r2 (SEQ ID NO.49) as template Primers, PCR amplification to obtain the Pfnrs-TyrB-gdh2 fragment, about 4779bp.
  • the digested fragment (plasmid backbone) recovered from the gel, the upstream and downstream homology arm PCR product and the Pfnrs-TyrB-gdh2 fragment recovered by purification were used
  • the MultiS one-step cloning kit was used for ligation, transformed into Top10 competent cells, and screened with an ampicillin-resistant plate (100 ⁇ g/mL) to obtain the PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA recombinant plasmid.
  • lacZ-sgRNA-f SEQ ID NO.56
  • lacZ-sgRNA-r SEQ ID NO.57
  • the endonuclease PstI and speI were digested, and the PCR product was purified and recovered.
  • PstI and speI were used to digest the PCBT003 plasmid, and the digested fragment (about 3123bp) recovered from the gel was ligated with the PCR product recovered from purification and transformed into E.coli Top10 competent cells. Screen on the ampicillin resistance plate (100 ⁇ g/mL) to obtain the PCBT003-lacZ-sgRNA plasmid.
  • PCBT003-lacZ-sgRNA plasmid was digested with restriction endonucleases PstI and XbaI, and the digested large fragment (plasmid backbone) was gel-recovered.
  • lacZ-LH-f SEQ ID NO.60
  • lacZ-LH-r SEQ ID NO.61
  • lacZ-RH-f SEQ ID NO.62
  • lacZ-RH-r SEQ ID NO.63
  • the digested fragment (plasmid backbone) recovered from the gel, the upstream and downstream homology arm PCR product and the Pfnrs-ARO10-YahK fragment recovered by purification were used
  • the MultiS one-step cloning kit was used for ligation, transformed into Top10 competent cells, and screened with an ampicillin resistance plate (100 ⁇ g/mL) to obtain the PCBT003-Pfnrs-ARO10-YahK-lacZ-sgRNA recombinant plasmid.
  • the engineering bacteria CBT2002 was obtained through the above-mentioned examples, which is a derivative of CBT2001, by integrating a copy of the aromatic amino acid transaminase gene, glutamic acid dehydrogenase gene, aldehyde reductase gene and phenylpyruvate decarboxylation respectively on the CBT2001 genome enzyme gene.
  • kefB-sgRNA-f SEQ ID NO.66
  • kefB-sgRNA-r SEQ ID NO.67
  • PstI and speI were used to digest the PCBT003 plasmid, and the digested fragment (about 3123bp) recovered from the gel was ligated with the PCR product recovered from purification and transformed into E.coli Top10 competent cells. Screen on the ampicillin resistance plate (100 ⁇ g/mL) to obtain the PCBT003-kefB-sgRNA plasmid.
  • the PCBT003-kefB-sgRNA plasmid was digested with restriction endonucleases PstI and XbaI, and the large fragment (plasmid backbone) digested was gel-recovered. Utilize amplification primer kefB-LH-f (SEQ ID NO.70) and kefB-LH-r (SEQ ID NO.71) and kefB-RH-f (SEQ ID NO.72) and kefB-RH-r (SEQ ID NO.73) Amplify the upstream homology arm fragment and the downstream homology arm fragment of the kefB site from the EcN genome template, about 500bp respectively.
  • the digested fragment (plasmid backbone) recovered from the gel, the upstream and downstream homology arm PCR products and the Pfnrs-PheP fragment recovered by purification were used
  • the MultiS one-step cloning kit was used for ligation, transformed into Top10 competent cells, and screened with an ampicillin resistance plate (100 ⁇ g/mL) to obtain the PCBT003-Pfnrs-PheP-kefB-sgRNA recombinant plasmid.
  • the engineering bacteria CBT2003 which is a derivative of CBT2002, was finally obtained through the above examples.
  • a single copy of the phenylalanine transporter gene was further integrated into the genome of CBT2002 to enhance the uptake of phenylalanine by the strain.
  • the engineering strain introduces two copies of phenylalanine aminotransferase TyrB and a single copy of phenylalanine dehydrogenase pheDH (from Bacillus sp. SLBN-3, GenBank: TQJ42029.1) at different selected sites in the chassis bacterial genome.
  • the Pfnrs-TyrB-gdh2 expression cassette (SEQ ID NO: 104) was synthesized by GeneScript, and the sgRNA sequences targeting yghX and araAB are shown in Table 2. According to the methods of Examples 1 and 4, the sgRNA sequence and the expression cassette were sequentially inserted into the pCBT003 vector, thereby obtaining recombinant plasmids PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA and PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA; Subsequently, the above-mentioned recombinant plasmids and the pCBT001 plasmid expressing the Cas9 protein were electrotransformed into the chassis bacterium EcN ⁇ thyA and the redundant plasmids were removed to obtain the recombinant bacteria EcN ⁇ thyA ⁇ yghX::Pfnr
  • the Pfnrs-Aro10-yahk expression cassette (SEQ ID NO: 105) was synthesized by GeneScript, and the sgRNA sequences targeting ldhA and lacZ are shown in Table 2. According to the method of Examples 1 and 4, the sgRNA sequence and the expression cassette Pfnrs-Aro10-yahk were sequentially inserted into the pCBT003 vector, thereby obtaining recombinant plasmids PCBT003-Pfnrs-Aro10-yahk-ldhA-sgRNA and PCBT003-Pfnrs-Aro10-yahk -lacZ-sgRNA; then the above recombinant plasmids were co-electrotransformed with the pCBT001 plasmid expressing Cas9 protein into the engineered bacteria obtained in the previous step and the redundant plasmids were removed to obtain the recombinant bacteria EcN ⁇ thyA ⁇ yghX::Pfn
  • the PfnrS_pheDH_pheP expression cassette (SEQ ID NO: 106) was synthesized by GeneScript.
  • the codon-optimized pheDH gene sequence is shown in SEQ ID NO: 17, and the sgRNA sequence targeting kefB is shown in Table 2.
  • the sgRNA sequence and the expression cassette were sequentially inserted into the pCBT003 vector to obtain the recombinant plasmid PCBT003-PfnrS_pheDH_pheP-kefB-sgRNA;
  • the engineered bacteria obtained in one step and the excess plasmid removed the recombinant bacteria EcN ⁇ thyA ⁇ yghX::Pfnrs-TyrB-gdh2 ⁇ ldhA::Pfnrs-ARO10-yahk ⁇ araAB::Pfnrs-TyrB-gdh2 ⁇ lacZ::Pfnrs-ARO10-yahk ⁇ kefB::Pfnrs-pheDH-phep .
  • the PBAD_LAAD expression cassette (SEQ ID NO: 110) was synthesized by GeneScript, and the sgRNA sequence targeting rhtB/C is shown in Table 2. According to the method of Examples 1 and 4, the sgRNA sequence and the expression cassette were sequentially inserted into the pCBT003 vector to obtain the recombinant plasmid PCBT003-PBAD_LAAD-rhtB/C-sgRNA; then the above-mentioned recombinant plasmid and the pCBT001 plasmid expressing the Cas9 protein were co-electroporated After inserting the engineering bacteria obtained in the previous step and removing the redundant plasmid, the recombinant bacteria EcN ⁇ thyA ⁇ yghX::Pfnrs-TyrB-gdh2 ⁇ ldhA::Pfnrs-ARO10-yahk ⁇ araAB::Pfnrs-TyrB-gdh2 ⁇ lacZ:
  • CBT-201 strain on the genome of the chassis bacterium EcN ⁇ thyA ⁇ bscA ⁇ fliC, the same enzyme with the same copy number as that of the CBT-201 strain was transferred to obtain the CBT-203 strain with the following genotype:
  • the engineering strains CBT-202, CBT-209, and CBT-210 were constructed.
  • the genotypes of the above strains are as follows:
  • Genotype of CBT-202 EcN ⁇ thyA ⁇ ldhA::Pfnrs-pheDH-phep ⁇ yghX::Pfnrs-ARO10-yahk ⁇ maeB::Pfnrs-pheDH-phep ⁇ lacZ::Pfnrs-ARO10-yahk ⁇ rhtB/C::PBAD_LAAD
  • Genotype of CBT-210 EcN ⁇ thyA ⁇ yghX::Pfnrs-TyrB-gdh2 ⁇ ldhA::Pfnrs-ARO10-yahk ⁇ kefB::Pfnrs-pheP
  • the promoter used in all expression cassettes of the CBT-206 strain is Pvan.
  • the specific construction process and strain structure are as follows:
  • the Pvan-TyrB-gdh2 expression cassette (SEQ ID NO: 107) was synthesized by GeneScript, and the sgRNA sequences targeting yghX and araAB are shown in Table 2. According to the method of Examples 1 and 4, the sgRNA sequence and the expression cassette (Pvan-TyrB-gdh2 expression cassette and the vanRAM expression cassette shown in SEQ ID NO: 103) were sequentially inserted into the pCBT003 vector, thereby obtaining the recombinant plasmid PCBT003- Pvan-TyrB-gdh2-yghX-sgRNA and PCBT003-Pvan-TyrB-gdh2-araAB-sgRNA; then the above recombinant plasmids were co-electroporated with the pCBT001 plasmid expressing Cas9 protein and the chassis bacteria EcN ⁇ thyA ⁇ bscA ⁇ fliC was removed, and the recombinant bacteria EcN ⁇
  • the Pfnrs-Aro10-yahk expression cassette (SEQ ID NO: 108) was synthesized by GeneScript, and the sgRNA sequences targeting ldhA, agaI/rsml and yjcS are listed in Table 2.
  • the sgRNA sequence and the expression cassette (Pvan-Aro10-yahk expression cassette and the vanRAM expression cassette shown in SEQ ID NO: 103) were sequentially inserted into the pCBT003 vector to obtain the recombinant plasmid PCBT003- Pvan-Aro10-yahk-ldhA-sgRNA, PCBT003-Pvan-Aro10-yahk-agaI/rsml-sgRNA and PCBT003-Pvan-Aro10-yahk-yjcS-sgRNA; Then, the above-mentioned recombinant plasmids and the pCBT001 plasmid expressing the Cas9 protein were electrotransferred into the engineering bacteria obtained in the previous step and the redundant plasmids were removed to obtain the recombinant bacteria EcN ⁇ thyA ⁇ bscA ⁇ fliC ⁇ yghX::Pvan-TyrB-gd
  • the Pvan_pheDH_pheP expression cassette (SEQ ID NO: 109) was synthesized by GeneScript, and the sgRNA sequence targeting nth/tppB is shown in Table 2. According to the method of Examples 1 and 4, the sgRNA sequence and the expression cassette (Pvan_pheDH_pheP expression cassette and the vanRAM expression cassette shown in SEQ ID NO: 103) were sequentially inserted into the pCBT003 vector to obtain the recombinant plasmid PCBT003-Pvan_pheDH_pheP-nth/tppB -sgRNA; then the above-mentioned recombinant plasmid and the pCBT001 plasmid expressing Cas9 protein were co-electrically transferred into the engineering bacteria obtained in the previous step and the redundant plasmid was removed to obtain the recombinant bacteria EcN ⁇ thyA ⁇ bscA ⁇ fliC ⁇ yghX::Pvan-TyrB-gdh2 ⁇ y
  • the PBAD_LAAD expression cassette (SEQ ID NO: 110) was synthesized by GeneScript, and the sgRNA sequence targeting rhtB/C is shown in Table 2. According to the method of Example 1, the sgRNA sequence and the expression cassette (PBAD_LAAD expression cassette and the vanRAM expression cassette shown in SEQ ID NO: 103) were sequentially inserted into the pCBT003 vector to obtain the recombinant plasmid PCBT003-PBAD_LAAD-rhtB/C-sgRNA ; Subsequently, the above-mentioned recombinant plasmid and the pCBT001 plasmid expressing the Cas9 protein were jointly electrotransferred into the engineering bacteria obtained in the previous step and after removing the redundant plasmid, the recombinant bacteria EcN ⁇ thyA ⁇ bscA ⁇ fliC ⁇ yghX::Pvan-TyrB-gdh2 ⁇ yjcS::Pvan-ARO
  • the genotypes of the CBT-205, CBT-207, and CBT-208 strains are as follows:
  • Genotype of CBT-205 strain EcN ⁇ thyA ⁇ yghX::Pvan-TyrB-gdh2 ⁇ yjcS::Pvan-ARO10-yahk ⁇ araAB::Pvan-TyrB-gdh2 ⁇ ldhA::Pvan-ARO10-yahk ⁇ nth/tppB::Pvan-pheDH-phep ⁇ agaI/rsml:: Pvan-ARO10-yahk ⁇ rhtB/C::PBAD_LAAD
  • Genotype of CBT-207 strain EcN ⁇ thyA ⁇ yghX::Pvan-TyrB-gdh2 ⁇ yjcS::Pvan-ARO10-yahk ⁇ araAB::Pvan-TyrB-gdh2 ⁇ ldhA::Pvan-ARO10-yahk ⁇ nth/tppB::Pvan-pheDH-phep ⁇ agaI/rsml:: Pvan-ARO10-yahk ⁇ tkrA::Pvan-pheDH-phep ⁇ rhtB/C::PBAD_LAAD
  • strains CBT-212 and CBT-213 with promoters of Pvan and Pfnrs were constructed, and the genotype information is as follows:
  • Genotype of CBT-212 strain EcN ⁇ thyA ⁇ yghX::Pvan-TyrB-gdh2 ⁇ yjcS::Pvan-ARO10-yahk ⁇ araAB::Pfnrs-TyrB-gdh2 ⁇ lacZ::Pfnrs-ARO10-yahk ⁇ maeB::Pfnrs-pheDH-phep ⁇ agaI/rsml::Pfnrs- ARO10-yahk ⁇ rhtB/C::PBAD_LAAD
  • Genotype of CBT-213 strain EcN ⁇ thyA ⁇ yghX::Pvan-TyrB-gdh2 ⁇ yjcS::Pvan-ARO10-yahk ⁇ araAB::Pvan-TyrB-gdh2 ⁇ ldhA::Pvan-ARO10-yahk ⁇ maeB::Pfnrs-pheDH-phep ⁇ agaI/rsml::Pfnrs- ARO10-yahk ⁇ kefB::Pfnrs-pheDH- phep ⁇ rhtB/C::PBAD_LAAD
  • Example 5 Testing method for the ability of engineered bacteria to degrade phenylalanine in vitro
  • Chromatographic column Athena C18 liquid chromatographic column (250mm ⁇ 4.6mm, 5 ⁇ m); column temperature 35°C; gradient elution with 0.1% phosphoric acid water (V/V) and methanol as mobile phase; UV detector, detection wavelength 254nm, injection volume 10 ⁇ L.
  • Inoculate Glycerolbacterium EcN and each engineered bacteria in 5mL LB medium, culture overnight at 37°C. The next day, transfer the bacterial solution to 200mL LB medium with an inoculum of 1% (v/v), culture at 37°C, 220rpm for 5.5h; measure the OD 600 , centrifuge at 4000g for 8min, collect the bacterial cells, and wash with PBS buffer (containing 4mM phenylalanine) to resuspend the bacteria, adjust the OD 600 of the bacteria concentration to 2 or 0.5; take 10mL of the bacterial suspension in a 14mL shaking tube, position T 0h, put it at 37°C, 220rpm and incubate for 3h , sample 1 mL. Centrifuge at 12000rpm for 10min, collect supernatant samples for HPLC detection.
  • Example 6 The engineering bacteria transferred to the phenylalanine degradation pathway can effectively degrade phenylalanine
  • This example uses the method in Example 5 to test the ability of CBT2001/CBT2002 and CBT2003 to degrade phenylalanine in vitro.
  • CBT2001 is an engineering bacterium that integrates a single copy of the phenylalanine metabolism pathway gene in the present disclosure on the genome of the probiotic E.
  • CBT2002 integrated the double-copy phenylalanine metabolism pathway gene in CBT2001, and CBT2003 further strengthened the uptake of phenylalanine in the environment by the strain in CBT2002 A single copy of the phenylalanine transporter gene was integrated.
  • CBT2001, CBT2002 and CBT2003 engineering strains can all degrade phenylalanine to produce phenylethyl alcohol.
  • the in vitro detection results of each strain degrading phenylalanine are shown in Table 3 and Figure 3.
  • Table 3 The maximum production rate of phenylethanol produced by strains metabolizing phenylalanine
  • Table 4 The maximum production rate of phenylethanol produced by bacterial strains metabolizing phenylalanine
  • CBT2002 integrating two copies of related metabolic pathway genes is stronger than that of single copy CBT2001, and the addition of the phenylalanine transporter that can promote the uptake of phenylalanine makes CBT2003 have Stronger ability to metabolize phenylalanine.
  • Phenylalanine dehydrogenase has better metabolic activity
  • the engineered strain CBT-209 which uses phenylalanine dehydrogenase to replace the aromatic amino acid transaminase gene TyrB, was constructed, wherein phenylalanine dehydrogenase is the only metabolic enzyme that directly converts phenylalanine. Since the transamination of TyrB no longer occurs, there is no need to add the transamination reaction for coupling phenylalanine and the reduction reaction of phenylacetaldehyde. amino acid dehydrogenase.
  • the CBT-209 strain contained single copies of phenylalanine dehydrogenase (pheDH), phenylpyruvate decarboxylase (Aro10), aldehyde reductase yahk, and phenylalanine transporter (pheP).
  • pheDH phenylalanine dehydrogenase
  • Aro10 phenylpyruvate decarboxylase
  • pheP phenylalanine transporter
  • this example also constructed the CBT-210 engineering strain, which contained a single copy of aromatic amino acid transaminase (TyrB), phenylpyruvate decarboxylase (Aro10), glutamate dehydrogenase (Gdh2), aldehyde reducing The enzyme yahk and the phenylalanine transporter (pheP), of which phenylalanine aminotransferase (TyrB) is the only enzyme that directly converts phenylalanine.
  • TyrB aromatic amino acid transaminase
  • Aro10 phenylpyruvate decarboxylase
  • Gdh2 glutamate dehydrogenase
  • pheP phenylalanine transporter
  • LAAD L-amino acid deaminase
  • Flagella is related to the movement of Escherichia coli. Studies have shown that knocking out the flagellar synthesis/structure-related genes of Escherichia coli will cause defects in the flagellar structure of Escherichia coli, which will affect its motility and increase the residence time of Escherichia coli in the body (Zachary J.S. Mays, Todd C. Chappell, and Nikhil U. Nair ACS Synthetic Biology 2020 9(2), 356-367. DOI: 10.1021/acssynbio.9b00356).
  • knocking out flagella-related genes in the genome of engineered bacteria is expected to be an effective means to prolong the residence time of phenylalanine-metabolizing engineered bacteria in the intestinal tract of patients, thereby enhancing the persistence of phenylalanine degradation. Therefore, this example uses the method in Example 5 to test whether the knockout of flagella-related genes has adverse effects on the ability of engineering bacteria to degrade phenylalanine in vitro.
  • CBT-205 strains and CBT-207 strains are engineering bacteria with normal flagella function
  • CBT-206 strains and CBT-208 strains have knocked out the flagella-related genes bscA and fliC, from the results It can be seen (compare CBT-205 and CBT-206 in Figure 8, and CBT-207 and CBT-208 in Figure 9, respectively) that the knockout of flagella-related genes does not affect the function and activity of the strain.
  • Pvan promoter can endow engineered bacteria with stronger metabolic activity
  • This example uses the method in Example 5 to test the effect of the Pvan promoter compared with the Pfnrs promoter on the metabolic capacity of engineered bacteria.
  • Both CBT-201 and CBT-211 have the same enzyme and copy number, the only difference is that all expression cassettes/copies of CBT-201 use the PfnrS promoter, while all expression cassettes/copies of CBT-211 use Pvan promoter, the results are shown in Figure 10 (ie, the top two curves), the activity of CBT-211 is higher than that of CBT-201.
  • CBT-205 and CBT-212 are also transferred to the same enzyme and expression cassette copy number, of which CBT-205 uses 6 Pvan promoters, and CBT-212 uses 2 Pvan promoters promoter and 4 PfnrS promoters
  • the results showed that all the activities of CBT-205 using the Pvan promoter was higher than that of CBT-212 using both Pvan and PfnrS promoters (compare CBT-205 and CBT-212); the same phenomenon also occurred in CBT-207 and CBT-213 strains (see also Figure 11, compare CBT-207 and CBT-213).
  • the above results show that the Pfnrs promoter has shown satisfactory activity, and the Pvan promoter can endow the engineered bacteria with stronger in vitro metabolic activity than the Pfnrs promoter.
  • Example 5 the method in Example 5 was used to test the effect of the copy number of different expression cassettes on the metabolic capacity of engineered bacteria.
  • Both CBT-205 and CBT-207 used a better Pvan promoter, and CBT-207 added a copy of the pheDH expression cassette on the basis of CBT-205, as can be seen from Figure 10 (compare CBT-207 and CBT- 205), the metabolic efficiency of CBT-207 is significantly improved compared with CBT-205.
  • Comparative example 1 Construction of phenylalanine metabolism engineering bacteria SYNB1934
  • Synlogic's phenylalanine metabolism engineered bacterium SYNB1934 which is also constructed with EcN as the chassis bacteria, is currently in the phase III clinical research phase. According to previously reported data, SYBN1934 has achieved good metabolism in both animals and humans active.
