Method for synthesizing phenylethylamine and application thereof

Abstract

The technical scheme provides a method for synthesizing phenylethylamine by taking acetophenone as a substrate based on a multienzyme system. The reaction system realizes efficient synthesis of phenylethylamine by synergistically utilizing various dehydrogenase and transaminase.

Description

Technical Field

[0001] This invention relates to the design of catalytic pathways, the construction of gene-edited microbial strains, and their applications in the field of biocatalysis. Specifically, it relates to a method for synthesizing R-phenylethylamine or S-phenylethylamine from acetophenone using the synergistic catalytic action of multiple enzymes, and its applications. Background Technology

[0002] Chiral amines are a crucial class of pharmaceutical intermediates with wide applications in medicine, agrochemicals, and cosmetics. Statistics show that approximately 40% of the world's top 200 best-selling drugs contain chiral amine units in their molecular structure, and this structure often plays a key role in drug activity and pharmacodynamic characteristics. In 2013 alone, the total market value of these chiral amine-containing drugs exceeded US$88 billion, highlighting the strategic importance of chiral amines in the modern pharmaceutical industry. Therefore, the synthesis and preparation of chiral amine intermediates have become an indispensable core link in drug development and industrial production. They are widely used not only in the development of drugs for the central nervous system, cardiovascular diseases, chronic obstructive pulmonary disease, antihypertensive drugs, and hypoglycemic agents, but also demonstrate significant value in research on anti-infectives, antitumor agents, and immunomodulators.

[0003] Chiral amines typically exist in two enantiomers: (S)- and (R)-. The activity, selectivity, and safety of drug molecules are often highly dependent on their specific enantiomeric configuration. For example, α-phenylethylamine (CAS: 618-36-0) is an important organic synthetic intermediate, and it and its derivatives are widely used in dyes, pharmaceuticals, emulsifiers, and fragrances. α-Phenyleneethylamine has two enantiomers: (S)-(-)-α-phenylethylamine (CAS: 2627-86-3) and (R)-(+)-α-phenylethylamine (CAS: 3886-69-9). Physiologically, (R)-(+)-phenylethylamine can promote the release of monoamine neurotransmitters such as dopamine and norepinephrine and interact with receptors in the central nervous system, thereby enhancing mood, attention, and excitability. Its mechanism of action is similar to that of amphetamines, hence it is called "endogenous amphetamine." In contrast, (S)-(-)-phenylethylamine, despite its weaker activity, still holds unique value in neuropharmacology, drug development, and research on stereoselective receptor mechanisms. It has shown potential in lead structure optimization studies for antidepressants, anxiolytics, and drugs for neurodegenerative diseases, and its enantiomeric differences have also found applications in functional foods and nutritional supplements.

[0004] Therefore, the efficient, economical, and environmentally friendly synthesis of enantiomeric pure chiral amines has become a key research direction in synthetic chemistry and biocatalysis. Traditional chemical methods (such as asymmetric reduction) suffer from complex steps and heavy environmental burdens, while enzyme-based biocatalysis, due to its mild conditions, high enantioselectivity, and sustainability, is becoming the mainstream strategy. Current research focuses on utilizing amine dehydrogenases (AmDHs) and transaminases (ATAs) to achieve the conversion of ketones to chiral amines.

[0005] However, these two types of enzyme catalytic systems still face significant challenges: amine dehydrogenases require cofactors such as NAD(P)H, and the availability of suitable high-efficiency enzyme molecules is limited, resulting in poor substrate adaptability and a narrow application range. Transaminase reactions typically require alanine as an amino donor, with (R)- and (S)-selective transaminases depending on D-alanine or L-alanine, respectively. The reaction produces α-ketoglutarate as a byproduct, which must be recovered via alanine dehydrogenase cycling to maintain system operation. However, this cycle has several drawbacks: firstly, transaminase reactions are reversible and easily subject to equilibrium inhibition in the later stages; secondly, alanine dehydrogenase has low catalytic efficiency, leading to limited cycling efficiency; and thirdly, the system lacks sufficient thermodynamic driving force. In summary, existing enzyme-catalyzed preparation methods have limitations in terms of efficiency, substrate range, and driving force. Therefore, developing new high-efficiency enzyme systems or coupled reaction modes to overcome the bottlenecks of existing methods is of significant scientific and industrial value for achieving the green and efficient preparation of chiral amines. Summary of the Invention

[0006] The technical problem solved by this invention is how to use an enzyme reaction to synthesize phenylethylamine from acetophenone, while reducing the use of co-substrate and overcoming the limitations of reaction efficiency.

[0007] To address the aforementioned technical problems, in a first aspect, the present invention provides an enzyme composition for the production of S-phenylethylamine, comprising a protein or polypeptide having the following functions: A) S-transaminase, B) alanine aminotransferase, C) glutamate dehydrogenase, and D) formate dehydrogenase.

[0008] Furthermore, A) S-transaminase can be derived from Ochrobactrum anthropi, B) alanine aminotransferase can be derived from barley (Hordeum vulgare), C) glutamate dehydrogenase can be derived from Pyrobaculum calidifontis, and D) formate dehydrogenase can be derived from Candida boidinii.

[0009] Furthermore, the amino acid sequence of A) S-transaminase comprises the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of B) alanine aminotransferase comprises the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:8.

[0010] Alternatively or alternatively, the amino acid sequences of A), B), C), and D above can also be modified as shown in any of the following ways, according to the sequences shown in SEQ ID:2, SEQ ID:4, SEQ ID:6, and SEQ ID:8:

[0011] (a1) A protein with the same function is obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0012] (a2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

[0013] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0014] Furthermore, A)-D) can all be obtained by conventional methods, such as prokaryotic expression, cell disruption, or protein purification. Even further, cell disruption can be achieved through ultrasonic disruption or high-pressure homogenization. Still further, protein purification can be performed using nickel column affinity chromatography.

[0015] Furthermore, the recipient bacterium for prokaryotic expression in this invention is preferably Escherichia coli; more preferably Escherichia coli MC02.

[0016] Secondly, the present invention provides a method for synthesizing S-phenylethylamine using acetophenone and formate as raw materials. The method comprises the following reaction system: (a1) the enzyme composition described in the first aspect; (a2) acetophenone; (a3) ​​formate; (a4) L-alanine; (a5) L-glutamic acid; (a6) NAD+. + 、;(a7)Water.

[0017] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0018] Furthermore, in the reaction, the final concentration of formate is 50 mM-1 M, the final concentration of acetophenone is 50 mM-1 M, the final concentration of L-alanine is 1-5 mM, the final concentration of L-glutamic acid is 2-10 mM, and the final concentration of NAD+ is 0.5-1 mM.

[0019] Furthermore, after the reaction is completed, the supernatant of the reaction system is collected to obtain S-phenylethylamine.

[0020] Thirdly, the present invention provides a group of recombinant engineered bacteria, including: I) recombinant Escherichia coli S01, containing an introduced or enhanced expression of a gene encoding A) S-transaminase; II) recombinant Escherichia coli CO2, containing an introduced or enhanced expression of a gene encoding B) alanine aminotransferase, a gene encoding C) glutamate dehydrogenase and a gene encoding D) formate dehydrogenase.

[0021] Furthermore, A) S-transaminase can be derived from Ochrobactrum anthropi, B) alanine aminotransferase can be derived from barley (Hordeum vulgare), C) glutamate dehydrogenase can be derived from Pyrobaculum calidifontis, and D) formate dehydrogenase can be derived from Candida boidinii.