  • This comparative example is based on the method published by Synlogic (for example, Isabella, VM, BNHa, MJCastillo, DJLubkowicz, SERowe, YAMillet, CLAnderson, N.Li, ABFisher, KAWest, PJReeder, MMMomin, CGBergeron, SEGuilmain, PFMiller, CBKurtz and D. Falb (2018).
  • SYNB1934 sexual reference to compare the metabolic activity of the disclosed engineering bacteria and SYNB1934.
  • the genotype information of SYNB1934 is as follows:
  • Embodiment 8 In vivo activity experiment of EcN and engineered bacterial strain
  • mice were subcutaneously injected with 0.05 mg/g (BW) phenylalanine solution (5 mg/ml), and 200 ⁇ L ( 5 ⁇ 10 10 cfu/mouse) probiotic EcN or engineering bacteria (engineering bacteria include engineering bacteria CBT-201, CBT-201-AA and CBT-205 of the present disclosure and SYNB1934 constructed in Comparative Example 1 as an activity reference) , 5 mice in each group, 4 hours after the injection of phenylalanine, serum was collected to detect the concentration of phenylalanine in the blood.
  • BW phenylalanine solution
  • 200 ⁇ L 5 ⁇ 10 10 cfu/mouse probiotic EcN or engineering bacteria
  • probiotic EcN or engineering bacteria engineering bacteria include engineering bacteria CBT-201, CBT-201-AA and CBT-205 of the present disclosure and SYNB1934 constructed in Comparative Example 1 as an activity reference
  • the changes in the concentration of phenylalanine in the blood of mice are shown in Figure 12.
  • the increase in the concentration of phenylalanine in the blood of mice administered with all selected engineered bacteria was significantly lower than that of mice administered with EcN, indicating that the engineered bacteria Can effectively control serum phenylalanine concentration.
  • the phenylalanine in the blood of the mice fed with CBT-201 engineering bacteria was reduced by as much as 55%, and its phenylalanine degradation ability was higher than that of the SYNB1934 engineering bacteria used as an active reference About 50% (SYNB1934 engineering bacteria can achieve 37% reduction in phenylalanine concentration).
  • CBT-201-AA and CBT-205 reached 44% and 53.1%, respectively, which were also significantly higher than the SYNB1934 engineering bacteria. It can be seen that the engineering bacteria constructed in the present disclosure exhibit Strong phenylalanine metabolic activity.
  • CBT-201-AA increased two copies of the Pfnrs-ARO10-yahk expression cassette compared with CBT-201, but the concentration of phenylalanine was reduced On the contrary, the amplitude decreased slightly, which also indicated that the copy number/expression of related enzymes in the phenylalanine metabolic pathway of the engineered bacteria was not the more the better.
  • the engineered bacteria constructed in the present disclosure can effectively degrade phenylalanine both in vitro and in vivo, and can be used to treat phenylketonuria.
  • the engineering bacteria constructed in the present disclosure can effectively degrade phenylalanine and/or phenylpyruvate.
  • the engineering bacteria can effectively degrade phenylalanine and phenylalanine metabolites in PKU mice, showing good results. application prospects.

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Abstract

一种工程微生物，其包含编码芳香族氨基酸转氨酶的基因、编码苯丙氨酸脱氢酶的基因、编码苯丙酮酸脱羧酶的基因、编码醛还原酶的基因、编码谷氨酸脱氢酶的基因和编码苯丙氨酸转运蛋白的基因中的一种或多种或其功能等效物。还涉及包含工程微生物的组合物；使用工程微生物或组合物来缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的方法；以及工程微生物或组合物在制备用于治疗与高苯丙氨酸血症相关的疾病和/或病症的药物或保健品中的用途。

Description

一种用于治疗高苯丙氨酸血症的工程微生物及其用途 技术领域
本公开涉及基因工程领域，具体涉及一种工程微生物以及工程微生物组合物，包含该工程微生物或工程微生物组合物的组合物，使用所述工程微生物或工程微生物组合物或来缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的方法，所述工程微生物或工程微生物组合物在制备用于缓解或治疗与高苯丙氨酸血症相关的疾病和/或病症的药物或保健品中的用途。
背景技术
高苯丙氨酸血症(hyperphenylalaninemia，HPA)是指由于苯丙氨酸代谢途径中的酶发生缺陷，例如苯丙氨酸羟化酶(PAH)、四氢生物喋呤合成酶或二氢生物喋呤还原酶等产生突变，导致血液中苯丙氨酸(Phe)水平过高的一类先天性苯丙氨酸代谢障碍疾病。其中苯丙酮尿症(phenylketonuria,PKU)是由于苯丙氨酸代谢途径中苯丙氨酸羟化酶(PAH)发生突变导致，使得苯丙氨酸不能转变成为酪氨酸，致使苯丙氨酸与α-酮戊二酸在血液与组织中堆积，并从尿中大量排出。健康人血液中苯丙氨酸的含量在100μM左右。通常认为人血液中的苯丙氨酸的含量在360μM～600μM以上，便会有苯丙酮尿症症状。在中国，苯丙酮尿症在新生儿中的发病率大概在1/11000，苯丙酮尿症患儿出生若不及时诊治，将会严重影响患儿的智力发育，引起癫痫和运动障碍等症状，并伴随黑色素合成障碍。
目前对于这种疾病的治疗主要是对食物的控制。苯丙酮尿症患者通过干预饮食或治疗，可以使血清苯丙氨酸水平保持在120-360μM范围内，可能有助于缓解与疾病相关的认知障碍。但这种治疗方式往往影响新生儿或青少年的成长，以及孕妇腹中胎儿的发育；饮 食治疗需要长期、不间断的治疗，给病人和家庭造成极大的经济负担。除了控制饮食外，近些年出现的经批准的苯丙酮尿症治疗药物有盐酸沙丙蝶呤和苯丙氨酸解氨酶。然而，盐酸沙丙蝶呤不能作为一种单独的治疗方法，需要与控制饮食结合使用；苯丙氨酸解氨酶是最近被批准用于治疗成人苯丙酮尿症患者，依靠全身注射苯丙氨酸氨裂解酶(PAL)，然而，该疗法常伴随严重的过敏反应和免疫介导的不良反应。科学家也在尝试基因疗法，将携带表达苯丙氨酸羟化酶基因(PAH)的cDNA重组腺病毒置于小鼠体内，以恢复肝脏PAH活性，但目前主要存在转运效率低的问题，且基因治疗的研发成本高，治疗费用昂贵，对于大多数病人来说接受程度低。
当前临床治疗方法比如饮食控制、酶辅助因子、酶制剂都没有达到很好的临床效果，亟需新的治疗方法。在新药研发的尝试中，AAV基因治疗由于显示出潜在的肝脏致癌毒性而部分公司暂停了临床试验，红细胞递送酶基因的方法显示出较低的表达水平而部分公司暂停了临床前研究。因此，本领域迫切需要开发得到一种新型有效的能够治疗或缓解苯丙氨酸代谢障碍相关疾病和/或病症的药物或保健品。
发明内容
为了寻求更新更有效的能够治疗或缓解苯丙氨酸代谢障碍相关疾病和/或病症的药物或保健品，本公开尝试使用工程微生物作为递送苯丙氨酸相关降解酶的载体，利用基因工程技术来改造出发微生物(例如肠道益生菌)，构建能够降解苯丙氨酸使之生成无毒害的中间代谢物、能够有效降低动物(包括人)体内苯丙氨酸水平的工程微生物。
根据本公开的一种实施方式，可以提供一种导入外源基因的工程微生物，其特征在于，所述外源基因包括：一种或更多种编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；一种或更多种编码能够将苯丙酮酸转化为苯乙醛的酶的基因；一种或更多种编码能够将苯乙醛转化为苯乙醇的酶的基因；以及一种或更多种编码能够将苯丙 氨酸转运至所述工程微生物体内的蛋白的基因。。
根据本公开的一种实施方式，可以提供一种工程微生物组合物，其包含多于一种工程微生物，所述多于一种工程微生物中的每一种彼此独立地包含以下基因中的任一种或更多种：
编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；
编码能够将苯丙酮酸转化为苯乙醛的酶的基因；
编码能够将苯乙醛转化为苯乙醇的酶的基因；
其中所述工程微生物组合物包含编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、编码能够将苯丙酮酸转化为苯乙醛的酶的基因以及编码能够将苯乙醛转化为苯乙醇的酶的基因。
根据本公开的一种实施方式，可以提供一种组合物，其包含根据本公开所述的工程微生物或工程微生物组合物和药学上、营养学上或生理学上可接受的载体。
根据本公开的一种实施方式，可以提供一种试剂盒，其包含根据本公开所述的工程微生物或根据本公开所述的工程微生物组合物或根据本公开所述的组合物。
根据本公开的一种实施方式，可以提供一种用于缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的方法，所述方法包括向有此需要的患者施用根据本公开所述的工程微生物或根据本公开所述的工程微生物组合物或根据本公开所述的组合物或根据本公开所述的试剂盒。
根据本公开的一种实施方式，可以提供本公开所述的工程微生物或根据本公开所述的工程微生物组合物或根据本公开所述的组合物在制备用于缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的药物或保健品中的用途。
附图说明
图1描绘了质粒PCBT003的图谱。
图2描绘了质粒PCBT001的图谱。
图3描绘了有氧条件下工程菌株EcN、CBT2001、CBT2002和 CBT2003降解苯丙氨酸的体外检测结果。
图4描绘了厌氧条件下工程菌株EcN、CBT2001、CBT2002和CBT2003降解苯丙氨酸的体外检测结果。
图5描绘了工程菌株CBT209和CBT210降解苯丙氨酸的体外检测结果。
图6描绘了工程菌株CBT201降解苯丙氨酸的体外检测结果。
图7描绘了工程菌株CBT202降解苯丙氨酸的体外检测结果。
图8描绘了工程菌株CBT205和CBT206降解苯丙氨酸的体外检测结果。
图9描绘了工程菌株CBT207和CBT208降解苯丙氨酸的体外检测结果。
图10描绘了工程菌株CBT201、CBT205、CBT207和CBT211降解苯丙氨酸的体外检测结果。
图11描绘了工程菌株CBT-201、CBT-212、CBT-213、CBT-205和CBT-207体外降解苯丙氨酸的检测结果。
图12描绘了工程菌株CBT-201、CBT-201-AA、CBT-205、阴性对照组EcN和活性参照组SYNB1934体内降解苯丙氨酸的结果。
具体实施方式
除非另有说明或与上下文相矛盾，否则本文中使用的术语或表述应结合本文整体内容并且如本领域普通技术人员所理解的进行阅读。除非另有限定，本文使用的所有技术和科学术语具有与由本领域的普通技术人员通常所理解的相同的意思。
本公开中所述“微生物”是指通常由单细胞组成的显微、亚显微或超微尺寸的生物体或微生物。微生物的例子包括但不限于细菌、病毒、寄生虫、真菌、某些藻类和原生动物。本公开中所述“工程微生物”是指经遗传修饰以表现出期望性能或特征的微生物。此外，在本公开中，术语“工程微生物”与“基因工程微生物”或“经遗传修饰的微生物”可互换使用。
本公开中所述“苯丙氨酸”用于指具有分子式C6H5CH2CH(NH2) COOH的氨基酸。苯丙氨酸是酪氨酸、多巴胺、去甲肾上腺素和肾上腺素的前体。L-苯丙氨酸是必需氨基酸，主要存在于膳食蛋白中的苯丙氨酸形式；发现立体异构体D-苯丙氨酸在膳食蛋白中的含量较低；DL-苯丙氨酸是两种形式的组合。苯丙氨酸可以指L-苯丙氨酸，D-苯丙氨酸和DL-苯丙氨酸中的一种或多种。
除非上下文特意说明或有明显矛盾，否则本公开中提及“苯丙酮酸”时还旨在涵盖苯丙酮酸盐。除非上下文特意说明或有明显矛盾，否则本公开中使用的“苯丙酮酸脱羧酶”与“苯丙酮酸盐脱羧酶”具有相同含义。
本公开中所述“芳香族氨基酸转氨酶(Aromatic amino acids transaminase)”指将苯丙氨酸转化或加工成苯丙酮酸的苯丙氨酸代谢酶。
本公开中所述“L-氨基酸脱氨酶(L-amino acid deaminase)”指将苯丙氨酸转化或加工成苯丙酮酸的苯丙氨酸代谢酶。
本公开中所述“苯丙氨酸脱氢酶(Phenylalanine dehydrogenase)”指将苯丙氨酸转化或加工成苯丙酮酸的苯丙氨酸代谢酶。
本公开中所述“苯丙氨酸代谢酶”指能够降解或转化苯丙氨酸的酶。本领域已知的任何苯丙氨酸代谢酶均能够由工程微生物编码。苯丙氨酸代谢酶包括但不限于芳香族氨基酸转氨酶、苯丙氨酸脱氢酶和L-氨基酸脱氨酶等。
本公开中所述“苯丙氨酸代谢物”是指由于苯丙氨酸降解而产生的代谢物。代谢物能够通过以苯丙氨酸为底物的酶直接催化苯丙氨酸产生，或者代谢物能够通过作用于苯丙氨酸代谢物的代谢途径下游不同的酶间接产生。苯丙氨酸代谢物可以由编码苯丙氨酸代谢酶的工程微生物产生。
本公开中所述“苯丙酮酸脱羧酶(Phenylpyruvate decarboxylase)”指将苯丙酮酸转化或加工成苯乙醛的苯丙酮酸代谢酶。
本公开中所述“α-酮酸脱羧酶(α-keto acid decarboxylase)”指将苯丙酮酸转化或加工成苯乙醛的苯丙酮酸代谢酶。
本公开中所述“醛还原酶(Aldehyde dehydrogenase)”指将苯乙 醛转化或加工成苯乙醇的苯乙醛代谢酶。
本公开中所述“谷氨酸脱氢酶(Glutamate dehydrogenase)”指将谷氨酸或谷氨酸盐转化或加工成α-酮戊二酸或α-酮戊二酸盐的谷氨酸(盐)代谢酶。
本公开中所述“苯丙氨酸转运蛋白(Phenylalanine transporter)”用于指能够将苯丙氨酸转运到细胞中的膜转运蛋白。在大肠杆菌中，苯丙氨酸转运蛋白基因编码负责苯丙氨酸转运的高亲和力苯丙氨酸特异性苯丙氨酸转运蛋白。苯丙氨酸转运蛋白可以由来源于细菌的苯丙氨酸转运蛋白基因编码，所述细菌包括但不限于乙酸钙不动杆菌(Acinetobacter calcoaceticus)，肠沙门氏菌(Salmonellaenteritidis)和大肠杆菌(Escherichia coli)。其它苯丙氨酸转运蛋白包括由arop基因编码的通用氨基酸转运蛋白，其能够以高亲和力转运包括苯丙氨酸在内的三种芳族氨基酸，并且被认为与苯丙氨酸转运蛋白一起负责大部分苯丙氨酸输入。另外，低水平的苯丙氨酸转运活性已经溯源于LIV-I/LS系统的活性，LIV-I/LS系统是由两种周质结合蛋白，LIV-结合蛋白(LIV-I系统)和LS-binding蛋白(LS系统)，以及膜组分LIVHMGF组成的支链氨基酸转运蛋白。
至少在一些实施方式中，本公开中的能够将苯丙氨酸转化为苯丙酮酸的酶、能够将苯丙酮酸转化为苯乙醛的酶、能够将苯乙醛转化为苯乙醇的酶、苯丙氨酸转运蛋白以及谷氨酸脱氢酶或其编码基因还包括上述酶、蛋白或其编码基因的功能等效物；所述“功能等效物”是指尽管氨基酸序列或多核苷酸序列或化学结构具有差异，但仍至少部分保留天然存在基因/酶的一种或多种生物功能的任何相关变异体。
本公开中所述“表达盒”是指能够在适当的宿主细胞中指导至少一种目的多核苷酸/编码基因的表达的核酸分子，至少在一些实施方式中，所述核酸分子还包含可操作地连接至目的多核苷酸/编码基因的启动子和/或其它调控元件。
本公开中所述“可操作地连接”指核酸序列以允许表达的核酸序列的方式(例如以顺式作用)连接至调节区域序列。调节区是可以指 导目的基因转录的核酸，例如启动子，可选的还可以任选的包括增强子、响应元件、蛋白质识别位点、诱导元件、启动子控制元件、蛋白质结合序列、5'和3'非翻译区、转录起始位点、终止序列、聚腺苷酸化序列和/或内含子。
本公开中所述“启动子”是指可以控制编码序列转录的多核苷酸序列。启动子序列包括足以使RNA聚合酶识别、结合和转录启动的特定序列。此外，启动子序列可以包括任选地在本公开提供的工程微生物中调节RNA聚合酶的识别、结合和转录启动活性的序列。启动子可以影响与自身位于同一核酸分子上的基因或与自身位于不同核酸分子上的基因的转录。
本公开中所述“诱导型启动子”指可以通过外部刺激，如化学、光、激素、应激、厌氧条件或病原体，在一种或多种细胞类型中启动在其控制下的编码序列或基因的转录水平增加的受调控的启动子。诱导型启动子和变体是本领域中众所周知的，包括但不限于PLteto1、galP1、PLlacO1、Pfnrs、PBAD和Pvan。
本公开中所述“直接诱导型启动子”是指与编码蛋白质或多肽的基因可操作地连接的调节区域，在所述调节区域的诱导物的存在下蛋白质或多肽被表达或抑制。
本公开中所述“间接诱导型启动子”是指包含两个或更多个调节区域的调节系统，例如，被可操作地连接至编码第一分子的基因的第一调节区域，例如，转录调节物，其能够调节被可操作地连接至编码效应物分子的基因的第二调节区域。在第一调节区域的诱导物的存在下，第二调节区域可以被活化或阻遏，从而活化或阻遏效应物分子的表达。直接诱导型启动子和间接诱导型启动子两者均被“诱导型启动子”所包括。
本公开中所述“组成型启动子”是指能够促进在其控制下和/或与其可操作地连接的编码序列或基因的连续转录的启动子。大肠杆菌Nissle1917(简称EcN)的组成型启动子和变体是本领域中众所周知的，包括但不限于BBa_J23119、BBa_J23101、BBa_J23102、BBa_J23103、BBa_J23109、BBa_J23110、BBa_J23114、 BBa_J23117、USP45_promoter、OmpA_promoter、BBa_J23100、BBa_J23104、BBa_J23105、BBa_I14018、BBa_J45992、BBa_J23118、BBa_J23116、BBa_J23115、BBa_J23113、BBa_J23112、BBa_J23111、BBa_J23108、BBa_J23107、BBa_J23106、BBa_I14033、BBa_K256002、BBa_K1330002、BBa_J44002、BBa_J23150、BBa_I14034、Oxb19、oxb20、BBa_K088007、Ptet、Ptrc、PlacUV5、BBa_K292001、BBa_K292000、BBa_K137031和BBa_K137029。
如本文所述“外源启动子”是指与编码区可操作结合的启动子，其中所述启动子不是生物体基因组中与编码区天然相关的启动子。基因组中与编码区天然相关或相连的启动子被称为该编码区的“内源启动子”。
本公开中所述“外源环境条件”或“环境条件”是指本文所述启动子被直接或间接诱导的环境，指工程微生物外部的环境或环境条件，与微生物的体外培养条件有关。“外源环境条件”还可以指工程微生物生长，生产和制造期间的条件。这样的条件包括有氧培养条件，厌氧培养条件，低氧培养条件和设定氧浓度下的其他条件。这样的条件还包括在培养基中存在化学和/或营养诱导剂，例如四环素，阿拉伯糖，IPTG，鼠李糖等。这样的条件还包括工程微生物在哺乳动物体内的环境温度。
本公开中所述“肠道”是指负责食物的转移和消化，营养的吸收以及废物的排泄的器官，腺体，道和系统。在人类中，肠道包含胃肠道(GI)，该胃肠道始于口腔，终止于肛门，另外还包含食道、胃、小肠和大肠。肠道还包括附属器官和腺体，例如脾脏，肝脏，胆囊和胰腺。上消化道由小肠的食道，胃和十二指肠组成。胃肠道下部包括小肠的其余部分，即空肠和回肠，以及所有大肠的全部，即盲肠，结肠，直肠和肛管。细菌可以在整个肠道中发现，例如胃肠道。
本公开中所述“氧水平依赖性启动子”或“氧水平依赖性调节区”是指一种或多种氧水平感应转录因子能够结合的核酸序列，其中相应转录的结合和/或活化因子激活下游基因表达。
本公开中所述“非致病性细菌”指不能在宿主中引起疾病或有害响应的细菌。非致病性细菌可以是共生细菌。非致病性细菌的实例包括但不限于芽孢杆菌属(Bacillus)、拟杆菌属(Bacteroides)、双歧杆菌属(Bifidobacterium)、短杆菌属(Brevibacteria)、梭菌属(Clostridium)、肠球菌属(Enterococcus)、大肠杆菌(Escherichia coli)、乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、酵母属(Saccharomyces)和葡萄球菌属(Staphylococcus)，凝结芽孢杆菌(Bacillus coagulans)、枯草芽孢杆菌(Bacillus subtilis)、脆弱拟杆菌(Bacteroides fragilis)、枯草拟杆菌(Bacteroides subtili)、多形拟杆菌(Bacteroides thetaiotaomicron)、两歧双歧杆菌(Bifidobacterium bifidum)、婴儿双歧杆菌(Bifidobacterium infantis)、乳双歧杆菌(Bifidobacterium lactis)、长双歧杆菌(Bifidobacterium longum)、丁酸梭菌(Clostridium butyricum)、屎肠球菌(Enterococcus faecium)、嗜酸乳杆菌(Lactobacillus acidophilus)、保加利亚乳杆菌(Lactobacillus bulgaricus)、干酪乳杆菌(Lactobacillus casei)、约翰逊氏乳杆菌(Lactobacillus johnsonii)、副干酪乳杆菌(Lactobacillus paracasei)、植物乳杆菌(Lactobacillus plantarum)、罗伊乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、乳酸乳球菌(Lactococcus lactis)和鲍氏酵母菌(Saccharomyces boulardii)。天然致病性细菌能够被遗传工程化为提供降低或消除致病性。
本公开中所述“益生菌”用于指活的、非致病性微生物，例如细菌，其能够对含有适当量该微生物的宿主生物体赋予健康益处。宿主生物体可以是哺乳动物，特别地可以是人类。
本公开中所述“营养缺陷型”是指宿主细胞(例如微生物菌株)的生长需要特定代谢物的外部来源，所述代谢物由于获得性遗传缺陷而不能合成。如本文所用，术语“营养缺陷型相关基因”是指宿主细胞(例如微生物如细菌)生存所需的基因。营养缺陷型相关基因可以是微生物为生存或生长所必需的营养素生产所必需的，或者可以是检测环境中调节转录因子活性的信号所必需的，其中信号的缺 失将导致细胞死亡。
如本文所用，术语“缓解”和“治疗”及其同义词指的是疾病、病症和/或状况的改善。“缓解”和“治疗”可以是至少一个可测量的物理参数的改善，该参数不一定是患者能够识别的。“缓解”和“治疗”也可以是物理上(例如，稳定可识别的症状)、生理学上(例如，稳定物理参数)或两者都抑制疾病、病症和/或状况的发展。“缓解”和“治疗”还可以是减缓疾病、病症和/或状况的发展或逆转。
在本文中使用时，术语“治疗”旨在涵盖“预防”。本文所用的“预防”及其同义词是指延迟特定疾病、病症和/或状况或与这些疾病、病症和/或状况相关的症状的发作或降低获得这些疾病、病症和/或状况的风险。
本公开中所述“药物组合物”指本公开所述的工程微生物或工程微生物组合物与其他成分如药学上可接受的载体和/或赋形剂的制剂。
本公开中所述“药学上可接受的载体”指用于工程微生物或工程微生物组合物的成药或给药的载体或稀释剂或佐剂，其本身并不是必要的活性成分，且施用后没有过分的毒性。合适的药学上可接受的载体是本领域普通技术人员所熟知的，包括但不限于碳酸氢钙、磷酸钙、多种糖和淀粉类型、纤维素衍生物、明胶、植物油、聚乙二醇和表面活性剂，包括例如聚山梨醇酯20。
本公开中所述“生理上可接受的载体”指不会对生物体引起显著刺激并且不会消除所施用的工程微生物或工程微生物组合物的生物学活性和特性的载体或稀释剂或佐剂。
本公开中所述“与高苯丙氨酸血症相关的疾病和/或病症”包括但不限于：苯丙酮尿症(例如经典型或典型(classical or typical)苯丙酮尿症和非经典型(atypical)苯丙酮尿症)，轻度(mild)高苯丙氨酸血症，非苯丙酮尿症类的高苯丙氨酸血症，苯丙氨酸羟化酶缺陷，辅因子缺陷，二氢蝶呤还原酶缺陷，四氢蝶呤合酶缺陷，Segawa氏病和肝病。
本公开中所述“高苯丙氨酸血症”或“过量的苯丙氨酸”指体内苯丙氨酸浓度增加或异常高。在一些实施方式中，高苯丙氨酸血症的 诊断信号是血液苯丙氨酸水平至少2mg/dl，至少4mg/dl，至少6mg/dl，至少8mg/dl，至少10mg/dl，至少12mg/dl，至少14mg/dl，至少16mg/dl，至少18mg/dl，至少20mg/dl或至少25mg/dl。
本公开中所述“高苯丙氨酸血症”包括但不限于苯丙酮尿症(例如经典型或典型(classical or typical)苯丙酮尿症和非经典型(atypical)苯丙酮尿症)，永久性轻度(mild)高苯丙氨酸血症，非苯丙酮尿症类的高苯丙氨酸血症，苯丙氨酸羟化酶缺陷，辅因子缺陷，二氢蝶呤还原酶缺陷，四氢蝶呤合酶缺陷，Segawa氏病和肝病。
本公开中所述“苯丙氨酸代谢障碍相关疾病和/或病症”与“与高苯丙氨酸血症相关的疾病和/或病症”同义，包括：苯丙酮尿症(例如经典型或典型(classical or typical)苯丙酮尿症和非经典型(atypical)苯丙酮尿症)，永久性轻度(mild)高苯丙氨酸血症，非苯丙酮尿症类的高苯丙氨酸血症，苯丙氨酸羟化酶缺陷，辅因子缺陷，二氢蝶呤还原酶缺陷，四氢蝶呤合酶缺陷，Segawa氏病和肝病。
工程微生物
本公开所述的工程微生物能够减少过量的苯丙氨酸。在一些实施方式中，工程微生物是细菌或酵母。在一些实施方式中，工程微生物是非致病性细菌。在一些实施方式中，工程微生物是共生菌。在一些实施方式中，工程微生物是真菌。在一些实施方式中，工程微生物是益生菌。在一些实施方式中，工程微生物是天然致病菌，其被修饰或突变以降低或消除致病性。在一些实施方式中，工程微生物是革兰氏阴性菌。在一些实施方式中，工程微生物是革兰氏阳性菌。在一些实施方式中，工程微生物选自包括但不限于拟杆菌属(Bacteroides)、双歧杆菌属(Bifidobacterium)、梭菌属(Clostridium)、埃希氏菌属(Escherichia)、乳杆菌属(Lactobacillus)和乳球菌属(Lactococcus)。
在一些实施方式中，工程微生物是大肠杆菌(Escherichia coli)。在一些实施方式中，工程微生物是大肠杆菌Nissle1917(E.coli Nissle)，一种“已演化为最佳表征的益生菌之一”的肠杆菌科 (Enterobacteriaceae)的革兰氏阴性细菌。该菌株的特征在于其完全无害，并且具有GRAS(generally recognized as safe，一般认为是安全的)状态。基因组测序证实，大肠杆菌Nissle缺乏突出的毒力因子(例如，大肠杆菌α-溶血素、P-菌毛粘附素(P-fimbrial adhesin))。另外，已经显示大肠杆菌Nissle不携带病原性粘附因子，不产生任何肠毒素或细胞毒素，不是侵入性的，而且不是尿道病原性的(uropathogenic)。早在1917年，大肠杆菌Nissle被包装为药物胶囊(称为Mutaflor)用于治疗用途。从那时起，大肠杆菌Nissle已被用于体内治疗人类中的溃疡性结肠炎，用于体内治疗人类炎性肠病、克罗恩病和储袋炎(Pouchitis)，并在体外抑制肠侵入性沙门氏菌属(Salmonella)、军团菌属(Legionella)、耶尔森氏菌属(Yersinia)和志贺氏菌属(Shigella)。普遍认为，大肠杆菌Nissle作为治疗载体的安全性和实用性已得到令人信服地证实。
在本公开的一些实施方式中，工程微生物为营养缺陷型。在本公开的一些实施方式中，营养缺陷型为尿嘧啶、胸腺嘧啶、亮氨酸、组氨酸、色氨酸、赖氨酸、甲硫氨酸、腺嘌呤、非天然存在的氨基酸、二氨基丙烯酸营养缺陷型中的任何一种或其组合。在本公开的一些实施方式中，营养缺陷型为二氨基丙烯酸营养缺陷型和/或胸腺嘧啶营养缺陷型。在本公开的一些实施方式中，当工程微生物存在于哺乳动物肠道时，营养缺陷型得到补充。在本公开的一些实施方式中，其中哺乳动物肠道是人肠道。在一些实施方式中，工程微生物包含至少一个营养缺陷型相关基因的失活或缺失。在一些实施方式中，所述营养缺陷型相关基因选自：thyA、cysE、glnA、ilvD、leuB、lysA、serA、metA、glyA、hisB、ilvA、pheA、proA、thrC、trpC、tyrA、uraA、dapF、flhD、metB、metC、proAB、yhbV、yagG、hemB、secD、secF、ribD、ribE、thiL、dxs、ispA、dnaX、adk、hemH、IpxH、cysS、fold、rplT、infC、thrS、nadE、gapA、yeaZ、aspS、argS、pgsA、yeflA、metG、folE、yejM、gyrA、nrdA、nrdB、folC、accD、fabB、gltX、ligA、zipA、dapE、dapA、der、hisS、ispG、suhB、tadA、acpS、era、rnc、fisB、eno、pyrG、chpR、 Igt、pgk、yqgD、metK、yqgF、plsC、ygiT、pare、ribB、cca、ygjD、tdcF、yraL、yihA、ftsN、murl、murB、birA、secE、nusG、rplJ、rplL、rpoB、rpoC、ubiA、plsB、lexA、dnaB、ssb、alsK、groS、psd、orn、yjeE、rpsR、chpS、ppa、valS、yjgP、yjgQ、dnaC、ribF、IspA、ispH、dapB、folA、imp、yabQ、flsL、flsl、murE、murF、mraY、murD、ftsW、murG、murC、ftsQ、ftsA、ftsZ、IpxC、secM、secA、can、folK、hemL、yadR、dapD、map、rpsB、in/B、nusA、ftsH、obgE、rpmA、rplU、ispB、murA、yrbB、yrbK、yhbN、rpsl、rplM、degS、mreD、mreC、mreB、accB、accC、yrdC、def、fint、rplQ、rpoA、rpsD、rpsK、rpsM、entD、mrdB、mrdA、nadD、hlepB、rpoE、pssA、yfiO、rplS、trmD、rpsP、ffh、grpE、yfjB、csrA、ispF、ispD、rplW、rplD、rplC、rpsJ、fusA、rpsG、rpsL、trpS、yr/F、asd、rpoH、ftsX、ftsE、ftsY、frr、dxr、ispU、rfaK、kdtA、coaD、rpmB、djp、dut、gmk、spot、gyrB、dnaN、dnaA、rpmH、rnpA、yidC、tnaB、glmS、glmU、wzyE、hemD、hemC、yigP、ubiB、ubiD、hemG、secY、rplO、rpmD、rpsE、rplR、rplF、rpsH、rpsN、rplE、rplX、rplN、rpsQ、rpmC、rplP、rpsC、rplV、rpsS、rplB、cdsA、yaeL、yaeT、lpxD、fabZ、IpxA、IpxB、dnaE、accA、tilS、proS、yafF、tsf、pyrH、olA、rlpB、leuS、Int、glnS、fldA、cydA、in/A、cydC、ftsK、lolA、serS、rpsA、msbA、IpxK、kdsB、mukF、mukE、mukB、asnS、fabA、mviN、rne、yceQ、fabD、fabG、acpP、tmk、holB、lolC、lolD、lolE、purB、ymflC、minE、mind、pth、rsA、ispE、lolB、hemA、prfA、prmC、kdsA、topA、ribA、fabi、racR、dicA、yd B、tyrS、ribC、ydiL、pheT、pheS、yhhQ、bcsB、glyQ、yibJ和gpsA中的任何一个或其组合。
在本公开的一些实施方式中，所述工程微生物的鞭毛结构经过改造后出现功能缺陷或者丧失，使得所述工程微生物在肠道中的滞留时间增加，这将更有利于苯丙氨酸的体内代谢。在本公开的一些实施方式中，鞭毛结构的改造是通过部分或者全部鞭毛合成相关基因的突变或者缺失而实现的。在本公开的一些实施方式中，所述工 程微生物为大肠杆菌，鞭毛合成相关基因为bscA和/或fliC基因。
酶和外源基因
根据本公开的一种实施方式，可以提供一种导入外源基因的工程微生物，其中所述外源基因包括：一种或更多种编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；一种或更多种编码能够将苯丙酮酸转化为苯乙醛的酶的基因；一种或更多种编码能够将苯乙醛转化为苯乙醇的酶的基因；以及一种或更多种编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因。在本公开的一些实施方式中，所述工程微生物能够在人和/或哺乳动物肠道内代谢苯丙氨酸。在本公开的一些实施方式中，外源基因整合在基因组中或者位于表达质粒上。
在本公开的一些实施方式中，所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因选自编码转氨酶的基因、编码脱氢酶的基因、编码脱氨酶的基因、及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。
在本公开的一些实施方式中，所述编码脱氢酶的基因是编码苯丙氨酸脱氢酶的基因；在另一些实施方式中，所述能够将苯丙氨酸转化为苯丙酮酸的酶包括苯丙氨酸脱氢酶和转氨酶(例如芳香族氨基酸转氨酶)。本公开意外的发现，至少在一些实施方式中，使用苯丙氨酸脱氢酶相比转氨酶能够一定程度上赋予工程微生物更多的代谢优势。在本公开的一些实施方式中，所述编码苯丙氨酸脱氢酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码苯丙氨酸脱氢酶的基因源自脲芽孢八叠球菌和/或芽孢杆菌，例如脲芽孢八叠球菌SCRC-R04(Sporosarcina ureae SCRC-R04)、球形赖氨酸芽孢杆菌SCRC-R79a(Lysinibacillus sphaericus SCRC-R79a)、栗褐芽胞杆菌(Bacillus badius)、和/或芽孢杆菌SLBN-3(Bacillus sp.SLBN-3)。在本公开的一些实施方式中，所述编码苯丙氨酸脱氢酶的基因选自芽孢杆菌SLBN-3的PheDH(Phenylalanine Dehydrogenase)。在本公开的一些实施方式中，所述苯丙氨酸脱氢 酶具有如SEQ ID NO:6所示的氨基酸序列；或者编码所述苯丙氨酸脱氢酶的基因具有如SEQ ID NO:17所示的核苷酸序列。
在本公开的一些实施方式中，编码转氨酶的基因是编码芳香族氨基酸转氨酶的基因。在本公开的一些实施方式中，所述编码芳香族氨基酸转氨酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码芳香族氨基酸转氨酶的基因源自大肠杆菌和/或酵母，例如大肠杆菌BL21(DE3)(Escherichia coli BL21(DE3))和/或酿酒酵母S288C(Saccharomyces cerevisiae S288C)。在本公开的一些实施方式中，所述编码芳香族氨基酸转氨酶的基因选自大肠杆菌BL21(DE3)的TyrB(Tyrosine aminotransferase)或酿酒酵母S288C的ARO8(Bifunctional 2-aminoadipate transaminase/aromatic-amino-acid:2-oxoglutarate transaminase)。在本公开的一些实施方式中，所述芳香族氨基酸转氨酶具有如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；或者编码所述芳香族氨基酸转氨酶的基因具有如SEQ ID NO:15或SEQ ID NO:90所示的核苷酸序列。
在本公开的一些实施方式中，所述工程微生物还包含编码谷氨酸脱氢酶的基因及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性，特别是当工程微生物中包含转氨酶(例如芳香族氨基酸转氨酶)时。谷氨酸脱氢酶可以将苯丙氨酸的转氨反应与苯乙醛的还原反应偶联起来，实现了辅因子的再生，为转氨反应提供了更多的α-酮戊二酸(α-ketoglutaric acid)，进一步提高了苯丙氨酸代谢速率。在本公开的一些实施方式中，所述编码谷氨酸脱氢酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码谷氨酸脱氢酶的基因源自酵母和/或艰难梭菌，例如酿酒酵母S288C和/或艰难梭菌(Clostridioides difficile)。在本公开的一些实施方式中，所述编码谷氨酸脱氢酶的基因选自酿酒酵母S288C的或艰难梭菌的GDH2(Glutamate dehydrogenase(NAD+))。在本公开的一些实施方式中，所述谷氨酸脱氢酶具有如SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列；或者编码所述谷氨酸脱氢酶的基因具有如SEQ ID NO:20或SEQ ID NO:97所示的核苷酸序 列。
在本公开的一些实施方式中，所述编码脱氨酶的基因是编码L-氨基酸脱氨酶的基因。在本公开的一些实施方式中，所述编码L-氨基酸脱氨酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码L-氨基酸脱氨酶的基因源自大肠杆菌、酵母、和/或奇异变形杆菌，例如大肠杆菌BL21(DE3)、酿酒酵母S288C、和/或奇异变形杆菌HI4320(Proteus mirabilis HI4320)。在本公开的一些实施方式中，所述编码L-氨基酸脱氨酶的基因选自大肠杆菌BL21(DE3)、酿酒酵母S288C、或奇异变形杆菌HI4320的LAAD(L-amino acid deaminase)。在本公开的一些实施方式中，所述L-氨基酸脱氨酶具有如SEQ ID NO:89所示的氨基酸序列，或者编码所述L-氨基酸脱氨酶的基因具有如SEQ ID NO:91所示的核苷酸序列。
在本公开的一些实施方式中，所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因包括以下组中的至少一项：(1)编码苯丙氨酸脱氢酶的基因；(2)编码芳香族氨基酸转氨酶的基因；(3)编码芳香族氨基酸转氨酶的基因和编码苯丙氨酸脱氢酶的基因；(4)编码芳香族氨基酸转氨酶的基因和编码L-氨基酸脱氨酶的基因；(5)编码苯丙氨酸脱氢酶的基因和编码L-氨基酸脱氨酶的基因；或(6)编码芳香族氨基酸转氨酶的基因、编码苯丙氨酸脱氢酶的基因和编码L-氨基酸脱氨酶的基因。
在本公开的一些实施方式中，所述编码能够将苯丙酮酸转化为苯乙醛的酶的基因选自编码苯丙酮酸脱羧酶的基因、编码α-酮酸脱羧酶的基因、及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。在本公开的一些实施方式中，所述编码苯丙酮酸脱羧酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码苯丙酮酸脱羧酶的基因源自酵母，例如酿酒酵母S288C。在本公开的一些实施方式中，所述编码苯丙酮酸脱羧酶的基因是酿酒酵母S288C的ARO10(Phenylpyruvate decarboxylase)。在本公开的一些实施方式中，所述苯丙酮酸脱羧酶具有如SEQ ID NO:7所示的氨基酸序列；或者编码所述苯丙酮酸脱 羧酶的基因具有如SEQ ID NO:18所示的核苷酸序列。在本公开的一些实施方式中，所述编码α-酮酸脱羧酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码α-酮酸脱羧酶的基因源自酵母和/或奇异变形杆菌，例如酿酒酵母S288C和/或奇异变形杆菌JN458(Proteus mirabilis JN458)。在本公开的一些实施方式中，所述编码α-酮酸脱羧酶的基因选自酿酒酵母S288C的或奇异变形杆菌JN458的KDC(Alpha-keto-acid decarboxylase)的至少一种。在本公开的一些实施方式中，所述α-酮酸脱羧酶具有如SEQ ID NO:8所示的序列；或者编码所述α-酮酸脱羧酶的基因具有如SEQ ID NO:95所示的核苷酸序列。
在本公开的一些实施方式中，所述编码能够将苯乙醛转化为苯乙醇的酶的基因选自编码醛还原酶的基因及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。在本公开的一些实施方式中，所述编码醛还原酶的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，所述编码醛还原酶的基因源自大肠杆菌和/或短乳杆菌，例如大肠杆菌str.K-12substr.MG1655和/或短乳杆菌(Lactobacillus brevis)。在本公开的一些实施方式中，所述编码醛还原酶的基因选自大肠杆菌str.K-12substr.MG1655的YahK(NADPH-dependent aldehyde reductase)或短乳杆菌的ADH(Alcohol dehydrogenase)。在本公开的一些实施方式中，所述醛还原酶具有如SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列；或者编码所述醛还原酶的基因具有如SEQ ID NO:19或SEQ ID NO:96所示的核苷酸序列。