[0022] Furthermore, the amino acid sequence of A) S-transaminase comprises the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of B) alanine aminotransferase comprises the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:8.

[0023] Alternatively or alternatively, the amino acid sequences of A), B), C), and D above can also be modified as shown in any of the following ways, according to the sequences shown in SEQ ID:2, SEQ ID:4, SEQ ID:6, and SEQ ID:8:

[0024] (a1) A protein with the same function is obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0025] (a2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

[0026] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0027] Furthermore, in the recombinant engineered bacteria, the encoding genes of A)-D) can be introduced into the recipient bacteria in the form of a recombinant vector. Even further, a vector promoter may be optionally included.

[0028] Furthermore, the recipient bacteria of the recombinant engineered bacteria in this invention are preferably Escherichia coli; more preferably Escherichia coli MC02.

[0029] Fourthly, this invention provides a method for synthesizing S-phenylethylamine using acetophenone and formate as raw materials. The method comprises the following reaction system: (a1) the recombinant engineered bacteria described in the third aspect; (a2) acetophenone; (a3) ​​formate; (a4) L-alanine; (a5) L-glutamic acid; (a6) NAD+. + 、;(a7)Water.

[0030] Furthermore, the recombinant engineered bacteria are in the form of cells or cell fragments. Even further, the cell fragments can be obtained by ultrasonic disruption or high-pressure homogenization of cells.

[0031] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0032] Further, in the reaction, the final concentration of formate is 50 mM-1 M, the final concentration of acetophenone is 50 mM-1 M, the final concentration of L-alanine is 1-5 mM, the final concentration of L-glutamic acid is 2-10 mM, and NAD... + The final concentration is 0.5-1 mM.

[0033] Furthermore, after the reaction is completed, the supernatant of the reaction system is collected to obtain S-phenylethylamine.

[0034] Fifthly, the present invention provides an enzyme composition for the production of R-phenylethylamine, comprising a protein or polypeptide having the following functions: F) R-transaminase, G) alanine racemic enzyme, B) alanine aminotransferase, C) glutamate dehydrogenase, and D) formate dehydrogenase.

[0035] Furthermore, the F) R-transaminase can be derived from Fusarium graminearum, the G) alanine racemic enzyme can be derived from Bacillus amyloliquefaciens, the B) alanine aminotransferase can be derived from barley (Hordeum vulgare), the C) glutamate dehydrogenase can be derived from Pyrobaculum calidifontis, and the D) formate dehydrogenase can be derived from Candida boidinii.

[0036] Furthermore, the amino acid sequence of F) R-transaminase comprises the amino acid sequence shown in SEQ ID NO:10, the amino acid sequence of G) alanine racemic enzyme comprises the amino acid sequence shown in SEQ ID NO:12, the amino acid sequence of B) alanine aminotransferase comprises the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:8.

[0037] Alternatively or alternatively, the amino acid sequences of F), G), B), C), and D) above may also be modified as shown in any of the following ways: SEQ ID:10, SEQ ID:12, SEQ ID:4, SEQ ID:6, and SEQ ID:8.

[0038] (a1) A protein with the same function is obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0039] (a2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

[0040] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0041] Furthermore, the F)-D) can all be obtained by conventional methods, such as prokaryotic expression, cell disruption, or protein purification. Even further, cell disruption can be achieved through ultrasonic disruption or high-pressure homogenization. Still further, protein purification can be performed using nickel column affinity chromatography.

[0042] Furthermore, the recipient bacterium for prokaryotic expression in this invention is preferably Escherichia coli; more preferably Escherichia coli MC02.

[0043] Sixthly, the present invention provides a method for synthesizing R-phenylethylamine using acetophenone and formate as raw materials. The method comprises the following reaction system: (a1) the enzyme composition described in the first aspect; (a2) acetophenone; (a3) ​​formate; (a4) L-alanine; (a5) L-glutamic acid; (a6) NAD+. + 、;(a7)Water.

[0044] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0045] Further, in the reaction, the final concentration of formate is 50 mM-1 M, the final concentration of acetophenone is 50 mM-1 M, the final concentration of L-alanine is 1-5 mM, the final concentration of L-glutamic acid is 2-10 mM, and NAD... + The final concentration is 0.5-1 mM.

[0046] Further, after the reaction is completed, the supernatant of the reaction system is collected to obtain R-phenylethylamine.

[0047] In a seventh aspect, the present invention provides a group of recombinant engineered bacteria, including: III) recombinant Escherichia coli R01, comprising an introduced or enhanced expression of a gene encoding F) R-transaminase and a gene encoding G) alanine racemase; II) recombinant Escherichia coli CO2, comprising an introduced or enhanced expression of a gene encoding B) alanine aminotransferase, a gene encoding C) glutamate dehydrogenase and a gene encoding D) formate dehydrogenase.

[0048] Furthermore, the F) R-transaminase can be derived from Fusarium graminearum, the G) alanine racemic enzyme can be derived from Bacillus amyloliquefaciens, the B) alanine aminotransferase can be derived from barley (Hordeum vulgare), the C) glutamate dehydrogenase can be derived from Pyrobaculum calidifontis, and the D) formate dehydrogenase can be derived from Candida boidinii.

[0049] Furthermore, the amino acid sequence of F) R-transaminase comprises the amino acid sequence shown in SEQ ID NO:10, the amino acid sequence of G) alanine racemic enzyme comprises the amino acid sequence shown in SEQ ID NO:12, the amino acid sequence of B) alanine aminotransferase comprises the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:8.

[0050] Alternatively or alternatively, the amino acid sequences of F), G), B), C), and D) above may also be modified as shown in any of the following ways: SEQ ID:10, SEQ ID:12, SEQ ID:4, SEQ ID:6, and SEQ ID:8.

[0051] (a1) A protein with the same function is obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0052] (a2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus.

[0053] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0054] Furthermore, in the recombinant engineered bacteria, the encoding gene of the F)-D) can be introduced into the recipient bacteria in the form of a recombinant vector. Even further, a vector promoter may be optionally included.

[0055] Furthermore, the recipient bacteria of the recombinant engineered bacteria in this invention are preferably Escherichia coli; more preferably Escherichia coli MC02.

[0056] Eighthly, the present invention provides a method for synthesizing R-phenylethylamine using acetophenone and formate as raw materials. The method comprises the following reaction system: (a1) the recombinant engineered bacteria described in the third aspect; (a2) acetophenone; (a3) ​​formate; (a4) L-alanine; (a5) L-glutamic acid; (a6) NAD+. + 、;(a7)Water.

[0057] Furthermore, the recombinant engineered bacteria are in the form of cells or cell fragments. Even further, the cell fragments can be obtained by ultrasonic disruption or high-pressure homogenization of cells.

[0058] Furthermore, the reaction temperature is 30-40°C; preferably 37°C.

[0059] Further, in the reaction, the final concentration of formate is 50 mM-1 M, the final concentration of acetophenone is 50 mM-1 M, the final concentration of L-alanine is 1-5 mM, the final concentration of L-glutamic acid is 2-10 mM, and NAD... + The final concentration is 0.5-1 mM.

[0060] Further, after the reaction is completed, the supernatant of the reaction system is collected to obtain R-phenylethylamine.

[0061] In a ninth aspect, the present invention provides the use of an enzyme composition or recombinant engineered bacteria according to any one of the first to eighth aspects in the production of S-phenylethanol or R-phenylethanol.