在本公开的一些实施方式中，所述编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因选自编码苯丙氨酸转运蛋白的基因及其功能等效物中的一种或更多种，所述功能等效物至少保留相关酶的部分活性。在本公开的一些实施方式中，所述编码苯丙氨酸转运蛋白的基因源自病毒、真菌和/或细菌。在本公开的一些实施方式中，苯丙氨酸转运蛋白由来源于细菌的苯丙氨酸转运蛋白基因编码，所述细菌包括但不限于乙酸钙不动杆菌(Acinetobacter  calcoaceticus)，肠沙门氏菌(Salmonellaenteritidis)和大肠杆菌(Escherichia coli)。在本公开的一些实施方式中，所述编码苯丙氨酸转运蛋白的基因源自大肠杆菌。在一些实施方式中，苯丙氨酸转运蛋白由来源于细菌的PHEP基因编码。在一些实施方式中，苯丙氨酸转运蛋白由来源于细菌的AROP基因编码。在一些实施方式中，苯丙氨酸转运蛋白由来源于细菌的LIV-结合蛋白和LS-结合蛋白以及LIVHMGF基因编码。在一些实施方式中，工程微生物包含至少一种以上类型的苯丙氨酸转运蛋白，所述苯丙氨酸转运蛋白选自PHEP、AROP和LIV-I/LS。在本公开的一些实施方式中，所述编码苯丙氨酸转运蛋白的基因是大肠杆菌的PheP(Phenylalanine:H(+)symporter)。在本公开的一些实施方式中，所述苯丙氨酸转运蛋白具有如SEQ ID NO:13所示的氨基酸序列；或者编码所述苯丙氨酸转运蛋白的基因具有如SEQ ID NO:21所示的核苷酸序列。
在本公开的一些实施方式中，所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、所述编码能够将苯丙酮酸转化为苯乙醛的酶的基因、所述编码能够将苯乙醛转化为苯乙醇的酶的基因、所述编码谷氨酸脱氢酶的基因、和/或所述编码苯丙氨酸转运蛋白的基因中的任一个、两个、三个、四个、五个、六个、多个或全部可选地与一个或更多个相同或不同的启动子可操作地连接。在本公开的一些实施方式中，所述编码芳香族氨基酸转氨酶的基因和编码谷氨酸脱氢酶的基因共用同一启动子。在本公开的一些实施方式中，所述编码苯丙酮酸脱羧酶的基因和编码醛还原酶的基因共用同一启动子。在本公开的一些实施方式中，所述编码苯丙氨酸脱氢酶的基因和编码苯丙氨酸转运蛋白的基因共用同一启动子。在本公开的一些实施方式中，所述编码L-氨基酸脱氨酶的基因单独与启动子可操作地连接。在本公开的一些实施方式中，所述编码苯丙氨酸转运蛋白的基因单独与启动子可操作地连接。在本公开的一些实施方式中，所述启动子为内源启动子或外源启动子。在本公开的一些实施方式中，所述启动子为诱导型启动子或组成型启动子。在本公开的一些实施方式中，所述启动子为直接或间接诱导型启动子。在一些实施方式 中，所述启动子为pH依赖性启动子。在一些实施方式中，启动子为氧水平依赖性启动子。在本公开的一些实施方式中，所述启动子由外源环境条件直接或间接诱导。在本公开的一些实施方式中，外源环境条件是指哺乳动物肠道中的外源环境条件。在本公开的一些实施方式中，哺乳动物肠道是指人肠道。在一些实施方式中，外源环境条件是指哺乳动物的上胃肠道。在一些实施方式中，外源环境条件是指哺乳动物的下胃肠道。在一些实施方式中，外源性环境条件是指哺乳动物的小肠。在一些实施方式中，外源环境条件是指低氧、微需氧或厌氧条件，例如哺乳动物肠道的环境。在一些实施方式中，外源环境条件是指在健康或疾病状态中哺乳动物肠道中存在的分子或代谢物。在一些实施方式中，外源环境条件是组织特异性或疾病特异性代谢产物或分子。在一些实施方式中，外源环境条件是低pH环境。在一些实施方式中，所述启动子选自PfnrS、FDHF、Ptet、Pbba、Ptrc、Pvan、或PBAD中的至少一种。在本公开的一些实施方式中，所述PfnrS启动子的序列如SEQ ID NO.22所示。在本公开的一些实施方式中，所述FDHF启动子的序列如SEQ ID NO.24所示。在本公开的一些实施方式中，所述Ptet启动子的序列如SEQ ID NO.100所示。在本公开的一些实施方式中，所述Pbba启动子的序列如SEQ ID NO.98所示。在本公开的一些实施方式中，所述Ptrc启动子的序列如SEQ ID NO.99所示。在本公开的一些实施方式中，所述Pvan启动子为香草酸(vanillic acid)诱导的启动子，序列如SEQ ID NO:101所示。在本公开的一些实施方式中，所述PBAD启动子的序列如SEQ ID NO:102所示。已经知晓的是，香草酸启动子的活性可被抑制子表达盒VanRAM(其编码序列如SEQ ID NO:111所示)所抑制，通过添加香草酸可以解除上述抑制，从而使香草酸启动子表现出启动子的活性。通过这个机制，可以实现启动子的诱导调控，当工程微生物内源不存在VanRAM时，香草酸启动子就处于持续激活状态，此时，为了实现诱导性表达，就需要额外添加VanRAM。因此在本公开的一些实施方式中，当启动子是Pvan启动子时，还可以在工程微生物基因组中同时转入能够表达一个或更多个拷贝的香 草酸抑制子VanRAM的表达盒。
在本公开的一些实施方式中，所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、所述编码能够将苯丙酮酸转化为苯乙醛的酶的基因、所述编码能够将苯乙醛转化为苯乙醇的酶的基因、所述编码谷氨酸脱氢酶的基因、和/或所述编码苯丙氨酸转运蛋白的基因位于一个或多个表达盒中。在本公开的一些实施方式中，所述表达盒以单拷贝、双拷贝、三拷贝、四拷贝或更多拷贝的形式存在。在一些实施方式中，所述表达盒存在于质粒上。在本公开的一些实施方式中，所述表达盒稳定整合在所述工程微生物的一个、两个、或多个相同或者不同的基因组位点中。在本公开的一些实施方式中，所述基因组位点选自yicS位点、malPT位点、malE位点、exo位点、rhtB/C位点、agaI/rsml位点、araBD位点、yghx位点、ldhA位点、araAB位点、lacZ位点、kefB位点、maeB位点、nth/tppB位点、和/或tkrA位点中的至少一个。
在本公开的一些实施方式中，本公开的工程微生物包括第一表达盒和第二表达盒，其中第一表达盒包含第一启动子、以及与其可操作连接的所述编码芳香族氨基酸转氨酶的基因和所述编码谷氨酸脱氢酶的基因；第二表达盒包含第二启动子、以及与其可操作连接的所述编码苯丙酮酸脱羧酶的基因和编码醛还原酶的基因；所述第一或第二启动子是相同的或者不同的。在一些实施方式中，所述工程微生物还包含第三表达盒，所述第三表达盒包含可操作地连接至第三启动子的所述编码苯丙氨酸转运蛋白的基因，所述第三启动子可以与第一启动子和/或第二启动子相同，或者与第一启动子和第二启动子均不同。在本公开的一些实施方式中，所述第一表达盒以单拷贝或双拷贝的形式存在。在本公开的一些实施方式中，所述第二表达盒以单拷贝、双拷贝、三拷贝或四拷贝的形式存在。在本公开的一些实施方式中，所述第三表达盒以单拷贝的形式存在。在本公开的一些实施方式中，所述第一表达盒整合在在yghx位点和/或araAB位点。在本公开的一些实施方式中，所述第二表达盒整合在ldhA位点、lacZ位点、yghx位点、yjcS位点和/或agaI/rsml位点。 在本公开的一些实施方式中，所述第三表达盒整合在kefB位点。
在本公开的一些实施方式中，本公开的工程微生物包含第四表达盒和第二表达盒，其中第四表达盒包含第四启动子、以及与其可操作连接的所述编码苯丙氨酸脱氢酶的基因和编码苯丙氨酸转运蛋白的基因；第二表达盒包含第二启动子、以及与其可操作连接的所述编码苯丙酮酸脱羧酶的基因和编码醛还原酶的基因；所述第四或第二启动子是相同的或者不同的。在一些实施方式中，所述工程微生物还包含第一表达盒，所述第一表达盒包含第一启动子、与其可操作连接的所述编码芳香族氨基酸转氨酶的基因和所述编码谷氨酸脱氢酶的基因；所述第一启动子可以与第四启动子和/或第二启动子相同，或者与第四启动子和第二启动子均不同。在一些实施方式中，所述工程微生物还包含第五表达盒，所述第五表达盒包含第五启动子、与其可操作连接的所述编码L-氨基酸脱氨酶的基因；所述第五启动子与第四启动子、第二启动子和/或第一启动子相同或不同。在本公开的一些实施方式中，所述表达盒以单拷贝、双拷贝、三拷贝、四拷贝或更多拷贝的形式存在。在本公开的一些实施方式中，所述第四表达盒以单拷贝或双拷贝的形式存在。在本公开的一些实施方式中，所述第二表达盒以单拷贝、双拷贝、三拷贝或四拷贝的形式存在。在本公开的一些实施方式中，所述第一表达盒以单拷贝或双拷贝的形式存在。在本公开的一些实施方式中，所述第五表达盒以单拷贝的形式存在。在本公开的一些实施方式中，所述第四表达盒整合在kefB位点、ldhA位点、nth/tppB位点、maeB位点和/或tkrA位点。在本公开的一些实施方式中，所述第二表达盒整合在ldhA位点、yghx位点、lacZ位点、yjcS位点和/或agaI/rsml位点。在本公开的一些实施方式中，所述第一表达盒整合在yghX位点和/或araAB位点。在本公开的一些实施方式中，所述第五表达盒整合在rhtB/C位点。
在本公开的一些实施方式中，所述第一启动子、第二启动子、第三启动子、第四启动子的全部或者部分选自PfnrS(序列如SEQ ID NO:22-23中任一项所示)和/或Pvan(序列如SEQ ID NO:101所示) 中的至少一个，所述第五启动子是PBAD(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第四表达盒，所述第四表达盒整合在kefB位点；双拷贝的第二表达盒，所述第二表达盒分别整合在ldhA和lacZ位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)，第五表达盒中使用的启动子为PBAD启动子(序列如SEQ ID NO:101所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第四表达盒，所述第四表达盒整合在nth/tppB位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、ldhA和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为Pvan启动子(序列如SEQ ID NO:101所示)，第五表达盒中使用的启动子为PBAD启动子(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括双拷贝的第四表达盒，所述第四表达盒分别整合在nth/tppB和tkrA位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、ldhA和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为Pvan启动子(序列如SEQ ID NO:101所示)，第五表达盒中使用的启动子为PBAD启动子(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第四表达盒，所述第四表达盒整合在maeB位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、lacZ和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位 点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述单拷贝的第四表达盒使用PfnrS启动子，三拷贝的第二表达盒分别使用PfnrS启动子(序列如SEQ ID NO:22中任一项所示)、PfnrS启动子(序列如SEQ ID NO:22所示)和Pvan启动子(序列如SEQ ID NO:101所示)，双拷贝的第一表达盒分别使用PfnrS启动子(序列如SEQ ID NO:22所示)和Pvan启动子(序列如SEQ ID NO:101所示)，第五表达盒中使用的启动子为PBAD启动子(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括双拷贝的第四表达盒，所述第四表达盒整合在maeB和kefB位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、ldhA和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述双拷贝的第四表达盒使用PfnrS启动子，三拷贝的第二表达盒分别使用Pvan启动子、Pvan启动子和PfnrS启动子(序列如SEQ ID NO:22所示)，双拷贝的第一表达盒使用Pvan启动子(序列如SEQ ID NO:101所示)，第五表达盒使用的启动子为PBAD启动子(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第四表达盒，所述第四表达盒整合在kefB位点；四拷贝的第二表达盒，所述第二表达盒分别整合在ldhA和lacZ位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)，第五表达盒中使用的启动子为PBAD启动子(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括双拷贝的第四表达盒，所述第四表达盒整合在ldhA和maeB位点；双拷贝的第二表达盒，所述第二表达盒分别整合在yghX和lacZ位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中， 所述第四和第二表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)，第五表达盒中使用的启动子为PBAD启动子(序列如SEQ ID NO:102所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第四表达盒，所述第四表达盒整合在ldhA位点；以及单拷贝的第二表达盒，所述第二表达盒整合在yghX位点；其中，所述第四和第二表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第一表达盒，所述第一表达盒整合在yghX位点；单拷贝的第二表达盒，所述第二表达盒整合在ldhA位点；以及单拷贝的第三表达盒，所述第三表达盒整合在kefB位点；其中，所述第一、第二和第三表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)。
在本公开的一些实施方式中，本公开的工程微生物包括单拷贝的第一表达盒，所述第一表达盒整合在yghX位点；以及单拷贝的第二表达盒，所述第二表达盒整合在ldhA位点；其中，所述第一和第二表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)。
在本公开的一些实施方式中，本公开的工程微生物包括双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及双拷贝的第二表达盒，所述第二表达盒整合在ldhA和lacZ位点；其中，所述第一和第二表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)。
在本公开的一些实施方式中，本公开的工程微生物包括双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；双拷贝的第二表达盒，所述第二表达盒分别整合在ldhA和lacZ位点；以及单拷贝的第三表达盒，所述第三表达盒整合在kefB位点；其中，所述第一、第二和第三表达盒中使用的启动子为PfnrS启动子(序列如SEQ ID NO:22所示)。
组合物和试剂盒
根据本公开的一种实施方式，提供了一种工程微生物组合物，其包含多于一种工程微生物，所述多于一种工程微生物中的每一种彼此独立地包含以下基因中的任一种或更多种：编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；编码能够将苯丙酮酸转化为苯乙醛的酶的基因；编码能够将苯乙醛转化为苯乙醇的酶的基因；编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因；其中所述工程微生物组合物包含编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、编码能够将苯丙酮酸转化为苯乙醛的酶的基因、编码能够将苯乙醛转化为苯乙醇的酶的基因、以及编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因。在本公开的一些实施方式中，所述多于一种工程微生物中的每一种彼此独立地包含以下基因中的任一种或更多种：编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；编码能够将苯丙酮酸转化为苯乙醛的酶的基因；编码能够将苯乙醛转化为苯乙醇的酶的基因；编码谷氨酸脱氢酶的基因；编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因；其中所述工程微生物组合物包含编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、编码能够将苯丙酮酸转化为苯乙醛的酶的基因、编码能够将苯乙醛转化为苯乙醇的酶的基因、编码谷氨酸脱氢酶的基因以及编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因。
根据本公开的一种实施方式，提供了一种组合物，所述组合物包含本公开所述的工程微生物或工程微生物组合物和药学上、营养学上或生理学上可接受的载体。在本公开的一些实施方式中，所述的组合物为药物组合物，可用于治疗、缓解、管理、改善和/或预防与高苯丙氨酸血症相关的疾病和/或病症。
工程微生物可以约104到约1013个菌落形成单位(CFU)范围内的量存在于组合物中。举例来说，工程微生物的有效量可以是约105CFU到约1013CFU，优选地约106CFU到约1013CFU，优选地约107CFU到约1012CFU，更优选地约108CFU到约1012CFU的量。工程 微生物可以是活细胞或可以是死细胞。
在本公开的一些实施方式中，所述组合物是可食用组合物。在本公开的一些实施方式中，所述组合物是益生菌组合物。在本公开的一些实施方式中，所述组合物是食品增补剂。在一些实施方式中，本文所公开的组合物可以经配制用于口服施用，并且可以是营养或滋养组合物，例如食品、食品增补剂、饲料或饲料增补剂，如乳制品，例如发酵乳制品，如酸奶或酸奶饮料。在此情况下，组合物可包含营养学上可接受的载剂，其可为适合的食物基。所属领域的技术人员知道可涵盖活或死微生物体且可呈现为食品增补剂(例如，丸剂、片剂等)或功能性食品(例如饮料、发酵酸奶等)的多种配方。本文所公开的组合物还可以在胶囊、丸剂、液体溶液中配制为药剂，例如配制为囊封的冻干细菌等。在一些实施方式中，所述的组合物是益生菌组合物。
本文所公开的组合物可以被配制成单次施用或多次施用而对给定个体有效。举例来说，单次施用实质上有效降低施用组合物的哺乳动物个体的靶向疾病病状的监测症状。
在一些实施方式中，配制组合物以使得单一口服剂量含有至少约1×104、1×105、1×106、1×107、1×108、1×109、1×1010、1×1011、1×1012、1×1013CFU的工程微生物(例如，细菌实体和/或真菌实体)。在一些实施方式中，所述组合物中包含1×108–1×1012CFU的本发明所述的工程微生物。
在一些配方中，按质量计，组合物含有至少约0.5％、1％、2％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％或大于90％本公开的微生物。在一些配方中，按质量计，所施用的剂量不超过200、300、400、500、600、700、800、900毫克或1、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8或1.9克的本申请提供的工程微生物。
用于本文所公开的组合物的生理学上可接受的载剂可包含例如生理学上可接受的液体、凝胶或固体载剂、水性媒剂、非水性媒剂、抗微生物剂、等渗剂、缓冲剂、抗氧化剂、悬浮剂/分散剂、螯合剂 (sequestering/chelating agent)、稀释剂、佐剂、赋形剂或无毒辅助物质、所属领域中已知的其它组分或其各种组合。在一些实施方式中，本公开所述的组合物为液态制剂、固态制剂、半固态制剂。在一些实施方式中，所述的液态制剂选自下组：溶液制品或悬浮液制品。
为了进一步说明，水性媒剂可包含如氯化钠注射液、林格注射液、等渗右旋糖注射液、无菌水注射液或右旋糖和乳酸林格注射液；非水性媒剂可包含如植物来源的不挥发性油、棉籽油、玉米油、芝麻油或花生油；抗微生物剂可为抑细菌或抑真菌浓度，和/或可以添加到在多剂量容器中的组合物中，所述容器包含苯酚或甲酚、汞剂、苯甲醇、氯丁醇、甲基和丙基对羟基苯甲酸酯、硫柳汞、苯扎氯铵和苄索氯铵。等渗剂可以包含如氯化钠或右旋糖；缓冲剂可以包含如磷酸盐或柠檬酸盐缓冲剂；抗氧化剂可以包含如硫酸氢钠；悬浮剂和分散剂可以包含如羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮；螯合剂可以包含如乙二胺四乙酸(EDTA)或乙二醇四乙酸(EGTA)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。适合的赋形剂可包含例如水、生理盐水、右旋糖、甘油或乙醇。适合的无毒辅助物质可包含例如润湿剂或乳化剂、pH缓冲剂、稳定剂、溶解性增强剂或如乙酸钠、脱水山梨糖醇单月桂酸酯、三乙醇胺油酸酯或环糊精的试剂。
在一些实施方式中，本文所提供的组合物可为药物组合物。在一些实施方式中，本文所提供的组合物可以是食品增补剂。本文所提供的组合物除本文所提供的经遗传修饰的工程微生物之外可以包含药学上、营养学上(nutritionally/alimentarily)或生理学上可接受的载剂。优选形式将取决于预期施用模式和(治疗性)应用。载剂可以是适合于将本文提供的经遗传修饰的工程微生物递送到哺乳动物(例如人类)的胃肠道，优选地哺乳动物的肠粘膜屏障(更优选地结肠粘膜屏障)附近或内的任何相容性生理学上可接受的无毒物质。在一些实施方式中，所述药物组合物的剂型选自下组：粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、 滴剂、舌下含片、或其组合。
组合物可为液体溶液、悬浮液、乳液、丸剂、胶囊、片剂、持续释放制剂或散剂。口服制剂可包含标准载剂，如药物级甘露糖醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯烷酮、糖精钠、纤维素、碳酸镁等。
本公开的组合物可使用一种或更多种生理学上可接受的载体(包括赋形剂和辅剂)以常规方式来配制，该载体有利于将活性成分加工为用于药物使用的组合物。在一些实施方式中，将药物组合物经历压片、冻干、直接压制、常规混合、溶解、制粒、研磨、乳化、包封、包埋或喷雾干燥以形成片剂、粒剂、纳米颗粒、纳米胶囊、微胶囊、微片剂、药丸、或粉末，其可以是肠溶包衣或未包衣的。适当的制剂取决于其施用途径。
本公开所述的工程微生物可以被配制为以任何合适的剂型(例如，用于口服施用的液体、胶囊、小药囊、硬胶囊、软胶囊、片剂、肠溶包衣片剂、悬浮粉剂、粒剂或基质持续释放制剂)和任何合适类型的施用(例如，口服、立即释放、脉冲释放、延迟释放或持续释放)的药物组合物。组合物可以每天、每周或每月一次或多次被施用。工程微生物可以被配制为包含一种或更多种药学上可接受的载体、增稠剂、稀释剂、缓冲剂、表面活性剂、中性或阳离子脂质、脂质复合物、脂质体、渗透增强剂、载体化合物和其他药学上可接受的载体或剂的药物组合物。
本公开所述的工程微生物可以被口服施用并且被配制为片剂、丸剂、糖衣丸、胶囊、液体、凝胶、糖浆剂、浆体、悬浮液等。用于口服使用的药物组合物可以使用固体赋形剂制造，任选地研磨所得的混合物，和(如果需要)在添加合适的辅剂之后，加工颗粒的混合物，以获得片剂或糖衣丸芯。适合的赋形剂包括但不限于填充剂诸如糖，包括乳糖、蔗糖、甘露醇或山梨醇；纤维素组合物诸如玉米淀粉、小麦淀粉、大米淀粉、马铃薯淀粉、明胶、黄蓍胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠；和/或生理上可接受的聚合物诸如聚乙烯吡咯烷酮(PVP)或聚乙二醇(PEG)。还可 添加崩解剂，诸如交联的聚乙烯吡咯烷酮、琼脂、海藻酸或其盐诸如藻酸钠。
片剂或胶囊可以与药学上可接受的赋形剂通过常规方法一起制备，所述药学上可接受的赋形剂诸如结合剂(例如，预胶化的玉米淀粉、聚乙烯吡咯烷酮、羟丙基甲基纤维素、羧甲基纤维素、聚乙二醇、蔗糖、葡萄糖、山梨醇、淀粉、树胶、高岭土和黄蓍胶)；填充剂(例如，乳糖、微晶纤维素或磷酸氢钙)；润滑剂(例如，钙、铝、锌、硬脂酸、聚乙二醇、月桂基硫酸钠、淀粉、苯甲酸钠、L-亮氨酸、硬脂酸镁、滑石或二氧化硅)；崩解剂(例如，淀粉、马铃薯淀粉、羟基乙酸淀粉钠、糖类、纤维素衍生物、二氧化硅粉末)；或润湿剂(例如，十二烷基硫酸钠)。片剂可通过本领域熟知的方法包衣。可存在包衣壳，且常见的膜包括但不限于，聚丙交酯、聚乙醇酸、聚酸酐、其他可生物降解聚合物、藻酸盐-聚赖氨酸-藻酸盐(alginate-polylysine-alginate)(APA)、藻酸盐-聚亚甲基-共-胍-藻酸盐(A-PMCG-A)、羟基甲基丙烯酸-甲基丙烯酸甲酯(hydroxymethyl acrylate-methyl methacrylate)(HEMA-MMA)、多层HEMA-MMA-MAA、聚丙烯腈氯乙烯(polyacrylonitrilevinylchloride)(PAN-PVC)、丙烯腈/甲代烯丙基磺酸钠(acrylonitrile/sodium methallylsulfonate)(AN-69)、聚乙二醇/聚五甲基环五硅氧烷/聚二甲基硅氧烷(PEG/PD5/PDMS)、聚N,N-二甲基丙烯酰胺(PDMAAm)、硅质包封物、硫酸纤维素/藻酸钠/聚亚甲基-共-胍(CS/A/PMCG)、邻苯二甲酸乙酸纤维素、藻酸钙、k-卡拉胶-槐豆胶凝胶珠、结冷胶(gellan)-黄原胶珠、聚(丙交酯-共-乙交酯)、卡拉胶、淀粉聚酸酐、淀粉聚甲基丙烯酸酯、聚氨基酸和肠溶包衣聚合物。
用于口服施用的液体制剂可以采用溶液、糖浆剂、悬浮液或在使用之前用水或其他合适的媒介物构成的干燥产品的形式。此类液体制剂可以与药学上可接受的剂通过常规方法一起制备：所述药学上可接受的剂诸如悬浮剂(例如，山梨醇糖浆剂、纤维素衍生物或氢化可食用脂肪)；乳化剂(例如，卵磷脂或阿拉伯胶)；非水性媒介物(例如，杏仁油、油性酯、乙醇或分级的植物油)；和防腐剂(例如， 对羟基苯甲酸甲酯或对羟基苯甲酸丙酯或山梨酸)。制剂还可以适当地包含缓冲盐、调味剂、着色剂和甜味剂。用于口服施用的制剂可以适当地被配制用于本公开所述的工程微生物的缓慢释放、控制释放或持续释放。
在一些实施方式中，本公开所述的工程微生物可以例如与惰性稀释剂或可同化的可食用载体一起口服施用。化合物也可以被封闭在硬壳或软壳明胶胶囊中，压制为片剂，或直接掺入到受试者的饮食中。对于口服治疗施用，化合物可以与赋形剂混合并以可摄入片剂、含服片剂、锭剂、胶囊、酏剂、悬浮液、糖浆剂、薄饼等形式使用。为了通过除了肠胃外施用以外的方式施用本公开的化合物，可能需要将化合物用材料包衣或将化合物与材料共同施用，以防止其失活。
在一些实施方式中，组合物被配制为经由纳米颗粒、纳米胶囊、微胶囊或微片剂(其被肠溶包衣或未包衣)用于肠内施用、空肠内施用、十二指肠内施用、回肠内施用、胃分流施用或结肠内施用。本公开的药物组合物也可以使用例如常规栓剂基质诸如可可脂或其他甘油酯被配制为直肠组合物诸如栓剂或保留灌肠剂。组合物可以是在油性或水性媒介物中的悬浮液、溶液或乳状液，并且可以包含悬浮剂、稳定剂和/或分散剂。
在一些实施方式中，本公开提供呈单一剂型的药学上可接受的组合物。单一剂型可以呈液体或固体形式。单一剂型可以被直接施用至患者而不经修改，或者可以在施用之前被稀释或重构。在某些实施方式中，单一剂型可以以推注形式被施用，例如单次注射、单次口服剂量，包括包含多个片剂、胶囊、丸剂等的口服剂量。在替代实施方式中，单一剂型可以例如通过输注在一定时间段内被施用。
本公开的药物组合物的单一剂型可以通过以下来制备：将药物组合物分配为较小的等分试样、分配到单剂量容器、分配为单剂量液体形式或单剂量固体形式，诸如片剂、粒剂、纳米颗粒、纳米胶囊、微胶囊、微片剂、小药丸或粉末，其可以是肠溶包衣或未包衣的。呈固体形式的单一剂量可以在施用至患者之前通过加入液体(通 常是无菌水或盐水溶液)来重构。
可以调整剂量方案以提供治疗响应。例如，可以在一次施用单次推注，可以在预定时间段内施用几个分开的剂量，或者可以如治疗情况所指示的降低或增加剂量。剂量的规格由活性化合物的独特特征和待实现的具体治疗效果决定。剂量值可以随着待缓解的状况的类型和严重程度而变化。对于任何特定的受试者，可以根据个体需要和治疗临床医师的专业判断随时间调整具体的剂量方案。
在另一个实施方式中，组合物可以在受控释放或持续释放系统中递送。在一个实施方式中，可以使用泵来实现受控或持续释放。在另一个实施方式中，可以使用聚合物材料来实现本公开内容的疗法的受控或持续释放。用于持续释放制剂的聚合物的实例包括但不限于，聚(甲基丙烯酸2-羟基乙酯)、聚(甲基丙烯酸甲酯)、聚(丙烯酸)、聚(乙烯-共-乙酸乙烯酯)、聚(甲基丙烯酸)、聚乙交酯(PLG)、聚酸酐、聚(N-乙烯基吡咯烷酮)、聚(乙烯醇)、聚丙烯酰胺、聚(乙二醇)、聚丙交酯(PLA)、聚(丙交酯-共-乙交酯)(PLGA)和聚原酸酯。用于持续释放制剂中的聚合物可以是惰性的，不含可浸出(leachable)的杂质，在储存时是稳定的、无菌的和可生物降解的。在一些实施方式中，控制释放或持续释放系统可以被置于预防或治疗靶附近，因此仅需要全身剂量的一部分。可使用本领域技术人员已知的任何合适的技术。
剂量可以取决于几个因素，包括疾病的严重程度和响应性、施用途径、治疗时间(几天至几个月至几年)、以及到改善疾病的时间。
在另一方面，本公开提供了一种试剂盒，其包含本公开所述的工程微生物或本公开所述的工程微生物组合物或本公开所述的组合物。
必要时，所述试剂盒可进一步包含各种常规药物试剂盒组分中的一种或多种，例如具有一种或多种药学上可接受的载剂的容器、额外容器等，如对所属领域的技术人员将显而易见。试剂盒中还可以包含作为插页或标签的说明书，指示应施用的组分的量、施用指南和/或混合组分的指南。
治疗方法和用途
本公开所述的工程微生物包含编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、编码能够将苯丙酮酸转化为苯乙醛的酶的基因、编码能够将苯乙醛转化为苯乙醇的酶的基因、编码谷氨酸脱氢酶的基因、和/或编码苯丙氨酸转运蛋白的基因中的一种或多种，其能够缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症。
本公开构建的工程微生物能够有效地降解苯丙氨酸和/或苯丙酮酸，实验证明，该工程微生物有效降解了苯丙酮尿症小鼠PahR408W的体内苯丙氨酸和苯丙氨酸代谢物，显示出良好的应用前景。
本公开提供了一种用于缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的方法，所述方法包括向有此需要的患者施用根据本公开所述的工程微生物或根据本公开所述的工程微生物组合物或根据本公开所述的组合物或根据本公开所述的试剂盒。
根据本公开的一种实施方式，可以提供本公开所述的工程微生物或根据本公开所述的工程微生物组合物在制备用于缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的药物或保健品中的用途。
在本公开的一些实施方式中，所述与高苯丙氨酸血症相关的疾病和/或病症包括但不限于：苯丙酮尿症(例如经典型或典型(classical or typical)苯丙酮尿症和非经典型(atypical)苯丙酮尿症)，永久性轻度(mild)高苯丙氨酸血症，非苯丙酮尿症类的高苯丙氨酸血症，苯丙氨酸羟化酶缺陷，辅因子缺陷，二氢蝶呤还原酶缺陷，四氢蝶呤合酶缺陷，Segawa氏病和肝病。
实施例
以下，说明本申请的实施例。下面描述的实施例是示例性的，仅用于解释本申请，而不能理解为对本申请的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
表1：实施例中所用酶、启动子和表达盒的具体序列信息

表2基因编辑中插入位点对应的sgRNA序列信息

实施例1：细菌基因编辑的方案
1.1 sgRNA设计
在目标整合位点序列的两条链上寻找连接NGG PAM序列的20bp序列(N20NGG)并且针对EcN基因组进行blast处理。选择独特的20bp序列作为目标整合位点的sgRNA。选择sgRNA上游和下游的300-500bp序列作为左同源臂(LHA)和右同源臂(RHA)。
1.2 sgRNA质粒构建
将sgRNA序列添加到gRNA骨架反向引物的5’端，通过PCR扩增并用限制性内切酶PstI和SpeI进行酶切，将酶切后的PCR产物片段与同样酶切的质粒pCBT003(SEQ ID NO：84，质粒图谱如图1所示)连接，形成pCBT003_sgRNA质粒。
1.3供体基因片段的制备
待整合到EcN基因组的目标基因由金斯瑞(GeneScript)在克隆质粒(例如pUC57)上合成。目标基因以合成的质粒为模板进行扩增，所选整合位点的LHA和RHA以EcN的基因组为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起来，得到的包含LHA-目 标基因-RHA的PCR产物用作供体基因片段。
1.4供体质粒PCBT003-目标基因-sgRNA制备
将从1.2中获得的表达整合位点的sgRNA的pCBT003_sgRNA质粒、从1.3中获得的包含LHA-目标基因-RHA的PCR产物，使用MultiS一步克隆试剂盒进行连接，转化进Top10感受态细胞中，用氨苄青霉素抗性平板(100μg/mL)筛选，挑取转化子进行菌落PCR验证及测序，选出测序正确的PCBT003-目标基因-sgRNA重组质粒。
1.5 EcN/pCBT001电转感受态细胞的制备
将PCBT001质粒(表达Cas9蛋白的质粒，SEQ ID NO.85，质粒图谱如图2所示)电转化进大肠杆菌EcN化学感受态细胞中，在含盐酸壮观霉素和链霉素(50μg/mL)的LB固体平板上进行筛选，获得EcN/PCBT001转化子。
挑取EcN/PCBT001单菌落于含壮观霉素和链霉素(50μg/mL)的LB液体培养基，30℃220rpm过夜培养。次日，菌液按1:100接种量(V/V)接入30mL LB液体培养基，在菌浓OD600为0.2时，添加终浓度为1mM的IPTG进行诱导，继续培养2-3h至OD值为0.6-0.8，制备电转感受态细胞。
1.6电转
将PCBT003-目的基因-sgRNA质粒电转入EcN/PCBT001感受态细胞中(电转化条件：2.5kV，200Ω，25μF)，涂布于氨苄青霉素(50μg/mL)、壮观霉素和链霉素(50μg/mL)LB平板上，培养1d，挑取在抗性平板上生长的单菌落。
1.7 PCBT003-目标基因-sgRNA质粒和pCBT001质粒的消除
挑取已成功整合目标基因表达盒的单菌落接种于添加壮观霉素和链霉素(50μg/mL)的LB液体培养基中，加入10mM的L-阿拉伯糖在30℃下诱导20-24h，涂布壮观霉素和链霉素(50μg/mL)LB平板；将长出的单菌落分别点于氨苄青霉素以及壮观霉素和链霉素(50μg/mL)平板，观察菌株生长情况。在氨苄青 霉素抗性平板上不能生长，在壮观霉素和链霉素平板上生长的菌落即为PCBT003-目的基因-sgRNA消除的菌株。
挑取PCBT003-目标基因-sgRNA质粒消除的菌落，接种于LB试管中，42℃培养24h，梯度稀释，涂布LB平板，过夜培养。次日挑取单菌落，划线于壮观霉素和链霉素抗性平板，观察菌落生长情况。在壮观霉素和链霉素平板上不能生长的菌落即为PCBT001质粒消除的菌株。
实施例2：营养缺陷型菌株的构建方案
通过CRISPR/Cas9技术敲除thyA基因，实现EcN菌株中thyA基因的缺失。以构建EcN_ΔthyA菌株为例，具体构建方案为，按照实施例1的构建方案，首先构建包含供体基因片段thyA-HA和靶向thyA位点的sgRNA的重组质粒PCBT003-thyA-HA-sgRNA(thyA-HA-sgRNA的序列如SEQ ID NO:131所示)，然后将其与表达Cas9蛋白的pCBT001质粒共同电转感受态EcN菌株，并去除多余的质粒后，获得营养缺陷型EcN_ΔthyA菌株。
thyA-HA-sgRNA的序列(SEQ ID NO:131)：

大写无下划线部分为左同源臂，大写加下划线部分为右同源臂，大写加框部分为sgRNA序列。
实施例3：鞭毛基因的敲除方案
为了延长菌株在肠道中的滞留时间，利用CRISPR/Cas9基因编辑系统，敲除鞭毛系统的关键基因fliC及黏附相关基因(纤维素合酶基因)bscA。以构建EcN_ΔthyAΔbscAΔfliC为例，具体构建方案为，首先制备感受态的EcN_ΔthyA菌株，然后分别构建包含供体片段以及靶向fliC或bscA的sgRNA的重组质粒PCBT003-fliC-HA-sgRNA/PCBT003-bscA-HA-sgRNA(fliC-HA-sgRNA的序列SEQ ID NO:132所示，bscA-HA-sgRNA的序列如SEQ ID NO:133所示)，与表达Cas9蛋白的pCBT001质粒共同电转EcN菌株并去除多余的质粒后，获得营养缺陷型EcN_ΔthyAΔbscAΔfliC菌株。
fliC-HA-sgRNA的序列(SEQ ID NO:132)：


大写无下划线部分为左同源臂，大写加下划线部分为右同源臂，大写加框部分为sgRNA序列。
bscA-HA-sgRNA的序列(SEQ ID NO:133)：

大写无下划线部分为左同源臂，大写加下划线部分为右同源臂，大写加框部分为sgRNA序列。
实施例4：降解苯丙氨酸的工程菌株的构建
以EcN为底盘菌或者以实施例2或3构建的已敲除鞭毛基因和/或营养缺陷型的菌株为底盘菌，敲入能够将苯丙氨酸转化为无毒副作用的产物的相关基因，从而得到能够降解苯丙氨酸的工程菌株。
构建过程中使用表达盒的序列如下所示：vanRAM抑制子(SEQ ID NO:103)，Pfnrs-TyrB-gdh2(SEQ ID NO:104)，Pfnrs-Aro10-yahk(SEQ ID NO:105)，Pfnrs-pheDH-pheP(SEQ ID NO:106)，Pvan-TyrB-gdh2(SEQ ID NO:107)，Pvan-Aro10-yahk(SEQ ID NO:108)，Pvan-pheDH-pheP(SEQ ID NO:109)，PBAD-LAAD(SEQ ID NO:110)。
目标基因表达盒在EcN中可选的插入位点选自：yicS位点、malPT位点、maeB位点、nth/tppB位点、tkrA位点、malE位点、exo位点、rhtB/C位点、agaI/rsml位点、araBD位点、yghX位点、ldhA位点、araAB位点、lacZ位点和/或kefB位点中的一个或更多个。
4.1降解苯丙氨酸的工程菌株CBT2001的构建
对菌株E.coli Nissle1917(EcN)基因组进行改造，包括如下步骤：
4.1.1 yghX位点敲入Pfnrs-TyrB-gdh2表达盒
(1)PCBT003-yghX-sgRNA质粒的构建
构建目的：将PCBT003质粒中的原靶位点X的序列“catcgccgcagcggtttcag”替换为新的含有靶位点X的N20序列“cgtatatcgaagatgtgacg”(yghX)，从而构建出含有“cgtatatcgaagatgtgacg”的PCBT003质粒(PCBT003-yghX-sgRNA)。
构建方法：
1)设计引物yghX-sgRNA-f(PstI)：SEQ ID NO.25
(加粗小写字体为PstI酶切位点序列，其他序列为PCBT003质粒上的序列)
2)设计引物yghX-sgRNA-r(SpeI)：SEQ ID NO.26
(加粗小写字体为SpeI酶切位点序列，加粗大写字体为yghX位点的N20序列，其他序列为PCBT003质粒上的序列)
3)利用引物yghX-sgRNA-f(SEQ ID NO.25)和yghX-sgRNA-r(SEQ ID NO.26)从PCBT003质粒(SEQ ID NO.84)DNA模板中PCR扩增含有靶位点X(yghX)的N20序列片段，将所得的片段使用限制性内切酶PstI和speI酶切，纯化回收PCR产物(121bp)。使用PstI酶和speI酶切PCBT003质粒，将胶回收的酶切片段(3123bp)与纯化回收的PCR产物连接，构建出含有靶位点X(yghX)序列的PCBT003质粒(PCBT003-yghX-sgRNA)，然后转化至E.coli Top10感受态细胞。用氨苄青霉素抗性平板(100μg/mL)筛选，挑取转化子摇菌培养，抽提质粒，利用引物PCBT003-seq-r(SEQ ID NO.27)进行测序。分别选出测序正确的sgRNA质粒：PCBT003-yghX-sgRNA。
(2)电转质粒PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA制备：
使用限制性内切酶PstI和XbaI酶切前述得到的PCBT003-yghX-sgRNA质粒，对酶切的大片段(质粒骨架)进行胶回收。 利用扩增引物yghX-LH-f(SEQ ID NO.30)和yghX-LH-r1(SEQ ID NO.31)和yghX-RH-f1(SEQ ID NO.32)和yghX-RH-r(SEQ ID NO.33)从菌株E.coli Nissle1917基因组模板中扩增yghX位点的上游同源臂片段和下游同源臂片段，纯化PCR产物。以pUC-fnrs-TyrB-gdh2质粒(SEQ ID NO.86)(金斯瑞合成)为模板，以fnrs-TyrB-f1(SEQ ID NO.28)和gdh2-r1(SEQ ID NO.29)为引物，PCR扩增获得Pfnrs-TyrB-gdh2片段，约5260bp；将胶回收的酶切片段(质粒骨架)、纯化回收的上下游同源臂PCR产物和Pfnrs-TyrB-gdh2片段，使用MultiS一步克隆试剂盒进行连接，转化进Top10感受态细胞中，用氨苄青霉素抗性平板(100μg/mL)筛选，挑取转化子进行菌落PCR验证及测序，选出测序正确的PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA重组质粒。
(3)电转感受态细胞制备方法：
挑取EcN单菌落于5mL LB液体培养基，37℃220rpm过夜培养。次日，菌液按1:100接种量接入30mL LB液体培养基，振荡培养2-3h至OD值为0.6-0.8，将菌液倒入预冷的离心管中，冰浴15-20min；6000rpm，4℃离心7min，弃上清，加入20mL10％预冷甘油，均匀悬浮菌体；离心，弃上清，重复三次；弃上清，加入300μL 10％预冷甘油，将菌体悬浮均匀，按每管100μL分装于离心管中。
将PCBT001质粒(SEQ ID NO.85)电转化进大肠杆菌EcN化学感受态细胞中，在含盐酸壮观霉素(50μg/mL)的LB固体平板上进行筛选，获得EcN/PCBT001转化子。
挑取EcN/PCBT001单菌落于含壮观霉素(50μg/mL)的LB液体培养基，30℃220rpm过夜培养。次日，菌液按1:100接种量(V/V)接入30mL LB液体培养基，在菌浓OD600为0.2时，添加终浓度为1mM的IPTG进行诱导，继续培养2-3h至OD值为0.6-0.8，制备电转感受态细胞。
(4)电转：
将PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA质粒电转入EcN/PCBT001感受态细胞中(电转化条件：2.5kV，200Ω，25μF)，涂布于氨苄青霉素(50μg/mL)和壮观霉素(50μg/mL)LB平板上，培养1d。挑取在抗性平板上生长的单菌落，利用引物yghX-verify-f(SEQ ID NO.34)和yghX-verify-r(SEQ ID NO.35)通过菌落PCR的方法验证基因的整合情况，阳性片段约6662bp；
(5)PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA质粒消除：
挑取已成功整合Pfnrs-TyrB-gdh2表达盒的单菌落接种于添加壮观霉素(50μg/mL)的LB液体培养基中，加入10mM的L-阿拉伯糖诱导20-24h，涂布壮观霉素LB平板；将长出的单菌落分别点于氨苄青霉素和壮观霉素平板，观察菌株生长情况。在氨苄青霉素抗性平板上不能生长，在壮观霉素抗性平板上生长的菌落即为PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA消除的菌株，获得EcN_Δyghx::Pfnrs-TyrB-gdh2/PCBT001。
4.1.2 ldhA位点敲入Pfnrs-ARO10-YahK表达盒
(1)PCBT003-ldhA-sgRNA质粒的构建