[0062] According to the above aspects of the present invention, the method of the present invention uses acetophenone as a raw material and carries out a catalytic reaction through an enzyme composition or recombinant engineered bacteria, which can efficiently synthesize S-phenylethanol or R-phenylethanol, which is beneficial for industrial application. Attached Figure Description

[0063] Figure 1 The images show the HPLC chromatograms of S-phenylethylamine and R-phenylethylamine samples during quantitative detection (A. S-phenylethylamine sample; B. R-phenylethylamine sample). Detailed Implementation

[0064] definition

[0065] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0066] Throughout this specification and the appended claims, the terms "comprising" and "including," as well as variations thereof, shall be interpreted inclusively. That is, where the context permits, these terms are intended to express the possibility of including other elements or integers not specifically listed.

[0067] The article “a / an” is used in this text to refer to one / an or more than one / an (i.e., one / an or at least one / at least one of the grammatical objects of the article). For example, “an element / an element” can mean one element / an element or more than one element / an element. When a noun (e.g., compound, additive, etc.) is mentioned in the singular form, it is intended to include the plural form. Therefore, when referring to a specific part (e.g., “gene”), unless otherwise specified, it means “at least one” of the genes, e.g., “at least one gene”.

[0068] Unless otherwise expressly indicated, the various embodiments of the invention described herein can be combined in various ways.

[0069] The term "carbon source" refers to a source of carbon, preferably a compound or molecule containing carbon. Preferably, the carbon source is a carbohydrate, amino acid, or its derivative. In this document, a carbon source is understood as an organic compound composed of elements such as carbon, oxygen, and hydrogen.

[0070] The term "cell" refers to a eukaryotic or prokaryotic organism, preferably existing as a single cell. In this invention, the cell can be recombinant *Escherichia coli*. That is, the recombinant cell is selected from a group of cells belonging to the genus *Escherichia coli*.

[0071] As used herein, the term “recombinant” / “engineered” (e.g., references to “recombinant E. coli,” “recombinant cell,” “recombinant microorganism,” and / or “recombinant strain”) can refer to a cell, microorganism, or strain containing nucleic acids as a result of one or more gene modifications. Simply put, a cell, microorganism, or strain contains different combinations of nucleic acids from one or more of its parents (any one of them). To construct recombinant cells, microorganisms, or strains, one or more recombinant DNA techniques and / or another one or more mutagenesis techniques can be used. For example, recombinant E. coli and / or recombinant E. coli cells may contain nucleic acids not present in the corresponding wild-type E. coli and / or cells, which have been introduced into the E. coli or E. coli cells using recombinant DNA techniques, or the absence of the nucleic acid in the wild-type E. coli and / or cells is the result of one or more mutations in the nucleic acid sequence (such as a gene encoding a wild-type polypeptide) present in the wild-type E. coli and / or E. coli cells (e.g., using recombinant DNA techniques or another mutagenesis technique such as UV irradiation). Furthermore, the term “recombinant” can suitably refer to, for example, cells, microorganisms, or strains from which nucleic acid sequences have been removed using recombinant DNA techniques.

[0072] In this paper, "recombinant E. coli containing or having a certain activity" is understood to mean that recombinant E. coli can contain one or more nucleic acid sequences encoding proteins having such activity. Therefore, recombinant E. coli is permitted to functionally express such proteins or enzymes.

[0073] The term "functional expression" refers to functional transcription in which the relevant nucleic acid sequence is present, allowing the nucleic acid sequence to actually be transcribed, for example, leading to protein synthesis.

[0074] As used herein with respect to proteins or peptides, the term "mutation" means that, compared to the wild-type or naturally occurring protein or peptide sequence, at least one amino acid has been substituted, inserted into, or deleted from the amino acid sequence. Amino acid substitutions, insertions, or deletions can be achieved, for example, by mutagenesis of the nucleic acids encoding those amino acids. Mutagenesis is a method well known in the art and includes, for example, site-directed mutagenesis by means of PCR or by oligonucleotide-mediated mutagenesis, as described in Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., vols. 1–3 (1989), published by Cold Spring Harbor Publishing.

[0075] As used herein with respect to genes, the term "mutation" means that, compared to a wild-type or naturally occurring nucleic acid sequence, at least one nucleotide in the nucleic acid sequence of a gene or its regulatory sequence has been replaced, inserted into, or deleted from the nucleic acid sequence by a different nucleotide. The substitution, insertion, or deletion of amino acids can be achieved, for example, via mutagenesis, resulting in transcription of a protein sequence having a qualitatively or quantitatively altered function, or the knockout of the gene. In the context of this invention, "altered gene" has the same meaning as a mutated gene.

[0076] As used herein, the term "gene" refers to a nucleic acid sequence of mRNA that can be transcribed and then translated into a protein. A gene that encodes a protein is one or more nucleic acid sequences that encode that protein.

[0077] As used herein, the term "nucleic acid" or "nucleotide" refers to a monomeric unit in a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer (i.e., a polynucleotide), and unless otherwise limited, encompasses known analogs that have the inherent properties of natural nucleotides because they hybridize with single-stranded nucleic acids (e.g., peptide nucleic acids) in a manner similar to naturally occurring nucleotides. For example, an enzyme defined by the nucleotide sequence encoding an enzyme includes (unless otherwise limited) a nucleotide sequence that hybridizes with a reference nucleotide sequence encoding the enzyme. A polynucleotide can be a natural or heterologous structure or a full-length or subsequence of a regulatory gene. Unless otherwise indicated, the term includes references to the specified sequence and its complementary sequence. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as contemplated herein. Furthermore, DNA or RNA containing rare bases (such as inosine) or modified bases (such as triphenylmethylated bases) (to name just two examples) is a polynucleotide as used herein. It will be understood that DNA and RNA have been modified in a wide variety of ways for many useful purposes known to those skilled in the art. As used herein, the term polynucleotide includes such chemical, enzymatic, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA specific to viruses and cells (especially simple and complex cells).

[0078] The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably in this article. An example of a nucleic acid sequence is a DNA sequence.

[0079] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers, for example, those containing amino acid residues as shown in the amino acid sequence. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. An essential property of such analogs of naturally occurring amino acids is that, when incorporated into a protein, the protein exhibits a specific reactivity to antibodies induced by proteins composed entirely of the same, but entirely, naturally occurring amino acids. The terms “polypeptide,” “peptide,” and “protein” also include modifications, including but not limited to glycosylation, lipid attachment, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0080] The term "enzyme" in this document refers to a protein that has a catalytic function. In cases where a protein catalyzes a biological reaction, the terms "protein" and "enzyme" may be used interchangeably herein. When referring to enzymes in the Enzyme Classification (EC), an enzyme class is a category in which an enzyme is classified or can be classified according to the enzyme nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which can be found at http: / / www.chem.qmul.ac.uk / iubmb / enzyme / . It is intended to include other suitable enzymes that are not yet (not yet) classified in the specified category but can be so classified.

[0081] If a protein or nucleic acid sequence (such as a gene) is referred to in this document by a reference accession number, unless otherwise specified, that number is specifically used to refer to a protein or nucleic acid sequence (gene) that can be found via www.ncbi.nlm.nih.gov / (available as of October 1, 2020).

[0082] Each nucleic acid sequence encoding a polypeptide in this paper also includes variants of any conserved modifications thereof. This includes, by reference to the genetic code, every possible silent variant of the nucleic acid. The term “conserved variant” applies to both amino acids and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conserved variant refers to those nucleic acids that encode the same amino acid sequence or a variant of a conserved modified amino acid sequence due to the degeneracy of the genetic code. The term “degeneracy of the genetic code” refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where the codon specifies alanine, the codon can be changed to any of the described corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are “silent variants” and represent a variant of a conserved modification.

[0083] As used herein, the term "functional homolog" (or simply "homology") of a gene having a specific sequence (e.g., "SEQ ID NO:X") refers to a polypeptide and / or amino acid sequence containing the specific sequence, or a nucleic acid sequence containing a polypeptide and / or amino acid sequence encoding the specific sequence, provided that one or more amino acids are mutated, substituted, deleted, added, and / or inserted, and that the polypeptide has (qualitatively) the same enzymatic function for substrate transformation.

[0084] As used herein, the term "functional homolog" (or simply "homolog") of a polynucleotide and / or nucleic acid sequence having a specific sequence (e.g., "SEQ ID NO:X") refers to a polynucleotide and / or nucleic acid sequence containing said specific sequence, provided that one or more nucleic acids are mutated, substituted, deleted, added, and / or inserted, and that the polynucleotide encodes a polypeptide sequence having (qualitatively) the same enzymatic function for substrate transformation. Regarding nucleic acid sequences, the term "functional homolog" is intended to include nucleic acid sequences that differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0085] In this document, sequence identity is defined as the relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. Typically, sequence identity or similarity is compared over the entire length of the sequences being compared. In this art, “identity” also means the degree of sequence correlation between amino acid or nucleic acid sequences, as determined by matching strings of such sequences.

[0086] Amino acid or nucleotide sequences are said to be homologous when they exhibit a certain level of similarity. Two sequences being homologous indicates a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "identity percentage" or "similarity percentage," which are high or low, respectively. Although controversial, "homology level" or "homology percentage" is often used interchangeably to indicate "identity percentage" or "similarity percentage." Sequence comparisons and the determination of the identity percentage between two sequences can be accomplished using mathematical algorithms. Technicians will realize that several different computer programs can be used to align two sequences and determine their homology (Kruskal et al., "An overview of sequence comparison: Time warps, string edits, and macromolecules", (1983), Society for Industrial and Applied Mathematics (SIAM), Vol. 25, No. 2, pp. 201-237; and the handbook edited by D. Sankoff and J.B. Kruskal, "Time warps, string edits and macro molecules: the theory and practice of sequence comparison", (1983), pp. 1-44, published by Addison-Wesley Publishing Company, Massachusetts USA).

[0087] The Needleman and Wunsch algorithm can be used to align two sequences to determine the percentage of identity between the two amino acid sequences. (Needleman et al., "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins" (1970), J. Mol. Biol., Vol. 48, pp. 443-453). This algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or later, see Rice et al., "EMBOSS: The European Molecular Biology Open Software Suite", (2000), Trends in Genetics, Vol. 16, (6), pp. 276-277, http: / / emboss.bioinformatics.nl / ) was used. For protein sequences, EBLOSUM62 was used as the substitution matrix. For nucleotide sequences, EDNAFULL was used. Other matrices may be specified. Optional parameters for amino acid sequence alignment are a vacancy opening penalty of 10 and a vacancy expansion penalty of 0.5. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences does not change significantly when different algorithms are used.

[0088] Homology or identity is the percentage of identical matches between two complete sequences across the total aligned region, including any vacancies or extensions. Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in both sequences showing the same amino acid in the alignment divided by the total length of the alignment, including vacancies. Identity, as defined herein, is available from NEEDLE and is labeled "IDENTITY" in the program's output.

[0089] Homology or identity between two aligned sequences is calculated as follows: the number of positions in the alignment showing the same amino acid in both sequences is divided by the total length of the alignment after subtracting the total number of vacancies in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "longest-identity" in the program's output.

[0090] Variants of the nucleotide or amino acid sequences disclosed herein may also be defined as nucleotide or amino acid sequences having one or more mutations, substitutions, insertions, and / or deletions compared to the nucleotide or amino acid sequences specifically disclosed herein (e.g., in the sequence listing).

[0091] Optionally, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called “conservative” amino acid substitutions, which will be clear to them. Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In one embodiment, the group of conserved amino acid substitutions is: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are variants in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted at its position. Preferably, the amino acid changes are conserved. In one embodiment, the conserved substitutions for each naturally occurring amino acid are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0092] The nucleotide sequences of the present invention can also be defined by their ability to partially hybridize with specific nucleotide sequences disclosed herein, respectively, under moderate hybridization conditions or preferably under stringent hybridization conditions. Stringent hybridization conditions are defined herein as conditions that allow nucleic acid sequences of at least about 25 nucleotides, preferably about 50, 75, or 100 nucleotides, and most preferably about 200 or more nucleotides, to hybridize at about 65°C in a solution containing about 1 M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to be washed at 65°C in a solution containing about 0.1 M or less salt (preferably 0.2x SSC or any other solution with equivalent ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with about 90% or higher sequence identity. In this document, moderate conditions are defined as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at about 45°C in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength), and to wash at room temperature in a solution containing about 1M salt (preferably 6x SSC or any other solution with equivalent ionic strength). Preferably, hybridization is performed overnight, i.e., at least 10 hours; and preferably, washing is performed for at least one hour, wherein the washing solution is changed at least twice. These conditions will generally allow specific hybridization of sequences with up to 50% sequence identity. Those skilled in the art will be able to modify these hybridization conditions to specifically identify sequences with identity varying between 50% and 90%.

[0093] "Expression" refers to the transcription of genes into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), which is then translated into proteins.

[0094] "Overexpression" refers to the expression of a gene (corresponding nucleic acid sequence) in recombinant cells exceeding its expression in the corresponding wild-type cells. Such overexpression can be arranged, for example, by increasing the transcription frequency of one or more nucleic acid sequences, such as by operatively linking the nucleic acid sequence to a functional promoter in the recombinant cell; and / or by increasing the copy number of a nucleic acid sequence.

[0095] The term "upregulation" and its variations refer to the process by which cells increase the amount of cellular components, such as RNA or proteins. This upregulation can be in response to or caused by gene modification.

[0096] In this article, the term "pathway" or "metabolic pathway" is understood as a series of chemical reactions that build up and break down molecules in the cell.

[0097] Nucleic acid sequences (i.e., polynucleotides) or proteins (i.e., polypeptides) can be native or heterologous to the host cell's genome.

[0098] The terms "natural," "homologous," or "endogenous" in relation to a host cell mean that the nucleic acid sequence is indeed naturally present in the host cell's genome, or that the protein is naturally produced by that cell. The terms "natural," "homologous," and "endogenous" are used interchangeably in this document.

[0099] As used herein, “heterologous” or “exogenous” can refer to a nucleic acid sequence or a protein. For example, in relation to a host cell, “heterologous” can refer to a polynucleotide that is not naturally present in the genome of the host cell in this manner, or a polypeptide or protein that is not naturally produced by the cell in this manner. A heterologous nucleic acid sequence is a nucleic acid derived from a foreign species, or, if from the same species, substantially modified relative to its natural form in terms of composition and / or genomic loci through deliberate human intervention. For example, a promoter operatively linked to a natural structural gene is derived from a species different from the species from which the structural gene is derived, or, if from the same species, one or both are substantially modified relative to their original form. A heterologous protein can be derived from a foreign species, or, if from the same species, substantially modified relative to its original form through deliberate human intervention. That is, heterologous protein expression involves the expression of a protein that is not naturally expressed in the host cell in this manner. The term “heterologous expression” refers to the expression of a heterologous nucleic acid in a host cell. The expression of heterologous proteins in eukaryotic host cell systems (such as *Escherichia coli*) is well known to those skilled in the art. Polynucleotides containing nucleic acid sequences encoding genes for proteins or enzymes with specific activities can be expressed in such eukaryotic systems. In some embodiments, transformed / transfected cells can be used as expression systems for enzymes. The expression of heterologous proteins in *E. coli* is well known. *The Cold Spring Harbor Laboratory* is a recognized work describing various methods that can be used to express proteins in *E. coli*.