利用引物ldhA-sgRNA-f(SEQ ID NO.36)和ldhA-sgRNA-r(SEQ ID NO.37)从PCBT003质粒模板中PCR扩增含有靶位点X(ldhA)的N20序列片段，使用限制性内切酶PstI和speI酶切，纯化回收PCR产物。
使用PstI和speI酶切PCBT003质粒，将胶回收的酶切片段(约3123bp)与纯化回收的PCR产物连接、转化至E.coli Top10感受态细胞。在氨苄青霉素抗性平板(100μg/mL)筛选，获取PCBT003-ldhA-sgRNA质粒。
(2)电转质粒PCBT003-Pfnrs-ARO10-YahK-ldhA-sgRNA制备：
使用限制性内切酶PstI和XbaI酶切PCBT003-ldhA-sgRNA质粒，对酶切的大片段(质粒骨架)进行胶回收。
利用扩增引物ldhA-LH-f(SEQ ID NO.40)和ldhA-LH-r1 (SEQ ID NO.41)和ldhA-RH-f1(SEQ ID NO.42)和ldhA-RH-r(SEQ ID NO.43)从EcN基因组模板中扩增ldhA位点的上游同源臂片段和下游同源臂片段，分别约500bp。
以pUC-fnrs-ARO10-YahK质粒(SEQ ID NO.87)(金斯瑞合成)为模板，以fnrs-ARO10-f1(SEQ ID NO.38)和YahK-r1(SEQ ID NO.39)为引物，PCR扩增获得Pfnrs-ARO10-YahK片段，约4779bp。
将胶回收的酶切片段(质粒骨架)、纯化回收的上下游同源臂PCR产物和Pfnrs-ARO10-YahK片段，使用MultiS一步克隆试剂盒进行连接，转化进Top10感受态细胞中，用氨苄青霉素抗性平板(100μg/mL)筛选，获得PCBT003-Pfnrs-TyrB-gdh2-ldhA-sgRNA重组质粒。
(3)参考4.1.1(3)方法制备EcN_Δyghx::Pfnrs-TyrB-gdh2/PCBT001电转感受态细胞。
(4)参考4.1.1(4)方法将PCBT003-Pfnrs-ARO10-YahK-ldhA-sgRNA质粒电转入EcN_Δyghx::Pfnrs-TyrB-gdh2/PCBT001感受态细胞，转化子利用ldhA-verify-f(SEQ ID NO.44)和ldhA-verify-r(SEQ ID NO.45)进行菌落PCR验证
(5)参考4.1.1(5)方法将消除辅助质粒，获得EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK/PCBT001。
(6)PCBT001质粒的消除
挑取PCBT003-Pfnrs-TyrB-gdh2-kefB-sgRNA质粒消除的菌落，接种于LB试管中，42℃培养24h，梯度稀释，涂布LB平板，过夜培养。次日挑取单菌落，划线于壮观霉素抗性平板，观察菌落生长情况。在壮观霉素平板上不能生长的菌落即为PCBT001质粒消除的菌株EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK，命名为CBT2001。
通过上述实施例，最后得到工程菌CBT2001，其为大肠杆菌Nissle1917衍生菌，在基因组上分别整合单拷贝芳香族氨基酸转氨酶基因TyrB、谷氨酸脱氢酶基因gdh2、醛还原酶基因YahK和 苯丙酮酸脱羧酶基因ARO10。
工程菌CBT2001的基因型：Nissle_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK。
4.2降解苯丙氨酸的工程菌株CBT2002的构建
4.2.1 araAB位点敲入Pfnrs-TyrB-gdh2
(1)PCBT003-araAB-sgRNA质粒的构建
利用引物araAB-sgRNA-f(SEQ ID NO.46)和araAB-sgRNA-r(SEQ ID NO.47)从PCBT003质粒模板中PCR扩增含有靶位点X(araAB)的N20序列片段，使用限制性内切酶PstI和speI酶切，纯化回收PCR产物。
使用PstI和speI酶切PCBT003质粒，将胶回收的酶切片段(约3123bp)与纯化回收的PCR产物连接、转化至E.coli Top10感受态细胞。在氨苄青霉素抗性平板(100μg/mL)筛选，获取PCBT003-araAB-sgRNA质粒。
(2)电转质粒PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA制备：
使用限制性内切酶PstI和XbaI酶切PCBT003-araAB-sgRNA质粒，对酶切的大片段(质粒骨架)进行胶回收。
利用扩增引物araAB-LH-f(SEQ ID NO.50)和araAB-LH-r(SEQ ID NO.51)和araAB-RH-f(SEQ ID NO.52)和araAB-RH-r(SEQ ID NO.53)从EcN基因组模板中扩增araAB位点的上游同源臂片段和下游同源臂片段，分别约500bp。
以pUC-fnrs-TyrB-gdh2质粒(SEQ ID NO.86)(金斯瑞合成)为模板，以fnrs-TyrB-f2(SEQ ID NO.48)和gdh2-r2(SEQ ID NO.49)为引物，PCR扩增获得Pfnrs-TyrB-gdh2片段，约4779bp。将胶回收的酶切片段(质粒骨架)、纯化回收的上下游同源臂PCR产物和Pfnrs-TyrB-gdh2片段，使用MultiS一步克隆试剂盒进行连接，转化进Top10感受态细胞中，用氨苄青霉素抗性平板(100μg/mL)筛选，获得PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA重组质粒。
(3)参考4.1.1(3)方法制备EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK/PCBT001电转感受态细胞。
(4)参考4.1.1(4)方法将PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA质粒电转入EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK/PCBT001感受态细胞，转化子利用araAB-verify-f(SEQ ID NO.54)和araAB-verify-r(SEQ ID NO.55)进行菌落PCR验证
(5)参考4.1.1(5)方法将消除辅助质粒，获得EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2。
4.2.2 lacZ位点敲入Pfnrs-ARO10-YahK
(1)PCBT003-lacZ-sgRNA质粒的构建
利用引物lacZ-sgRNA-f(SEQ ID NO.56)和lacZ-sgRNA-r(SEQ ID NO.57)从PCBT003质粒模板中PCR扩增含有靶位点X(lacZ)的N20序列片段，使用限制性内切酶PstI和speI酶切，纯化回收PCR产物。使用PstI和speI酶切PCBT003质粒，将胶回收的酶切片段(约3123bp)与纯化回收的PCR产物连接、转化至E.coli Top10感受态细胞。在氨苄青霉素抗性平板(100μg/mL)筛选，获取PCBT003-lacZ-sgRNA质粒。
(2)电转质粒PCBT003-Pfnrs-ARO10-YahK-lacZ-sgRNA制备：
使用限制性内切酶PstI和XbaI酶切PCBT003-lacZ-sgRNA质粒，对酶切的大片段(质粒骨架)进行胶回收。
利用扩增引物lacZ-LH-f(SEQ ID NO.60)和lacZ-LH-r(SEQ ID NO.61)和lacZ-RH-f(SEQ ID NO.62)和lacZ-RH-r(SEQ ID NO.63)从EcN基因组模板中扩增lacZ位点的上游同源臂片段和下游同源臂片段，分别约500bp。
以pUC-fnrs-ARO10-YahK质粒(SEQ ID NO.87)(金斯瑞合成)为模板，以fnrs-ARO10-f2(SEQ ID NO.58)和YahK-r2(SEQ ID NO.59)为引物，PCR扩增获得Pfnrs-ARO10-YahK片 段，约4819bp。将胶回收的酶切片段(质粒骨架)、纯化回收的上下游同源臂PCR产物和Pfnrs-ARO10-YahK片段，使用MultiS一步克隆试剂盒进行连接，转化进Top10感受态细胞中，用氨苄青霉素抗性平板(100μg/mL)筛选，获得PCBT003-Pfnrs-ARO10-YahK-lacZ-sgRNA重组质粒。
(3)参考4.1.1(3)方法制备EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2/PCBT001电转感受态细胞。
(4)参考4.1.1(4)方法将PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA质粒电转入EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2感受态细胞，转化子利用lacZ-verify-f(SEQ ID NO.64)和lacZ-verify-r(SEQ ID NO.65)进行菌落PCR验证。
(5)参考4.1.1(5)方法将消除辅助质粒，获得EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10-YahK/PCBT001。
(6)参考4.1.2(6)方法将消除辅助质粒PCBT001，获得EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10-YahK，命名为CBT2002。
通过上述实施例最后得到工程菌CBT2002，其为CBT2001衍生菌，通过在CBT2001基因组上再分别整合一个拷贝的芳香族氨基酸转氨酶基因、谷氨酸脱氢酶基因、醛还原酶基因和苯丙酮酸脱羧酶基因。
工程菌CBT2002的基因型：
EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10-YahK
4.3降解苯丙氨酸的工程菌株CBT2003的构建
4.3.1 kefB位点敲入Pfnrs-PheP
(1)PCBT003-kefB-sgRNA质粒的构建
利用引物kefB-sgRNA-f(SEQ ID NO.66)和kefB-sgRNA-r(SEQ ID NO.67)从PCBT003质粒模板中PCR扩增含有靶位点X(kefB)的N20序列片段，使用限制性内切酶PstI和speI酶切，纯化回收PCR产物。
使用PstI和speI酶切PCBT003质粒，将胶回收的酶切片段(约3123bp)与纯化回收的PCR产物连接、转化至E.coli Top10感受态细胞。在氨苄青霉素抗性平板(100μg/mL)筛选，获取PCBT003-kefB-sgRNA质粒。
(2)电转质粒PCBT003-Pfnrs-PheP-kefB-sgRNA制备：
使用限制性内切酶PstI和XbaI酶切PCBT003-kefB-sgRNA质粒，对酶切的大片段(质粒骨架)进行胶回收。利用扩增引物kefB-LH-f(SEQ ID NO.70)和kefB-LH-r(SEQ ID NO.71)和kefB-RH-f(SEQ ID NO.72)和kefB-RH-r(SEQ ID NO.73)从EcN基因组模板中扩增kefB位点的上游同源臂片段和下游同源臂片段，分别约500bp。
以pUC-fnrs-PheP质粒(金斯瑞合成)为模板，以fnrs-PheP-f(SEQ ID NO.68)和PheP-r(SEQ ID NO.78)为引物，PCR扩增获得Pfnrs-PheP片段，约1406bp。将胶回收的酶切片段(质粒骨架)、纯化回收的上下游同源臂PCR产物和Pfnrs-PheP片段，使用MultiS一步克隆试剂盒进行连接，转化进Top10感受态细胞中，用氨苄青霉素抗性平板(100μg/mL)筛选，获得PCBT003-Pfnrs-PheP-kefB-sgRNA重组质粒。
(3)参考4.1.1(3)方法制备EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10-YahK/PCBT001电转感受态细胞。
(4)参考4.1.1(4)方法将PCBT003-Pfnrs-PheP-kefB-sgRNA质粒电转入EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10- YahK/PCBT001感受态细胞，转化子利用kefB-verify-f(SEQ ID NO.74)和kefB-verify-r(SEQ ID NO.75)进行菌落PCR验证。
(5)参考4.1.1(5)方法将消除辅助质粒PCBT003-Pfnrs-PheP-kefB-sgRNA，获得EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10-YahK_ΔkefB::Pfnrs-PheP/PCBT001。
(6)参考4.1.2(6)方法将消除辅助质粒PCBT001，获得EcN_Δyghx::Pfnrs-TyrB-gdh2-TPheP_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2_ΔlacZ::Pfnrs-ARO10-YahK_ΔkefB::Pfnrs-PheP，命名为CBT2003。
通过上述实施例最后得到的是工程菌CBT2003，其为CBT2002的衍生菌，在CBT2002的基因组进一步整合了单拷贝的苯丙氨酸转运蛋白基因，以强化菌株对苯丙氨酸的摄取。
工程菌CBT2003的基因型：
EcN_Δyghx::Pfnrs-TyrB-gdh2_Δldha::Pfnrs-ARO10-YahK_ΔaraAB::Pfnrs-TyrB-gdh2-_ΔlacZ::Pfnrs-ARO10-YahK_ΔkefB::Pfnrs-PheP
4.4 CBT-201菌株及衍生菌株的构建(启动子为Pfnrs)
该工程菌株在底盘细菌基因组不同选定位点处中导入2个拷贝的苯丙氨酸转氨酶TyrB、单拷贝的苯丙氨酸脱氢酶pheDH(来自芽孢杆菌SLBN-3，GenBank:TQJ42029.1)和单拷贝的L-氨基酸脱氨酶(LAAD)，从而将苯丙氨酸转化为苯丙酮酸；导入2个拷贝的苯丙酮酸脱羧酶Aro10，用于将上一步生成的苯丙酮酸转化为苯乙醛，醛还原酶Yahk进一步将苯乙醛转化为苯乙醇，谷氨酸脱氢酶GDH用于与TyrB在氨基代谢上配合发挥作用；苯丙氨酸转运蛋白pheP用于将菌体外的苯丙氨酸转入工程菌内。在具体构建过程中，部分基因前后串联，利用同一个启动子调控转录，从而实现对基因表达量的控制。表达盒中选用的启动子为PfnrS。具体构建过程和菌株结构如下：
4.4.1在yghX和araAB位点敲入Pfnrs-TyrB-gdh2表达盒
Pfnrs-TyrB-gdh2表达盒(SEQ ID NO：104)由金斯瑞(GeneScript)合成，靶向yghX和araAB的sgRNA序列见表2。按照实施例1和4的方法，分别将sgRNA序列和表达盒依次接入pCBT003载体，从而获得重组质粒PCBT003-Pfnrs-TyrB-gdh2-yghX-sgRNA和PCBT003-Pfnrs-TyrB-gdh2-araAB-sgRNA；随后将上述重组质粒分别与表达Cas9蛋白的pCBT001质粒共同电转底盘细菌EcNΔthyA并去除多余质粒后，得到重组菌EcNΔthyAΔyghX::Pfnrs-TyrB-gdh2ΔaraAB::Pfnrs-TyrB-gdh2。
4.4.2在ldhA和lacZ位点敲入Pfnrs-Aro10-yahk
Pfnrs-Aro10-yahk表达盒(SEQ ID NO：105)由金斯瑞(GeneScript)合成，靶向ldhA和lacZ的sgRNA序列见表2。按照实施例1和4的方法，分别将sgRNA序列和表达盒Pfnrs-Aro10-yahk依次接入pCBT003载体，从而获得重组质粒PCBT003-Pfnrs-Aro10-yahk-ldhA-sgRNA和PCBT003-Pfnrs-Aro10-yahk-lacZ-sgRNA；随后将上述重组质粒分别与表达Cas9蛋白的pCBT001质粒共同电转入上一步获得的工程菌并去除多余质粒后，得到重组菌EcNΔthyAΔyghX::Pfnrs-TyrB-gdh2ΔldhA::Pfnrs-ARO10-yahkΔaraAB::Pfnrs-TyrB-gdh2ΔlacZ::Pfnrs-ARO10-yahk。
4.4.3在kefB位点敲入PfnrS_pheDH_pheP
PfnrS_pheDH_pheP表达盒(SEQ ID NO：106)由金斯瑞(GeneScript)合成，其中经密码子优化后的pheDH基因序列如SEQ ID NO:17所示，靶向kefB的sgRNA序列见表2。按照实施例1和4的方法，将sgRNA序列和表达盒依次接入pCBT003载体，从而获得重组质粒PCBT003-PfnrS_pheDH_pheP-kefB-sgRNA；随后将上述重组质粒与表达Cas9蛋白的pCBT001质粒共同电转入上一步获得的工程菌并去除多余质粒后，得到重组菌EcNΔthyAΔyghX::Pfnrs-TyrB-gdh2ΔldhA::Pfnrs-ARO10-yahkΔaraAB::Pfnrs-TyrB-gdh2ΔlacZ::Pfnrs-ARO10-yahkΔkefB::Pfnrs-pheDH-phep。
4.4.4在rhtB/C位点敲入PBAD_LAAD
PBAD_LAAD表达盒(SEQ ID NO：110)由金斯瑞(GeneScript)合成，靶向rhtB/C的sgRNA序列见表2。按照实施例1和4的方法，将sgRNA序列和表达盒依次接入pCBT003载体，从而获得重组质粒PCBT003-PBAD_LAAD-rhtB/C-sgRNA；随后将上述重组质粒与表达Cas9蛋白的pCBT001质粒共同电转入上一步获得的工程菌并去除多余质粒后，得到重组菌EcNΔthyAΔyghX::Pfnrs-TyrB-gdh2ΔldhA::Pfnrs-ARO10-yahkΔaraAB::Pfnrs-TyrB-gdh2ΔlacZ::Pfnrs-ARO10-yahkΔkefB::Pfnrs-pheDH-phepΔrhtB/C::PBAD_LAAD，即CBT-201菌株。
CBT-201-AA的构建
在CBT-201菌株的基础上，进一步添加了两个拷贝的Pfnrs-ARO10-yahk表达盒，得到了菌株CBT-201-AA，基因型如下：
EcNΔthyAΔyghX::Pfnrs-TyrB-gdh2ΔldhA::Pfnrs-ARO10-yahkΔaraAB::Pfnrs-TyrB-gdh2ΔlacZ::Pfnrs-ARO10-yahkΔkefB::Pfnrs-pheDH-phepΔagaI/rsml::Pfnrs-ARO10-yahkΔyjcS::Pfnrs-ARO10-yahkΔrhtB/C::PBAD_LAAD
CBT-203菌株的构建
按照CBT-201菌株的构建方法，在底盘细菌EcNΔthyAΔbscAΔfliC的基因组上，转入与CBT-201菌株相同拷贝数的相同的酶，得到CBT-203菌株，基因型如下：
EcNΔthyAΔbscAΔfliCΔyghX::Pfnrs-TyrB-gdh2ΔldhA::Pfnrs-ARO10-yahkΔaraAB::Pfnrs-TyrB-gdh2ΔlacZ::Pfnrs-ARO10-yahkΔkefB::Pfnrs-pheDH-phepΔlacZ::Pfnrs-ARO10-yahkΔlacZ::Pfnrs-ARO10-yahkΔrhtB/C::PBAD_LAAD
4.5 CBT-202、CBT-209、CBT-210菌株的构建：
参照CBT-201菌株的构建过程，构建工程菌株CBT-202、CBT-209、CBT-210，上述菌株的基因型如下：
CBT-202的基因型：EcNΔthyAΔldhA::Pfnrs-pheDH-phepΔyghX::Pfnrs-ARO10-yahkΔmaeB::Pfnrs-pheDH-phepΔlacZ::Pfnrs-ARO10-yahkΔrhtB/C::PBAD_LAAD
CBT-209的基因型：EcNΔthyAΔldhA::Pfnrs-pheDH-phepΔyghX::Pfnrs-ARO10-yahk
CBT-210的基因型：EcNΔthyAΔyghX::Pfnrs-TyrB-gdh2ΔldhA::Pfnrs-ARO10-yahk△kefB::Pfnrs-pheP
4.6 CBT-206菌株的构建(启动子为Pvan)：
CBT-206菌株的所有表达盒中选用的启动子为Pvan。具体构建过程和菌株结构如下：
4.6.1在yghX和araAB位点敲入Pvan-TyrB-gdh2表达盒
Pvan-TyrB-gdh2表达盒(SEQ ID NO：107)由金斯瑞(GeneScript)合成，靶向yghX和araAB的sgRNA序列见表2。按照实施例1和4的方法，分别将sgRNA序列和表达盒(Pvan-TyrB-gdh2表达盒和如SEQ ID NO：103所示的vanRAM表达盒)依次接入pCBT003载体，从而获得重组质粒PCBT003-Pvan-TyrB-gdh2-yghX-sgRNA和PCBT003-Pvan-TyrB-gdh2-araAB-sgRNA；随后将上述重组质粒分别与表达Cas9蛋白的pCBT001质粒共同电转底盘细菌EcNΔthyAΔbscAΔfliC并去除多余质粒后，得到重组菌EcNΔthyAΔbscAΔfliCΔyghX::Pvan-TyrB-gdh2ΔaraAB::Pvan-TyrB-gdh2。
4.6.2在ldhA、agaI/rsml和yjcS位点敲入Pvan-Aro10-yahk
Pfnrs-Aro10-yahk表达盒(SEQ ID NO：108)由金斯瑞(GeneScript)合成，靶向ldhA、agaI/rsml和yjcS的sgRNA序列见表2。按照实施例1和4的方法，分别将sgRNA序列和表达盒(Pvan-Aro10-yahk表达盒和如SEQ ID NO：103所示的vanRAM表达盒)依次接入pCBT003载体，从而获得重组质粒PCBT003-Pvan-Aro10-yahk-ldhA-sgRNA、PCBT003-Pvan-Aro10-yahk-agaI/rsml-sgRNA和PCBT003-Pvan-Aro10-yahk-yjcS-sgRNA；随 后将上述重组质粒分别与表达Cas9蛋白的pCBT001质粒共同电转入上一步获得的工程菌并去除多余质粒后，得到重组菌EcNΔthyAΔbscAΔfliCΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔagaI/rsml::Pvan-ARO10-yahk。
4.6.3在nth/tppB位点敲入Pvan_pheDH_pheP