[0100] As used herein, a "promoter" is a DNA sequence that directs the transcription of a (structural) gene or other (partial) nucleic acid sequence. Appropriately, a promoter is located in the 5' region of a gene, near the transcription start site of the (structural) gene. Promoter sequences can be constitutive, inducible, or repressive. In one embodiment, no (external) inducer is required.

[0101] As used herein, the term "vector" includes references to autosomal expression vectors and integration vectors for integration into chromosomes.

[0102] The term "expression vector" refers to a linear or circular DNA molecule containing a segment encoding a target polypeptide, which is controlled (i.e., operatively linked to) additional nucleic acid segments that provide for its transcription. These additional segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both.

[0103] "Plasmid" refers to autonomously replicating extrachromosomal DNA that does not integrate into the genome of a microorganism and is usually circular in nature.

[0104] In this paper, "host cell" is understood to be a cell (such as an E. coli cell) that will be transformed with one or more nucleic acid sequences encoding one or more heterologous proteins to construct a transformed cell (also known as a recombinant cell). For example, the transformed cell may contain a vector and may support the replication and / or expression of the vector.

[0105] As used herein, “transformation” refers to the insertion of a foreign polynucleotide into a host cell, regardless of the method used for insertion, such as direct uptake, transduction, f-conjugation, or electroporation. The foreign polynucleotide may be maintained as a non-integrating vector (e.g., a plasmid) or alternatively integrated into the host cell genome.

[0106] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0107] Those skilled in the art will understand that the gene or its functional homologs disclosed herein comprise a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the exemplarily listed sequences, or having one or more mutated, substituted, inserted, and / or deleted amino acid sequences compared to the amino acid sequence it encodes.

[0108] Preferably, compared with the amino acid sequence encoded by a gene, the amino acid sequence encoded by any functional homolog of that gene has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10, or no more than 5 amino acid mutations, substitutions, insertions, and / or deletions.

[0109] Exemplary, and not limiting, the functional homolog of the OaTA gene shown in SEQ ID NO.1 comprises a nucleic acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of those nucleic acid sequences, or its functional homolog comprises a nucleic acid sequence having one or more mutations, substitutions, insertions, and / or deletions compared to any of those nucleic acid sequences; preferably, compared to such nucleic acid sequences, the nucleic acid sequence of any such functional homolog has no more than 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 10, or 5 nucleic acid mutations, substitutions, insertions, and / or deletions. More preferably, the functional homolog of the OaTA gene comprises an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the polypeptide shown by SEQ ID NO. 2.

[0110] Those skilled in the art will understand that other sequences include the above-described cases.

[0111] Unless otherwise specified herein, the terms and terminology used herein should be understood in accordance with the conventional knowledge and usage of those skilled in the art. Unless otherwise stated, the specific operational methods (including: preparation processes, experimental steps, detection methods, etc.) employed in this application utilize conventional techniques in the fields of biochemistry, cell biology, molecular biology, gene editing (e.g., recombinant DNA technology), zoology, and related areas. These techniques are well-described in existing literature. For details, please refer to Sam Brook et al., *Molecular Cloning: a Laboratory Manual*, 4th edition, Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., *Current Protocols in Molecular Biology*, Wiley Online Press, updated irregularly; Kursad Turksen et al., *Embryonic StemCell Protocols*, 3rd edition, Springer Press, 2016; P. Nagarajan et al., *Essentials of Laboratory Animal Science: Principles and Practices*, Springer Press, 2021; and Jann Hau et al., *Handbook of Laboratory Animal Science: Essential Principles and Practices*, 4th edition, CRC Press, 2021.

[0112] The acetophenone used in the following examples was purchased from Aladdin, catalog numbers A103669 and A800304.

[0113] Sodium formate: Purchased from Aladdin, catalog number S164504

[0114] L-Alanine: Purchased from Aladdin, catalog number A108263

[0115] L-Glutamic acid: Purchased from Macklin, catalog number L810368

[0116] NAD: Purchased from Aladdin, catalog number N111609

[0117] S-Phenylacetylamine: Purchased from Beijing Zeping Technology Co., Ltd., product catalog number 371450500

[0118] R-Phenylacetamine: Purchased from Sichuan Hengcheng Zhiyuan Biotechnology Co., Ltd., product catalog number C0115 Detailed Implementation

[0120] The present disclosure will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0121] The liquid LB medium (pH 7.0) in the following examples contains 1 g / 100 mL NaCl, 1 g / 100 mL tryptone, 0.5 g / 100 mL yeast extract, and the remainder is water. The solid medium is obtained by adding agarose to the liquid medium.

[0122] The reaction solution in the following examples is a 1XM9 salt solution containing the following components: acetophenone 20 g / L, formate 300 mM, L-alanine 3 mM, L-glutamic acid 10 mM, and NAD 1 mM.

[0123] The preparation process of phenylethylamine in the following examples is as follows:

[0124] (1) Preparation of crude enzyme solution: The constructed strain was inoculated into LB liquid medium and cultured overnight at 37℃ and 200 rpm to obtain seed solution. The seed solution was then inoculated into 100 mL of fresh LB medium at a ratio of 1:100 and cultured at 37℃ and 200 rpm until OD. 600 The concentration of L-arabinose was 0.8–1.0, and the mixture was induced overnight for 16 h. The bacterial culture was centrifuged at 5000 rpm for 10 min to collect the cells. At the same time, 1XM9 salt solution was added, and the cells were sonicated for 20 min using an ultrasonic disruptor with a power of 30 W, an alarm temperature of 15 ℃, a 2 s off-off time, and a 3 s on-off time to completely disrupt the cells and obtain the crude enzyme solution.

[0125] (2) Add the crude enzyme solution to the reaction solution to make the final concentration of acetophenone 20 g / L. React at 30 °C for 24 h. After the reaction, take 1 mL of the reaction solution, centrifuge at 12000 rpm for 10 min, and collect the supernatant for HPLC determination. Then filter through a 0.45 μm filter membrane and collect the filtrate.

[0126] The detection method for phenylethylamine in the following examples is as follows:

[0127] (1) The quantitative detection of acetophenone and phenylethylamine (including S-phenylethylamine and R-phenylethylamine) was performed by HPLC. The detection conditions were as follows: column: him-pack GIST C18 column; detector: PDA detector; detection wavelength: 204 nm; mobile phase: 0.1% formic acid: methanol = 65:35; flow rate: 1.0 mL / min; column temperature: 40℃; injection volume: 2 μL.

[0128] (2) The chirality of phenethylamine was determined by derivatization using HPLC. Detection conditions: column: Agilent Zorbax C18 column; detector: PDA detector; detection wavelength: 241 nm; mobile phase: methanol:PBS (pH 3.0) = 58:42; flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 20 μL. The sample was pre-treated with an equal volume of 5 g / L 2,3,4,6-tetra-O-β-D-glucopyranose isocyanate (GITC) at 30℃ for 30 min. The reaction was terminated by adding twice the volume of PBS (pH 3.0) before loading the sample.

[0129] The specific *E. coli* strain used in the following examples may be one of *E. coli* MG1655, BW25113, or MC02. *E. coli* MG1655 (CGSC#: 6300) and BW25113 (CGSC#: 7636) are products of the Yale University Genetic Collection Center for *E. coli*. *E. coli* MC02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) (accession number CGMCC No. 34378). The sequence information of the pYB1k plasmid is described in patent CN119799606A.