Pvan_pheDH_pheP表达盒(SEQ ID NO：109)由金斯瑞(GeneScript)合成，靶向nth/tppB的sgRNA序列见表2。按照实施例1和4的方法，将sgRNA序列和表达盒(Pvan_pheDH_pheP表达盒和如SEQ ID NO：103所示的vanRAM表达盒)依次接入pCBT003载体，从而获得重组质粒PCBT003-Pvan_pheDH_pheP-nth/tppB-sgRNA；随后将上述重组质粒与表达Cas9蛋白的pCBT001质粒共同电转入上一步获得的工程菌并去除多余质粒后，得到重组菌EcNΔthyAΔbscAΔfliCΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔnth/tppB::Pvan-pheDH-phepΔagaI/rsml::Pvan-ARO10-yahk。
4.6.4在rhtB/C位点敲入PBAD_LAAD
PBAD_LAAD表达盒(SEQ ID NO：110)由金斯瑞(GeneScript)合成，靶向rhtB/C的sgRNA序列见表2。按照实施例1的方法，将sgRNA序列和表达盒(PBAD_LAAD表达盒和如SEQ ID NO：103所示的vanRAM表达盒)依次接入pCBT003载体，从而获得重组质粒PCBT003-PBAD_LAAD-rhtB/C-sgRNA；随后将上述重组质粒与表达Cas9蛋白的pCBT001质粒共同电转入上一步获得的工程菌并去除多余质粒后，得到重组菌EcNΔthyAΔbscAΔfliCΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔnth/tppB::Pvan-pheDH-phepΔagaI/rsml::Pvan-ARO10-yahkΔrhtB/C::PBAD_LAAD，即CBT-206菌株。
4.7 CBT-205、CBT-207、CBT-208菌株的构建：
参照CBT-206菌株的构建过程，CBT-205、CBT-207、CBT-208菌株的基因型如下：
CBT-205菌株的基因型：EcNΔthyAΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔnth/tppB::Pvan-pheDH-phepΔagaI/rsml::Pvan-ARO10-yahkΔrhtB/C::PBAD_LAAD
CBT-207菌株的基因型：EcNΔthyAΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔnth/tppB::Pvan-pheDH-phepΔagaI/rsml::Pvan-ARO10-yahkΔtkrA::Pvan-pheDH-phepΔrhtB/C::PBAD_LAAD
CBT-208菌株的基因型：EcNΔthyAΔbscAΔfliCΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔnth/tppB::Pvan-pheDH-phepΔagaI/rsml::Pvan-ARO10-yahkΔtkrA::Pvan-pheDH-phepΔrhtB/C::PBAD_LAAD
4.8 CBT-212、CBT-213菌株的构建(启动子为Pvan和Pfnrs)
参考实施例4.4和4.6的构建方法，构建了启动子为Pvan和Pfnrs的菌株CBT-212、CBT-213，其基因型信息如下：
CBT-212菌株的基因型：EcNΔthyAΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pfnrs-TyrB-gdh2ΔlacZ::Pfnrs-ARO10-yahkΔmaeB::Pfnrs-pheDH-phepΔagaI/rsml::Pfnrs-ARO10-yahkΔrhtB/C::PBAD_LAAD
CBT-213菌株的基因型：EcNΔthyAΔyghX::Pvan-TyrB-gdh2ΔyjcS::Pvan-ARO10-yahkΔaraAB::Pvan-TyrB-gdh2ΔldhA::Pvan-ARO10-yahkΔmaeB::Pfnrs-pheDH-phepΔagaI/rsml::Pfnrs-ARO10-yahkΔkefB::Pfnrs-pheDH- phepΔrhtB/C::PBAD_LAAD
实施例5：工程菌体外降解苯丙氨酸的能力检测方法
5.1苯丙氨酸和苯乙醇的HPLC检测方法
色谱柱Athena C18液相色谱柱(250mm×4.6mm，5μm)；柱温35℃；以0.1％磷酸水(V/V)和甲醇为流动相，进行梯度洗脱；采用紫外检测器，检测波长254nm，进样量10μL。
5.2体外活性检测方法
分别接甘油菌EcN以及各工程菌于5mL LB培养基中，37℃过夜培养。次日，以1％(v/v)的接种量分别转接菌液于200mL LB培养基中，37℃，220rpm培养5.5h；测定OD600，4000g离心8min，收集菌体，用PBS缓冲液(含4mM苯丙氨酸)重悬菌体，将菌浓OD600调至2或0.5；分别取10mL菌悬液于14mL摇菌管中，定位T＝0h，放于37℃，220rpm培养3h，取样1mL。12000rpm，离心10min，收集上清样品进行HPLC检测。
实施例6转入了苯丙氨酸降解通路的工程菌能够有效降解苯丙氨酸
本实施例使用实施例5中的方法测试了CBT2001/CBT2002和CBT2003在体外降解苯丙氨酸的能力。其中CBT2001是益生菌大肠杆菌Nissle1917基因组上整合单拷贝的本公开中苯丙氨酸代谢通路基因的工程菌(包括单拷贝的芳香族氨基酸转氨酶基因TyrB、谷氨酸脱氢酶基因gdh2、醛还原酶基因YahK和苯丙酮酸脱羧酶基因ARO10)，CBT2002中则整合了双拷贝的CBT2001中苯丙氨酸代谢通路基因，CBT2003则为了强化菌株对环境中苯丙氨酸的摄取，在CBT2002中进一步整合了单拷贝的苯丙氨酸转运蛋白基因。
在有氧催化条件下，CBT2001、CBT2002和CBT2003工程菌株均能够降解苯丙氨酸生成苯乙醇，各个菌株降解苯丙氨酸的体外检测结果见表3和图3。
表3菌株代谢苯丙氨酸产生苯乙醇的最大生成速率
在厌氧催化条件下，工程菌株降解苯丙氨酸生成苯乙醇，各个菌株降解苯丙氨酸的体外检测结果见表4和图4。
表4菌株代谢苯丙氨酸产生苯乙醇的最大生成速率
从图3和图4可知，在有氧催化条件和厌氧催化条件下，原始菌株EcN均不能降解苯丙氨酸生成苯乙醇；而工程菌株CBT2001，CBT2002和CBT2003均有苯乙醇生成，表明本公开的工程菌能按照工程菌代谢通路的设计，降解苯丙氨酸并生成苯乙醇。并且从各菌株代谢能力的对比看，整合两拷贝相关代谢通路基因的CBT2002的代谢能力强于单拷贝的CBT2001，而能促进苯丙氨酸摄取的苯丙氨酸转运蛋白的加入使得CBT2003具有了更强的苯丙氨酸代谢能力。
实施例7苯丙氨酸代谢工程菌的优化
7.1苯丙氨酸脱氢酶具有更好的代谢活性
本实施例构建了利用苯丙氨酸脱氢酶替代芳香族氨基酸转氨酶基因TyrB的工程菌CBT-209，其中，苯丙氨酸脱氢酶是唯一直接转化苯丙氨酸的代谢酶。由于不再发生TyrB的转氨作用，因此也无需加入用于偶联苯丙氨酸的转氨反应与苯乙醛的还原反应谷 氨酸脱氢酶。具体而言，CBT-209菌株包含了单拷贝的苯丙氨酸脱氢酶(pheDH)、苯丙酮酸脱羧酶(Aro10)、醛还原酶yahk以及苯丙氨酸转运蛋白(pheP)。
作为比较，本实施例还构建了CBT-210工程菌株，其包含了单拷贝的芳香族氨基酸转氨酶(TyrB)、苯丙酮酸脱羧酶(Aro10)、谷氨酸脱氢酶(Gdh2)、醛还原酶yahk以及苯丙氨酸转运蛋白(pheP)，其中苯丙氨酸转氨酶(TyrB)是唯一直接转化苯丙氨酸的酶。通过使用实施例5中的方法比较其体外代谢能力，结果如图5所示(比较CBT-209和CBT-210)，申请人意外的发现，CBT-209菌株不但表现出了有效的苯丙氨酸降解能力，其效率甚至优于通过芳香族氨基酸转氨酶降解苯丙氨酸的CBT-210菌株。这个发现进一步丰富了工程菌在第一步苯丙氨酸代谢中酶的选择。后续发明人考虑到工程菌用于体内治疗时面临环境和条件的复杂性，构建了同时包含苯丙氨酸转氨酶(TyrB)和苯丙氨酸脱氢酶(pheDH)的工程菌(如实施例4的4.4至4.8构建的多个工程菌)，以便充分利用苯丙氨酸代谢通路中不同代谢酶的优势，提高工程菌株的苯丙氨酸降解效果。
7.2 L-氨基酸脱氨酶(LAAD)的加入进一步提高苯丙氨酸代谢效果
除了苯丙氨酸转氨酶(TyrB)和苯丙氨酸脱氢酶(pheDH)的工程菌，为了进一步提高苯丙氨酸代谢效果，发明人进一步在工程菌的基因组中整合了单拷贝的L-氨基酸脱氨酶(LAAD)，并使用实施例5中的方法进行检测。结果如图6至图9所示，CBT-201、CBT-202、CBT-205、CBT-206、CBT-207和CBT-208菌株在不用阿拉伯糖(Ara)诱导LAAD表达的情况下，已经能够较为高效的降解苯丙氨酸，这表明利用苯丙氨酸脱氢酶和/或苯丙氨酸转氨酶能够实现较好的代谢效果，然而通过阿拉伯糖的诱导表达苯丙氨酸脱氨酶LAAD后，菌株展现了更强的苯丙氨酸代谢能力。
7.3敲除鞭毛相关基因不会影响工程菌对苯丙氨酸的代谢能力
鞭毛与大肠杆菌的运动相关，已有研究表明通过敲除大肠杆菌的鞭毛合成/结构相关基因，会使得大肠杆菌的鞭毛结构出现缺陷，进而影响其运动能力，并提高大肠杆菌在体内的停留时间(Zachary J.S.Mays,Todd C.Chappell,and Nikhil U.Nair ACS Synthetic Biology 2020 9(2),356-367.DOI:10.1021/acssynbio.9b00356)。因此敲除工程菌基因组中鞭毛相关基因预期将是一种延长苯丙氨酸代谢工程菌在患者肠道的滞留时间，从而增强苯丙氨酸降解持续性的有效手段。于是本实施例使用实施例5中的方法测试了在体外环境下鞭毛相关基因的敲除是否对工程菌降解苯丙氨酸的能力产生不利影响。结果如图8和图9所示，CBT-205菌株和CBT-207菌株均是鞭毛功能正常的工程菌，而CBT-206菌株和CBT-208菌株均敲除了鞭毛相关基因bscA和fliC，从结果可以看出(分别比较图8中的CBT-205和CBT-206，以及图9中的CBT-207和CBT-208)，鞭毛相关基因的敲除并不会影响菌株的功能和活性。
7.4 Pvan启动子相比于Pfnrs启动子能赋予工程菌更强的代谢活性
本实施例使用实施例5中的方法测试了Pvan启动子相比于Pfnrs启动子对工程菌代谢能力的影响。CBT-201和CBT-211转入的酶和拷贝数量均相同，区别仅在于CBT-201的所有表达盒/拷贝均使用了PfnrS启动子，而CBT-211的所有表达盒/拷贝均使用了Pvan启动子，结果如图10所示(即最上面的两条曲线)，CBT-211的活性要高于CBT-201。此外从图11中也可以看出，CBT-205和CBT-212同样转入相同的酶和表达盒拷贝数，其中CBT-205使用了6个Pvan启动子，CBT-212使用了2个Pvan启动子和4个PfnrS启动子，结果全部使用Pvan启动子的CBT-205的活 性高于同时使用Pvan和PfnrS启动子的CBT-212(比较CBT-205和CBT-212)；同样的现象也发生在CBT-207和CBT-213菌株中(同样参见图11，比较CBT-207和CBT-213)。上述结果显示，Pfnrs启动子已经表现出令人满意的活性，而Pvan启动子相比于Pfnrs启动子能赋予工程菌更强的体外代谢活性。
7.5增加特定表达盒的拷贝数可以改善菌株的代谢活性
本实施例使用实施例5中的方法测试了不同表达盒的拷贝数对工程菌代谢能力的影响。CBT-205和CBT-207均使用了更优的Pvan启动子，CBT-207在CBT-205的基础上增加了一个拷贝的pheDH表达盒，从图10可以看出(比较CBT-207和CBT-205)，CBT-207较CBT-205的代谢效率具有大幅提升。
对比例1：构建苯丙氨酸代谢工程菌SYNB1934
Synlogic公司正在开发中的同样以EcN为底盘菌所构建的苯丙氨酸代谢工程菌SYNB1934目前正处于临床III期研究阶段，根据此前报道的数据，SYBN1934在动物和人体中均获得了良好的代谢活性。本对比例即根据Synlogic公布的方法(例如，Isabella,V.M.,B.N.Ha,M.J.Castillo,D.J.Lubkowicz,S.E.Rowe,Y.A.Millet,C.L.Anderson,N.Li,A.B.Fisher,K.A.West,P.J.Reeder,M.M.Momin,C.G.Bergeron,S.E.Guilmain,P.F.Miller,C.B.Kurtz and D.Falb(2018)."Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria."Nat Biotechnol 36(9):857-864.和Adolfsen,K.J.,I.Callihan,C.E.Monahan,P.J.Greisen,J.Spoonamore,M.Momin,L.E.Fitch,M.J.Castillo,L.Weng,L.Renaud,C.J.Weile,J.H.Konieczka,T.Mirabella,A.Abin-Fuentes,A.G.Lawrence and V.M.Isabella(2021)."Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering."Nat Commun 12(1):6215.)，构建获得SYNB1934，作为本公开的活 性参照，以比较本公开工程菌与SYNB1934的代谢活性。SYNB1934的基因型信息如下：
EcNΔrhtBC::Ptac-phePΔaraDC::Para-LAADΔ05580::Ptac-stlA1934ΔmalEK::Ptac-stlA1934ΔagaI::Ptac-stlA1934ΔyicS/nepI::Ptac-stlAΔdapA
实施例8：EcN和工程菌株的体内活性实验
苯丙酮尿症小鼠Pah KO(简称PKU小鼠)由江苏集萃药康生物科技股份有限公司提供。新生苯丙酮尿症小鼠，用无苯丙氨酸(Phe-free)饲料喂养，含苯丙氨酸(0.2g/L)的饮用水作为苯丙氨酸补给。8周龄以上的小鼠可用于检测工程菌降解苯丙氨酸的能力。动物实验开始前2小时(T＝-2h)，停止含苯丙氨酸饮用水补给，更换为普通饮用水，采血清检测血液中苯丙氨酸浓度。在实验开始时(T＝0)，小鼠皮下注射0.05mg/g(BW)苯丙氨酸溶液(5mg/ml)，在皮下注射苯丙氨酸后1、2及3小时灌胃200μL(5×1010cfu/只小鼠)益生菌EcN或工程菌(工程菌包括本公开的工程菌CBT-201、CBT-201-AA及CBT-205以及对比例1中构建的SYNB1934作为活性参照)，每组5只小鼠，在注射苯丙氨酸第4小时后，采血清检测血液中苯丙氨酸的浓度。
小鼠血液中苯丙氨酸浓度的变化如图12所示，灌胃所有选定工程菌的小鼠血液中苯丙氨酸浓度增长值均明显低于灌胃EcN的小鼠，说明工程菌能够有效的控制血清苯丙氨酸浓度。相比灌胃EcN的小鼠，灌胃CBT-201工程菌的小鼠血液中苯丙氨酸降低幅度高达55％，其苯丙氨酸降解能力比作为活性参照的SYNB1934工程菌还要高出约50％(SYNB1934工程菌能够达到37％的苯丙氨酸浓度降低)。此外，CBT-201-AA和CBT-205的苯丙氨酸浓度降低幅度分别达到了44％和53.1％，也均显著高于SYNB1934工程菌，由此可见，本公开构建的工程菌在体内展现了极强的苯丙氨酸代谢活性。此外，CBT-201-AA与CBT-201相比，增加了两个拷贝的Pfnrs-ARO10-yahk表达盒，但是苯丙氨酸浓度的降低 幅度反而略有下降，这也表明了转入工程菌的苯丙氨酸代谢通路中相关酶的拷贝数/表达量并非越多越好。
综上所述，本公开所构建的工程菌在体外和体内均有效降解了苯丙氨酸，能够用于治疗苯丙酮尿症。本公开构建的工程菌能够有效地降解苯丙氨酸和/或苯丙酮酸，实验证明，该工程菌有效降解了PKU小鼠的体内苯丙氨酸和苯丙氨酸代谢物，显示出良好的应用前景。
以上所述仅为本公开的实施例，并非因此限制本公开的保护范围；凡是利用本公开说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本公开的保护范围内。

Claims (115)

  1. 一种工程微生物，其包含下列外源基因：
    一种或更多种编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；
    一种或更多种编码能够将苯丙酮酸转化为苯乙醛的酶的基因；
    一种或更多种编码能够将苯乙醛转化为苯乙醇的酶的基因；以及
    一种或更多种编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因。
  2. 根据权利要求1所述的工程微生物，所述工程微生物能够在人和/或哺乳动物肠道内代谢苯丙氨酸。
  3. 根据权利要求1或2所述的工程微生物，其中所述外源基因整合在基因组中或者位于表达质粒上。
  4. 根据权利要求1至3中任一项所述的工程微生物，其中所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因选自编码转氨酶的基因、编码脱氢酶的基因、编码脱氨酶的基因、及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。
  5. 根据权利要求4所述的工程微生物，其中所述编码脱氢酶的基因是编码苯丙氨酸脱氢酶的基因。
  6. 根据权利要求5所述的工程微生物，其中所述编码苯丙氨酸脱氢酶的基因源自病毒、真菌和/或细菌。
  7. 根据权利要求6所述的工程微生物，其中所述编码苯丙氨酸 脱氢酶的基因源自脲芽孢八叠球菌和/或芽孢杆菌，例如脲芽孢八叠球菌SCRC-R04(Sporosarcina ureae SCRC-R04)、球形赖氨酸芽孢杆菌SCRC-R79a(Lysinibacillus sphaericus SCRC-R79a)、栗褐芽胞杆菌(Bacillus badius)、和/或芽孢杆菌SLBN-3(Bacillus sp.SLBN-3)。
  8. 根据权利要求7所述的工程微生物，其中所述编码苯丙氨酸脱氢酶的基因选自芽孢杆菌SLBN-3的PheDH(Phenylalanine Dehydrogenase)。
  9. 根据权利要求8所述的工程微生物，其中所述苯丙氨酸脱氢酶具有如SEQ ID NO:6所示的氨基酸序列；或者编码所述苯丙氨酸脱氢酶的基因具有如SEQ ID NO:17所示的核苷酸序列。
  10. 根据权利要求4-9中任一项所述的工程微生物，其中所述编码转氨酶的基因是编码芳香族氨基酸转氨酶的基因。
  11. 根据权利要求10所述的工程微生物，其中所述编码芳香族氨基酸转氨酶的基因源自病毒、真菌和/或细菌。
  12. 根据权利要求11所述的工程微生物，其中所述编码芳香族氨基酸转氨酶的基因源自大肠杆菌和/或酵母，例如大肠杆菌BL21(DE3)(Escherichia coli BL21(DE3))和/或酿酒酵母S288C(Saccharomyces cerevisiae S288C)。
  13. 根据权利要求12所述的工程微生物，其中所述编码芳香族氨基酸转氨酶的基因选自大肠杆菌BL21(DE3)的TyrB(Tyrosine aminotransferase)或酿酒酵母S288C的ARO8(Bifunctional 2-aminoadipate transaminase/aromatic-amino-acid:2-oxoglutarate transaminase)。
  14. 根据权利要求13所述的工程微生物，其中所述芳香族氨基酸转氨酶具有如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；或者编码所述芳香族氨基酸转氨酶的基因具有如SEQ ID NO:15或SEQ ID NO:90所示的核苷酸序列。
  15. 根据权利要求10-14中任一项所述的工程微生物，其中所述工程微生物还包含编码谷氨酸脱氢酶的基因及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。
  16. 根据权利要求15所述的工程微生物，其中所述编码谷氨酸脱氢酶的基因源自病毒、真菌和/或细菌。
  17. 根据权利要求16所述的工程微生物，其中所述编码谷氨酸脱氢酶的基因源自酵母和/或艰难梭菌，例如酿酒酵母S288C和/或艰难梭菌(Clostridioides difficile)。
  18. 根据权利要求17所述的工程微生物，其中所述编码谷氨酸脱氢酶的基因选自酿酒酵母S288C的或艰难梭菌的GDH2(Glutamate dehydrogenase(NAD+))。
  19. 根据权利要求18所述的工程微生物，其中所述谷氨酸脱氢酶具有如SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列；或者编码所述谷氨酸脱氢酶的基因具有如SEQ ID NO:20或SEQ ID NO:97所示的核苷酸序列。
  20. 根据权利要求4至19中任一项所述的工程微生物，其中所述编码脱氨酶的基因是编码L-氨基酸脱氨酶的基因。
  21. 根据权利要求20所述的工程微生物，其中所述编码L-氨基 酸脱氨酶的基因源自病毒、真菌和/或细菌。
  22. 根据权利要求21所述的工程微生物，其中所述编码L-氨基酸脱氨酶的基因源自大肠杆菌、酵母、和/或奇异变形杆菌，例如大肠杆菌BL21(DE3)、酿酒酵母S288C、和/或奇异变形杆菌HI4320(Proteus mirabilis HI4320)。
  23. 根据权利要求22所述的工程微生物，其中所述编码L-氨基酸脱氨酶的基因选自大肠杆菌BL21(DE3)、酿酒酵母S288C、或奇异变形杆菌HI4320的LAAD(L-amino acid deaminase)。
  24. 根据权利要求23所述的工程微生物，其中所述L-氨基酸脱氨酶具有如SEQ ID NO:89所示的氨基酸序列，或者编码所述L-氨基酸脱氨酶的基因具有如SEQ ID NO:91所示的核苷酸序列。
  25. 根据权利要求4-24中任一项所述的工程微生物，其中所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因选自以下组：
    (1)编码苯丙氨酸脱氢酶的基因；
    (2)编码芳香族氨基酸转氨酶的基因；
    (3)编码芳香族氨基酸转氨酶的基因和编码苯丙氨酸脱氢酶的基因；
    (4)编码苯丙氨酸脱氢酶的基因和编码L-氨基酸脱氨酶的基因；
    (5)编码芳香族氨基酸转氨酶的基因和编码L-氨基酸脱氨酶的基因；或