[0130] Table 1. List of Sequence Fragments

[0131] Serial Number Segment Name Sequence (5'-3') SEQ ID NO.1 OaTA gene atgactgctcagccaaactctcttgaagctcgcgatatccgttatcatctccattcttataccgatgctgtccgcctcgaagcggaaggtccgctcgtcatcgagcgtggcgatggcatttacgtcgaagatgtatcgggcaagcgctatatcgaagcgatgtcaggactgtggagtgttggcgtgggcttttccgaaccgcgtctggccgaagcagctgcacgacagatgaagaagctgcctttctaccatacattctcctaccgttcgcatggtcctgtcattgatctggcagaaaagcttgtctcaatggctcctgttccgatgagcaaggcctacttcaccaattcaggttccgaagccaacgatacggtcgtcaagttgatctggtatcgctccaatgcgctgggtgaaccggagcgcaagaaaatcatctcacgcaagcgcggctatcacggtgtgacgattgcctctgccagcctgaccggcttgcccaacaatcaccgttctttcgatctgccgatcgatcgtatcctgcatacgggctgcccgcatttttatcgcgaaggacaggctggcgagagtgaggaacaattcgcaacgcggctggcggatgagctggaacagttgatcatcgcggaaggtcctcacaccatcgctgctttcattggcgagccggtgatgggggctggcggcgtagtcgtgccgcccaaaacctattgggaaaaagtgcaggctgttctcaagcgctacgatattctgctgatcgccgacgaggttatttgcggcttcggacggacaggcaatctgttcggcagccagactttcgatatgaaaccggacattctggtgatgtcgaagcagctttcgtcatcctatctgccgatttcggccttcctcatcaacgagcgtgtgtacgcgccaattgccgaagaaagccacaagatcggcacgcttggcacgggcttcacggcatctggccatccggtggcggcagcggtagcgctggaaaacctcgccattattgaagagcgtgatctggtcgccaatgcgcgcgaccgcggcacctatatgcagaagcgcctgcgtgagttgcaggatcatcctctggtcggcgaagtgcgtggcgttggtctcatagccggtgtcgagcttgtcaccgacaagcaggccaagacgggccttgaaccaaccggcgctctgggcgcaaaggcaaacgccgttcttcaggagcgcggcgtcatttcccgcgcaatgggcgatacgcttgccttctgcccgccgctcatcatcaacgatcagcaggttgatacgatggtgtccgcgctcgaggcgacgctgaacgatgttcaggcaagcctcaccaggtaa SEQ ID NO.2 OaTA protein sequence MTAQPNSLEARDIRYHLHSYTDAVRLEAEGPLVIERGDGIYVEDVSGKRYIEAMSGLWSVGVGFSEPRLAEAAARQMKKLPFYHTFSYRSHGPVIDLAEKLVSMAPVPMSKAYF TNSGSEANDTVVKLIWYRSNALGEPERKKIISRKRGYHGVTIASASLTGLPNNHRSFDLPIDRILHTGCPHFYREGQAGESEEQFATRLADELEQLIIAEGPHTIAAFIGEPVM GAGGVVVPPKTYWEKVQAVLKRYDILLIADEVICGFGRTGNLFGSQTFDMKPDILVMSKQLSSSYLPISAFLINERVYAPIAEESHKIGTLGTGFTASGHPVAAAVALENLAII EERDLVANARDRGTYMQKRLRELQDHPLVGEVRGVGLIAGVELVTDKQAKTGLEPTGALGAKANAVLQERGVISRAMGDTLAFCPPLIINDQQVDTMVSALEATLNDVQASLTR SEQ ID NO.3 HvAlaAT gene atggctgccaccgtcgccgtggacaacctgaaccccaaggttttaaaatgtgagtatgctgtgcgtggagagattgtcatccatgctcagcgcttgcaggaacagctaaagactcaaccagggtctctaccttttgatgagatcctctattgtaacattgggaacccacaatctcttggtcagcaaccagttacattcttcagggaggttcttgccctttgtgatcatccagacctgttgcaaagagaggaaatcaaaacattgttcagtgctgattctatttctcgagcaaagcagattcttgccatgatacctggaagagcaacaggagcatacagccatagccagggtattaaaggacttcgtgatgcaattgcttctgggatcgcttcacgagatggattccctgctaatgctgatgacatttttctcacagatggagcaagtcctggggtgcacctgatgatgcaattactgataaggaatgagaaagatggcattcttgtcccgattcctcagtaccccttgtactcggcttccatagctcttcatggcggagctcttgtcccatactatctcaatgaatcgacgggctggggtttggaaacctctgatgttaagaagcaacttgaagatgctcggtcaagaggcatcaacgttagggctttggtggttatcaatccaggaaatccaactggacaggtacttgctgaagaaaaccaatatgacatagtgaagttctgcaaaaatgagggtcttgttcttctagctgatgaggtataccaagagaacatctatgttgacaacaagaaattccactctttcaagaagatagtgagatccttgggatacggcgaggaggatctccctctagtatcatatcaatctgtttctaagggatattatggtgagtgtggtaaaagaggtggttactttgagattactggcttcagtgctccagtaagagagcagatctacaaaatagcatcagtgaacctatgctccaatatcactggccagatccttgctagtcttgtcatgaacccaccaaaggctagtgatgaatcatacgcttcatacaaggcagaaaaagatggaatcctcgcatctttagctcgtcgtgcgaaggcattggagcatgcattcaataaacttgagggaattacttgcaacgaggctgaaggagcaatgtacgtgttccctcaaatctgtctgccacagaaggcaattgaggctgctaaagctgctaacaaagcacctgatgcattctatgctcttcgtctcctcgagtcgactggaatcgtcgttgtccctggatcaggatttggccaggttcctggcacatggcacttcaggtgcacgatccttccgcaggaggataagatcccggcagtcatctcccgcttcacggtgttccatgaggcgttcatgtcagagtatcgtgactaa SEQ ID NO.4 HvAlaAT protein sequence MAATVAVDNLNPKVLKCEYAVRGEIVIHAQRLQEQLKTQPGSLPFDEILYCNIGNPQSLGQQPVTFFREVLALCDHPDLLQREEIKTLFSADSISRAKQILAMIPGRATGAYSHSQGIKGLRDAIASGIASRDGFPANADDIFLTDGASPGVHLMMQLLIRNEKDGILVPIPQYPLYSASIALHGGALVPYYLNESTGWGLETSDVKKQLEDARSRGINVRALVVINPGNPTGQVLAEENQYDIVKFCKNEGLVLLADEVYQENIYVDNKKFHSFKKIVRSLGYGEEDLPLVSYQSVSKGYYGECGKRGGYFEITGFSAPVREQIYKIASVNLCSNITGQILASLVMNPPKASDESYASYKAEKDGILASLARRAKALEHAFNKLEGITCNEAEGAMYVFPQICLPQKAIEAAKAANKAPDAFYALRLLESTGIVVVPGSGFGQVPGTWHFRCTILPQEDKIPAVISRFTVFHEAFMSEYRD SEQ ID NO.5 PcGDH gene atgagcacaacctacatagtatcagacttcctaatcaatacgctactgacaattaagcgaggagtagaactggccggactaccgccagaattctacgaagcactagaaaagccaaagagaatactagtggtaaacataccggtgaaaatggacgacggaaagataaagtactttgagggctaccgtgttcagcataatgatgctcttggtccttttaagggtggtattcgttttcatccggaggtgactttggctgatgatattgctcttgccatgcttatgacgcttaagaatagcctcgcgggcctgccctacggcggggctaagggggctgtgagagtggacccgcggaggctctctaggcgggagcttgaggagttggctaggggctacgcgcgggccgtggcgcctctcataggcgagcagttggacatcccagccccagacgtggggacagactcccaggtaatggcctggatggtagacgagtactcaaggcttgtgggaagaaacgccccagcagtctttacctcaaagcccccagagctctggggaaacccagtcagagaatactccacaggcttcggcgtggcagtagcggcaagagaagtcgcgaagaggctatggggaggaatagtgggaaaaaccgcggctgtgcaaggcttaggcaatgtgggcagatgggccgcctattggcttgagaaaatgggcgccaaagtcgtggcagtttcggacgtcaacggcgtggtatacagagagagggggttagacgtggatctcattagagaaacaaaggcaaaaggcccgcagctactcgaaatgataagccagaaaaacggcgttgaaattgtgaaaaacccagaccagatcttctccctagacgtggacatcttggtgcctgccgctattgagaatgtggttagggaggacaatgtggatggggttagggctaggttggtggtggagggggctaatgggcccactacccctggggctgagaggaggctgtatgagaggggggttgtggtggtgccggatatcttggctaacgctggcggcgtgattatgtcttatctggagtgggtggagaatttgcagtggctgttttgggatgaggaggagactaggaggaggcttgaggccataatgtccaacaacgtggctagagtatacgccaggtgggagaaggagaagagctggaccatgagagacgccgcagtagtcacagccctagaaagaatatacaacgccatgaagacaagggggtggatctag