    (6)编码芳香族氨基酸转氨酶的基因、编码苯丙氨酸脱氢酶的基因、和编码L-氨基酸脱氨酶的基因。
  26. 根据权利要求1-25中任一项所述的工程微生物，其中所述编码能够将苯丙酮酸转化为苯乙醛的酶的基因选自编码苯丙酮酸脱 羧酶的基因、编码α-酮酸脱羧酶的基因、及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。
  27. 根据权利要求26所述的工程微生物，其中所述编码苯丙酮酸脱羧酶的基因源自病毒、真菌和/或细菌。
  28. 根据权利要求27所述的工程微生物，其中所述编码苯丙酮酸脱羧酶的基因源自酵母，例如酿酒酵母S288C。
  29. 根据权利要求28所述的工程微生物，其中所述编码苯丙酮酸脱羧酶的基因是酿酒酵母S288C的ARO10(Phenylpyruvate decarboxylase)。
  30. 根据权利要求29所述的工程微生物，其中所述苯丙酮酸脱羧酶具有如SEQ ID NO:7所示的氨基酸序列；或者编码所述苯丙酮酸脱羧酶的基因具有如SEQ ID NO:18所示的核苷酸序列。
  31. 根据权利要求26所述的工程微生物，其中所述编码α-酮酸脱羧酶的基因源自病毒、真菌和/或细菌。
  32. 根据权利要求31所述的工程微生物，其中所述编码α-酮酸脱羧酶的基因源自酵母和/或奇异变形杆菌，例如酿酒酵母S288C和/或奇异变形杆菌JN458(Proteus mirabilis JN458)。
  33. 根据权利要求32所述的工程微生物，其中所述编码α-酮酸脱羧酶的基因选自酿酒酵母S288C的或奇异变形杆菌JN458的KDC(Alpha-keto-acid decarboxylase)的至少一种。
  34. 根据权利要求33所述的工程微生物，其中所述α-酮酸脱羧酶具有如SEQ ID NO:8所示的序列；或者编码所述α-酮酸脱羧酶的 基因具有如SEQ ID NO:95所示的核苷酸序列。
  35. 根据权利要求1至34中任一项所述的工程微生物，其中所述编码能够将苯乙醛转化为苯乙醇的酶的基因选自编码醛还原酶的基因及其功能等效物中的一种或多种，所述功能等效物至少保留相关酶的部分活性。
  36. 根据权利要求35所述的工程微生物，其中所述编码醛还原酶的基因源自病毒、真菌和/或细菌。
  37. 根据权利要求36所述的工程微生物，其中所述编码醛还原酶的基因源自大肠杆菌和/或短乳杆菌，例如大肠杆菌str.K-12 substr.MG1655和/或短乳杆菌(Lactobacillus brevis)。
  38. 根据权利要求37所述的工程微生物，其中所述编码醛还原酶的基因选自大肠杆菌str.K-12 substr.MG1655的YahK(NADPH-dependent aldehyde reductase)或短乳杆菌的ADH(Alcohol dehydrogenase)。
  39. 根据权利要求38所述的工程微生物，其中所述醛还原酶具有如SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列；或者编码所述醛还原酶的基因具有如SEQ ID NO:19或SEQ ID NO:96所示的核苷酸序列。
  40. 根据权利要求1至39中任一项所述的工程微生物，其中所述编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因选自编码苯丙氨酸转运蛋白的基因及其功能等效物中的一种或更多种，所述功能等效物至少保留相关酶的部分活性。
  41. 根据权利要求40所述的工程微生物，其中所述编码苯丙氨 酸转运蛋白的基因源自病毒、真菌和/或细菌。
  42. 根据权利要求41所述的工程微生物，其中所述编码苯丙氨酸转运蛋白的基因源自大肠杆菌。
  43. 根据权利要求42所述的工程微生物，其中所述编码苯丙氨酸转运蛋白的基因是大肠杆菌的PheP(Phenylalanine:H(+)symporter)。
  44. 根据权利要求43所述的工程微生物，其中所述苯丙氨酸转运蛋白具有如SEQ ID NO:13所示的氨基酸序列；或者编码所述苯丙氨酸转运蛋白的基因具有如SEQ ID NO:21所示的核苷酸序列。
  45. 根据权利要求1-44中任一项所述的工程微生物，所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、所述编码能够将苯丙酮酸转化为苯乙醛的酶的基因、所述编码能够将苯乙醛转化为苯乙醇的酶的基因、所述编码谷氨酸脱氢酶的基因、和/或所述编码苯丙氨酸转运蛋白的基因中的任一个、两个、三个、四个、五个、六个、多个或全部可选地与一个或更多个相同或不同的启动子可操作地连接。
  46. 根据权利要求45所述的工程微生物，其中所述编码芳香族氨基酸转氨酶的基因和编码谷氨酸脱氢酶的基因共用同一启动子。
  47. 根据权利要求45所述的工程微生物，其中所述编码苯丙酮酸脱羧酶的基因和编码醛还原酶的基因共用同一启动子。
  48. 根据权利要求45所述的工程微生物，其中所述编码苯丙氨酸脱氢酶的基因和编码苯丙氨酸转运蛋白的基因共用同一启动子。
  49. 根据权利要求45所述的工程微生物，其中所述编码L-氨基酸脱氨酶的基因单独与启动子可操作地连接。
  50. 根据权利要求45所述的工程微生物，其中所述编码苯丙氨酸转运蛋白的基因单独与启动子可操作地连接。
  51. 根据权利要求45-50中任一项所述的工程微生物，其中所述启动子为内源启动子或外源启动子。
  52. 根据权利要求45-50中任一项所述的工程微生物，其中所述启动子为诱导型启动子或组成型启动子。
  53. 根据权利要求52所述的工程微生物，其中所述启动子为直接或间接诱导型启动子。
  54. 根据权利要求53所述的工程微生物，其中所述启动子由外源环境条件直接或间接诱导。
  55. 根据权利要求54所述的工程微生物，其中所述启动子由哺乳动物肠道中的外源环境条件直接或间接诱导。
  56. 根据权利要求51-55中任一项所述的工程微生物，其中所述启动子选自PfnrS、FDHF、Ptet、Pbba、Ptrc、Pvan、或PBAD中的至少一种。
  57. 根据权利要求56所述的工程微生物，其中所述PfnrS启动子的序列如SEQ ID NO.22所示。
  58. 根据权利要求56所述的工程微生物，其中所述FDHF启动子的序列如SEQ ID NO.24所示。
  59. 根据权利要求56所述的工程微生物，其中所述Ptet启动子的序列如SEQ ID NO.100所示。
  60. 根据权利要求56所述的工程微生物，其中所述Pbba启动子的序列如SEQ ID NO.98所示。
  61. 根据权利要求56所述的工程微生物，其中所述Ptrc启动子的序列如SEQ ID NO.99所示。
  62. 根据权利要求56所述的工程微生物，其中所述Pvan启动子的序列如SEQ ID NO:101所示。
  63. 根据权利要求56所述的工程微生物，其中所述PBAD启动子的序列如SEQ ID NO:102所示。
  64. 根据权利要求1-63中任一项所述的工程微生物，其中所述编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、所述编码能够将苯丙酮酸转化为苯乙醛的酶的基因、所述编码能够将苯乙醛转化为苯乙醇的酶的基因、所述编码谷氨酸脱氢酶的基因、和/或所述编码苯丙氨酸转运蛋白的基因位于一个或多个表达盒中。
  65. 根据权利要求64所述的工程微生物，其中所述工程微生物包含第一表达盒和第二表达盒，其中第一表达盒包含第一启动子、以及与其可操作连接的所述编码芳香族氨基酸转氨酶的基因和所述编码谷氨酸脱氢酶的基因；第二表达盒包含第二启动子、以及与其可操作连接的所述编码苯丙酮酸脱羧酶的基因和编码醛还原酶的基因；所述第一或第二启动子是相同的或者不同的。
  66. 根据权利要求65所述的工程微生物，其中所述工程微生物 还包含第三表达盒，所述第三表达盒包含可操作地连接至第三启动子的所述编码苯丙氨酸转运蛋白的基因，所述第三启动子可以与第一启动子和/或第二启动子相同，或者与第一启动子和第二启动子均不同。
  67. 根据权利要求64所述的工程微生物，其中所述工程微生物包含第四表达盒和第二表达盒，其中第四表达盒包含第四启动子、以及与其可操作连接的所述编码苯丙氨酸脱氢酶的基因和编码苯丙氨酸转运蛋白的基因；第二表达盒包含第二启动子、以及与其可操作连接的所述编码苯丙酮酸脱羧酶的基因和编码醛还原酶的基因；所述第四或第二启动子是相同的或者不同的。
  68. 根据权利要求67所述的工程微生物，其中所述工程微生物还包含第一表达盒，所述第一表达盒包含第一启动子、以及与其可操作连接的所述编码芳香族氨基酸转氨酶的基因和所述编码谷氨酸脱氢酶的基因；所述第一启动子可以与第四启动子和/或第二启动子相同，或者与第四启动子和第二启动子均不同。
  69. 根据权利要求67-68任一项所述的工程微生物，其中所述工程微生物还包含第五表达盒，所述第五表达盒包含第五启动子、与其可操作连接的所述编码L-氨基酸脱氨酶的基因；所述第五启动子与第四启动子、第二启动子和/或第一启动子相同或不同。
  70. 根据权利要求64-69任一项所述的工程微生物，其中所述第一启动子、第二启动子、第三启动子、第四启动子的全部或者部分选自PfnrS和/或Pvan中的至少一个，所述第五启动子是PBAD。
  71. 根据权利要求64-69任一项所述的工程微生物，其中所述表达盒以单拷贝、双拷贝、三拷贝、四拷贝或更多拷贝的形式存在。
  72. 根据权利要求71所述的工程微生物，其中所述第一表达盒以单拷贝或双拷贝的形式存在。
  73. 根据权利要求71所述的工程微生物，其中所述第二表达盒以单拷贝、双拷贝、三拷贝或四拷贝的形式存在。
  74. 根据权利要求71所述的工程微生物，其中所述第三表达盒以单拷贝的形式存在。
  75. 根据权利要求71所述的工程微生物，其中所述第四表达盒以单拷贝或双拷贝的形式存在。
  76. 根据权利要求71所述的工程微生物，其中所述第五表达盒以单拷贝的形式存在。
  77. 根据权利要求64-76任一项所述的工程微生物，其中所述表达盒存在于质粒上。
  78. 根据权利要求64-76任一项所述的工程微生物，其中所述表达盒稳定整合在所述工程微生物的一个、两个、或多个相同或者不同的基因组位点中。
  79. 根据权利要求78所述的工程微生物，其中所述基因组位点选自yicS位点、malPT位点、malE位点、exo位点、rhtB/C位点、agaI/rsml位点、araBD位点、yghx位点、ldhA位点、araAB位点、lacZ位点、kefB位点、maeB位点、nth/tppB位点、和/或tkrA位点中的至少一个。
  80. 根据权利要求79所述的工程微生物，其中所述第一表达盒整合在在yghx位点和/或araAB位点。
  81. 根据权利要求79所述的工程微生物，其中所述第二表达盒整合在ldhA位点、lacZ位点、yghx位点、yjcS位点和/或agaI/rsml位点。
  82. 根据权利要求79中所述的工程微生物，其中所述第三表达盒整合在kefB位点。
  83. 根据权利要求79中所述的工程微生物，其中所述第四表达盒整合在kefB位点、ldhA位点、nth/tppB位点、maeB位点和/或tkrA位点。
  84. 根据权利要求79中所述的工程微生物，其中所述第五表达盒整合在rhtB/C位点。
  85. 如前述权利要求中任一项所述的工程微生物，所述工程微生物包含：
    1)单拷贝的第四表达盒，所述第四表达盒整合在kefB位点；双拷贝的第二表达盒，所述第二表达盒分别整合在ldhA和lacZ位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为PfnrS启动子，第五表达盒中使用的启动子为PBAD启动子；
    2)单拷贝的第四表达盒，所述第四表达盒整合在nth/tppB位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、ldhA和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为Pvan启动子，第五表达盒中使用的启动子为PBAD启动子；
    3)双拷贝的第四表达盒，所述第四表达盒分别整合在 nth/tppB和tkrA位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、ldhA和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为Pvan启动子，第五表达盒中使用的启动子为PBAD启动子；
    4)单拷贝的第四表达盒，所述第四表达盒整合在maeB位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、lacZ和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述单拷贝的第四表达盒使用PfnrS启动子，三拷贝的第二表达盒分别使用PfnrS启动子、PfnrS启动子和Pvan启动子，双拷贝的第一表达盒分别使用PfnrS启动子和Pvan启动子，第五表达盒中使用的启动子为PBAD启动子；
    5)双拷贝的第四表达盒，所述第四表达盒整合在maeB和kefB位点；三拷贝的第二表达盒，所述第二表达盒分别整合在yjcS、ldhA和agaI/rsml位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述双拷贝的第四表达盒使用PfnrS启动子，三拷贝的第二表达盒分别使用Pvan启动子、Pvan启动子和PfnrS启动子，双拷贝的第一表达盒使用Pvan启动子，第五表达盒使用的启动子为PBAD启动子；
    6)单拷贝的第四表达盒，所述第四表达盒整合在kefB位点；四拷贝的第二表达盒，所述第二表达盒分别整合在ldhA、lacZ、agaI/rsml和yjcS位点；双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四、第二和第一表达盒中使用的启动子为PfnrS启动子，第五表达盒中使用的启动子为PBAD启动子；
    7)双拷贝的第四表达盒，所述第四表达盒整合在ldhA和 maeB位点；双拷贝的第二表达盒，所述第二表达盒分别整合在yghX和lacZ位点；以及单拷贝的第五表达盒，所述第五表达盒整合在rhtB/C位点；其中，所述第四和第二表达盒中使用的启动子为PfnrS启动子，第五表达盒中使用的启动子为PBAD启动子；
    8)单拷贝的第四表达盒，所述第四表达盒整合在ldhA位点；以及单拷贝的第二表达盒，所述第二表达盒整合在yghX位点；其中，所述第四和第二表达盒中使用的启动子为PfnrS启动子；
    9)单拷贝的第一表达盒，所述第一表达盒整合在yghX位点；单拷贝的第二表达盒，所述第二表达盒整合在ldhA位点；以及单拷贝的第三表达盒，所述第三表达盒整合在kefB位点；其中，所述第一、第二和第三表达盒中使用的启动子为PfnrS启动子；
    10)单拷贝的第一表达盒，所述第一表达盒整合在yghX位点；以及单拷贝的第二表达盒，所述第二表达盒整合在ldhA位点；其中，所述第一和第二表达盒中使用的启动子为PfnrS启动子；
    11)双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；以及双拷贝的第二表达盒，所述第二表达盒整合在ldhA和lacZ位点；其中，所述第一和第二表达盒中使用的启动子为PfnrS启动子；或
    12)双拷贝的第一表达盒，所述第一表达盒分别整合在yghX和araAB位点；双拷贝的第二表达盒，所述第二表达盒分别整合在ldhA和lacZ位点；以及单拷贝的第三表达盒，所述第三表达盒整合在kefB位点；其中，所述第一、第二和第三表达盒中使用的启动子为PfnrS启动子。
  86. 根据前述权利要求中任一项所述的工程微生物，其中所述工程微生物为细菌或酵母。
  87. 根据权利要求86所述的工程微生物，其中所述工程微生物为非致病性细菌。
  88. 根据权利要求87所述的工程微生物，其中所述工程微生物为益生菌。
  89. 根据权利要求88所述的工程微生物，其中所述工程微生物选自拟杆菌属、双歧杆菌属、梭菌属、埃希氏菌属、乳杆菌属和乳球菌属中的至少一种。
  90. 根据权利要求89所述的工程微生物，其中所述工程微生物为大肠杆菌。
  91. 根据权利要求90所述的工程微生物，其中所述工程微生物为大肠杆菌菌株Nissle1917。
  92. 根据权利要求86-91任一项所述的工程微生物，其中所述工程微生物为营养缺陷型。
  93. 根据权利要求92所述的工程微生物，其中所述营养缺陷型为二氨基丙烯酸营养缺陷型和/或胸腺嘧啶营养缺陷型。
  94. 根据权利要求93所述的工程微生物，其中当所述工程微生物存在于哺乳动物肠道时，所述营养缺陷型得到补充。
  95. 根据权利要求94所述的工程微生物，其中所述哺乳动物肠道是人肠道。
  96. 根据权利要求86-95任一项所述的工程微生物，其中所述工程微生物的鞭毛功能有缺陷或者丧失。
  97. 根据权利要求96所述的工程微生物，其中所述工程微生物的鞭毛合成相关基因具有突变或者缺失。
  98. 根据权利要求97所述的工程微生物，其中所述鞭毛合成相关基因为bscA和/或fliC基因。
  99. 一种工程微生物组合物，其包含多于一种工程微生物，所述多于一种工程微生物中的每一种彼此独立地包含以下基因中的任一种或更多种：
    编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；
    编码能够将苯丙酮酸转化为苯乙醛的酶的基因；
    编码能够将苯乙醛转化为苯乙醇的酶的基因；
    编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因；
    其中所述工程微生物组合物包含编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、编码能够将苯丙酮酸转化为苯乙醛的酶的基因、编码能够将苯乙醛转化为苯乙醇的酶的基因、以及编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因。
  100. 根据权利要求99所述的工程微生物组合物，其中所述多于一种工程微生物中的每一种彼此独立地包含以下基因中的任一种或更多种：
    编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因；
    编码能够将苯丙酮酸转化为苯乙醛的酶的基因；
    编码能够将苯乙醛转化为苯乙醇的酶的基因；
    编码谷氨酸脱氢酶的基因；
    编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因；
    其中所述工程微生物组合物包含编码能够将苯丙氨酸转化为苯丙酮酸的酶的基因、编码能够将苯丙酮酸转化为苯乙醛的酶的基因、编码能够将苯乙醛转化为苯乙醇的酶的基因、编码谷氨酸脱氢酶的基因以及编码能够将苯丙氨酸转运至所述工程微生物体内的蛋白的基因。
  101. 一种组合物，其包含如权利要求1-98中任一项所述的工程微生物或如权利要求99-100中任一项所述的工程微生物组合物和药学上、营养学上或生理学上可接受的载体。
  102. 根据权利要求101所述的组合物，其中所述的组合物为药物组合物。
  103. 根据权利要求101所述的组合物，其中所述组合物是可食用组合物。
  104. 根据权利要求101所述的组合物，其中所述组合物是益生菌组合物。
  105. 根据权利要求101所述的组合物，其中所述组合物是食品增补剂。
  106. 根据权利要求101所述的组合物，所述组合物被配制用于口服施用。
  107. 根据权利要求101所述的组合物，其中所述组合物为液态制剂、固态制剂或半固态制剂。
  108. 根据权利要求107所述的组合物，其中所述的液态制剂选自溶液制品或悬浮液制品中的至少一种。
  109. 根据权利要求101所述的组合物，其中所述组合物的剂型选自下组：粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂，或舌下含片。
  110. 根据权利要求101所述的组合物，其中所述组合物被配制 为经由纳米颗粒、纳米胶囊、微胶囊或微片剂(其被肠溶包衣或未包衣)用于肠内施用、空肠内施用、十二指肠内施用、回肠内施用、胃分流施用或结肠内施用。
  111. 一种试剂盒，其包含如权利要求1-98中任一项所述的工程微生物或如权利要求99-100中任一项所述的工程微生物组合物、或如权利要求101-110中任一项所述的组合物。
  112. 一种用于缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的方法，所述方法包括向有此需要的患者施用如权利要求1-98中任一项所述的工程微生物、如权利要求99-100中任一项所述的工程微生物组合物、如权利要求101-110中任一项所述的组合物、或如权利要求111所述的试剂盒。
  113. 根据权利要求112所述的方法，其中所述与高苯丙氨酸血症相关的疾病和/或病症包括：苯丙酮尿症(例如经典型或典型(classical or typical)苯丙酮尿症和非经典型(atypical)苯丙酮尿症)，永久性轻度(mild)高苯丙氨酸血症，非苯丙酮尿症类的高苯丙氨酸血症，苯丙氨酸羟化酶缺陷，辅因子缺陷，二氢蝶呤还原酶缺陷，四氢蝶呤合酶缺陷，Segawa氏病和肝病。
  114. 权利要求1-98中任一项所述的工程微生物、如权利要求99-100中任一项所述的工程微生物组合物、或如权利要求101-110中任一项所述的组合物在制备用于缓解和/或治疗与高苯丙氨酸血症相关的疾病和/或病症的药物或保健品中的用途。
  115. 根据权利要求114所述的用途，其中所述与高苯丙氨酸血症相关的疾病和/或病症包括：苯丙酮尿症(例如经典型或典型(classical or typical)苯丙酮尿症和非经典型(atypical)苯丙酮尿症)，永久性轻度(mild)高苯丙氨酸血症，非苯丙酮尿症类的高 苯丙氨酸血症，苯丙氨酸羟化酶缺陷，辅因子缺陷，二氢蝶呤还原酶缺陷，四氢蝶呤合酶缺陷，Segawa氏病和肝病。
PCT/CN2023/079034 2022-03-02 2023-03-01 一种用于治疗高苯丙氨酸血症的工程微生物及其用途 Ceased WO2023165521A1 (zh)

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