SEQ ID NO.6 PcGDH protein sequence MSTTYIVSDFLINTLLTIKRGVELAGLPPEFYEALEKPKRILVVNIPVKMDDGKIKYFEGYRVQHNDALGPFKGGIRFHPEVTLADDIALAMLMTLKNSLAGLPYGGAKGAVRVDPRRLSRRELEELARGYARAVAPLIGEQLDIPAPDVGTDSQVMAWMVDEYSRLVGRNAPAVFTSKPPELWGNPVREYSTGFGVAVAAREVAKRLWGGIVGKTAAVQGLGNVGRWAAYWLEKMGAKVVAVSDVNGVVYRERGLDVDLIRETKAKGPQLLEMISQKNGVEIVKNPDQIFSLDVDILVPAAIENVVREDNVDGVRARLVVEGANGPTTPGAERRLYERGVVVVPDILANAGGVIMSYLEWVENLQWLFWDEEETRRRLEAIMSNNVARVYARWEKEKSWTMRDAAVVTALERIYNAMKTRGWI SEQ ID NO.7 atgaagatcgttttagtcttatatggtgctggtaaacacgctgccgatgaagaaaaattatacggttgtactgaaaacaaattaggtattgccaattggttgaaagatcaaggacatgaactaatcaccacgtctgataaagaaggcggaaacagtgtgttggatcaacatataccagatgccgatattatcattacaactcctttccatcctgcttatatcactaaggaaagaatcgacaaggctaaaaaattgaaattagttgttgtcgctggtgtcggttctgatcatattgatttggattatatcaaccaaacaggtaggaaaatctccgtcttggaagttaccggttctaatgttgtctctgttgcagaacacgttgtcatgaccatgcttgtcttggttagaaattttgttccagctcacgaacaaaacattaaccacgattgggaggttgctgctatcgctaaggatgcttacgatatcgaaggtaaaactatcgccaccattggtgccggtagaattggttacagagtcttggaaagattagtcccattcaatcctaaagaattattatactacgattatcaagctttaccaaaagatgctgaagaaaaagttggtgctagaagggttgaaaatattgaagaattggttgcccaagctgatatagttacagttaatgctccattacacgctggtacaaaaggtttaattaacaaggaattattgtctaaattcaagaaaggtgcttggttagtcaatactgcaagaggtgccatttgtgttgccgaagatgttgctgcagctttagaatctggtcaattaagaggttatggtggtgatgtttggttcccacaaccagctccaaaagatcacccatggagagatatgagaaacaaatatggtgctggtaacgccacgactcctcattactctggtactactttagatgctcaaactagatacgctcaaggtactaaaaatatcttggagtcattctttactggtaagtttgattacagaccacaagatatcatcttattaaacggtgaatacgttaccaaagcttacggtaaacacgataagaaataa CbFDH gene SEQ ID NO.8 CbFDH protein sequence MKIVLVLYGAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGGNSVLDQHIPDADIIITTPFHPAYITKERIDKAKKLKLVVVAGVGSDHIDLDYINQTGRKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQNINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLERLVPFNPKELLYYDYQALPKDAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNATTPHYSGTTLDAQTRYAQGTKNILESFFTGKFDYRPQDIILLNGEYVTKAYGKHDKK SEQ ID NO.9 FgTA gene atgtcgaccatggacaagatcttcgccggccacgcccagcgccaagccaccctcgtcgcaagcgacaacatcttcgccaacggcattgcctggatccaaggcgagctcgtccccctcaatgaagcccgcatccccctcatggaccaaggtttcatgcacggcgacttgacctacgatgtccctgcagtctgggatggtcgtttcttccgtcttgatgaccatctcgaccgtctcgaggcaagcgtcaagaagatgcgaatgcaattccccattccccgcgatgagatcagaatgactcttctcgacatgctcgccaagagtggaatcaaggatgcttttgttgagctcattgtcactcgtggcttgaagcctgttcgtgaggccaagcctggtgaggtcttgaacaaccacctctacttgatcgtccaaccctacgtctgggtcatgagccccgaagctcagtacgtcggcggtaatgccgttatcgcacgaactgttcgtcgaatccctcctggatccatggatcccaccatcaagaacctccaatggagtgatttcacccgcggcatgttcgaagcatacgatcgtggagcacaataccccttcctcaccgacggcgacacaaacatcaccgaaggatctggtttcaacgttgtctttgtcaagaacaacgttatttacaccccgaaccgaggagttttgcagggaattaccagaaagagtgtgatcgacgctgccaagtggtgtggtcatgaagttcgagtggagtatgtccctgttgagatggcctatgaagctgatgagatcttcatgtgtactactgctggaggaatcatgcctatcaccaccatggatggaaagccagtcaaggacggaaaggtcgggcctgtcacaaaggccatctgggatcggtactgggcgatgcactgggaggatgagttcagtttcaagattgactaccagaaactgaagctgtag SEQ IDNO.10 FgTA protein sequence MSTMDKIFAGHAQRQATLVASDNIFANGIAWIQGELVPLNEARIPLMDQGFMHGDLTYDVPAVWDGRFFRLDDHLDRLEASVKKMRMQFPIPRDEIRMTLLDMLAKSGIKDAFVELIVTRGLKPVREAKPGEVLNNHLYLIVQPYVWVMSPEAQYVGGNAVIARTVRRIPPGSMDPTIKNLQWSDFTRGMFEAYDRGAQYPFLTDGDTNITEGSGFNVVFVKNNVIYTPNRGVLQGITRKSVIDAAKWCGHEVRVEYVPVEMAYEADEIFMCTTAGGIMPITTMDGKPVKDGKVGPVTKAIWDRYWAMHWEDEFSFKIDYQKLKL SEQ IDNO.11 BaAR gene atgaacacagcatccttttacagagaaacgtgggcggaaatcaacctgtccgcgattaaagaaaatgtcacgcatatgaaaaaacacatcgggcaaaacgtccatctcatggcggttgtaaaggcgaacgcttacggccacggagatttggaagttgggaaagccgcgcttgaagcgggtgcctcttgtcttgccgtagccatactggatgaagcgatttcattgagaaaacggggcattacagcgccgattctcgtgcttggcgccgtgccgcccgaatatgtacaagcagctgccgagtatgacgtaacactcacggggtattctgttgaatggcttcaggaagctgcgcgccacctcggaagtgcgacagtgccgtttcatctgaaagtcgacaccggcatgaacagactcggagtcaaaacggaggaagaaatccaaagcgtgttaaaaatcctcagccaaaatcccggtttagtctgtaaaggcgtattcacccactttgctaccgcggatgagaaaaacagagattatttcctcttccagtttgaccgctttaaaacattgatcgctccgcttcctttaaaagaattgatggtgcactgcgcgaacagcgctgccggacttcgtcttaaaaaaggcttctttaacgcggttcgtttcggcatcagtatgtacgggcttcgtccttctgctgacatccaaagcgaaattccgtttcaattaaagccggcttttgcgctgcactctgttttatcccatgtgaaaaagatccgcaaaggggaaagcgtcagctacggcgcaacatatacggcagagaaggatcaatggatcggaacggttcccatcggctatgcggacgggtggctccgcaagctgagcggcacgtctgtgctgatcggcggcaaacggatgaacatcgcgggaagaatctgcatggatcaattgatggtcgaattagatcaatcttatccgccgggcaccaaagtcaccctgatcggcagccagaagggagaaacgatcacgatggatgaaatcgcagggcggcttgagacgattaattacgaggtcccttgtaccattagttcccgcgttccccgtatgtttttggaaaatgagagtataatggaagtaagaaatcctttactgcaagataatacaaacaagtag SEQ IDNO.12 BaAR protein sequence MNTASFYRETWAEINLSAIKENVTHMKKHIGQNVHLMAVVKANAYGHGDLEVGKAALEAGASCLAVAILDEAISLRKRGITAPILVLGAVPPEYVQAAAEYDVTLTGYSVEWLQEAARHLGSATVPFHLKVDTGMNRLGVKTEEEIQSVLKILSQNPGLVCKGVFTHFATADEKNRDYFLFQFDRFKTLIAPLPLKELMVHCANSAAGLRLKKGFFNAVRFGISMYGLRPSADIQSEIPFQLKPAFALHSVLSHVKKIRKGESVSYGATYTAEKDQWIGTVPIGYADGWLRKLSGTSVLIGGKRMNIAGRICMDQLMVELDQSYPPGTKVTLIGSQKGETITMDEIAGRLETINYEVPCTISSRVPRMFLENESIMEVRNPLLQDNTNK SEQ IDNO.13 RBS aggaggaattaacc

[0132] Table 2. Primer sequence list

[0133]

[0134] Example 1 Construction of strain S01

[0135] (1) Construction and preparation of pS-1 plasmid

[0136] The pY fragment of the plasmid backbone was prepared by PCR amplification using pYB1k plasmid as a template and primers pSY-F / pSY-R.

[0137] The OaTA fragment was synthesized by Qingke Biotechnology. The sequence is the OaTA gene, which encodes S-transaminase from human Ochrobacter rumanthropi. The fragment was prepared by PCR amplification using primers SF / SR.

[0138] All the above PCRs were performed using high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221), and the target fragments were recovered by agarose gel electrophoresis.

[0139] The OaTA fragment and pY fragment were ligated using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to selpxMeral hundred kilobases. Nat. methods. 2009; 6(5):343-345) to obtain the ligation product. *E. coli* DH5α competent cells (purchased from Beijing TransGen Biotech Co., Ltd., product catalog: CD201) were transformed using the CaCl2 method. The cells were evenly spread on LB agar plates containing kanamycin and incubated overnight at 37°C. Clones were selected, and those capable of amplifying the target fragment were identified using primers ara-F / rrn-R and sequenced. Positive clones were selected, and plasmids were extracted to obtain a positive plasmid named pS-1. The pS-1 plasmid was transformed into MC02 strain using the calcium chloride transformation method, named S01, and stored at -80°C.

[0140] Example 2 Construction of strains CO2 and R01

[0141] The construction process of strains CO2 and R01 is exactly the same as that of strain S01 in Example 1. The difference is that in the construction of CO2, the pS-1 plasmid is replaced with the pC-2 plasmid; in the construction of R01, the pS-1 plasmid is replaced with the pR-1 plasmid.

[0142] The construction process of plasmids pC-2 and pR-1 is exactly the same as that of plasmid pS-1 in Example 1. The difference is that in the construction of pC-2, the OaTA fragment is replaced with the HvAlaAT-PcGDH-CbFDH fragment, which contains the HvAlaAT gene (encoding alanine aminotransferase from barley (Hordeum vulgare), the PcGDH gene (encoding glutamate dehydrogenase from Pyrobaculum calidifontis), and the CbFDH gene (encoding formate dehydrogenase from Candida boidinii).

[0143] During the construction of pR-1, the OaTA fragment was replaced with the FgTA-BaAR fragment. This fragment contains the FgTA gene (an R-transaminase from Fusarium graminearum) and the BaAR gene (an alanine racemase from Bacillus amyloliquefaciens).

[0144] Specific plasmid information is shown in Table 3.

[0145] Table 3. Plasmid Construction Information

[0146]

[0147] Example 3 Protein Expression and Preparation of Crude Enzyme Solution

[0148] The test strains were S01, CO2, and R01.

[0149] (1) Preparation of crude enzyme solution: The constructed strain was inoculated into LB liquid medium and cultured overnight at 37℃ and 200 rpm to obtain seed solution.

[0150] (2) Inoculate the seed culture into 100 mL of fresh LB medium at a ratio of 1:100, and incubate at 37 ℃ and 200 rpm until OD. 600 The concentration was 0.8–1.0, and L-arabinose was added to a final concentration of 0.2%, and the mixture was induced overnight for 16 h.

[0151] (3) Centrifuge the bacterial culture at 8000 rpm for 10 min to collect the bacterial cells. Dilute the collected cell pellet to 160 OD using 1XM9 salt solution according to the cell concentration of each recombinant bacterial culture system.

[0152] (4) Use an ultrasonic disruptor to disrupt the cells for 20 minutes according to the program of power 30 W, alarm temperature 15 ℃, off for 2 s, on for 3 s, to completely disrupt the cells and obtain crude enzyme solution.

[0153] The crude enzyme solution obtained from recombinant bacteria S01 is named crude enzyme solution S01.

[0154] The crude enzyme solution obtained from recombinant bacteria CO2 is named crude enzyme solution CO2.

[0155] The crude enzyme solution obtained from recombinant bacteria R01 is named crude enzyme solution R01.

[0156] Example 4: Preparation of S-phenylethylamine

[0157] Three experimental groups were set up. Experimental group 1-1 was supplemented with 2 mL of crude enzyme solution SO1 and 2 mL of crude enzyme solution CO2. Experimental group 1-2 was supplemented with 2 mL of crude enzyme solution SO1. The remaining components were obtained by adding mother liquor to achieve the final concentration of the reaction solution, with a total volume of 10 mL. The reaction was carried out at 30 °C for 24 h. After the reaction, 1 mL of the reaction solution was taken, centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The concentration of S-phenylethylamine was determined using the HPLC method described above. Three replicate experiments were conducted, with three parallel experiments set up for each experimental group in each replicate. The results showed that the S-phenylethylamine yield of experimental group 1-1 reached 18.03 ± 2.72 g / L, which was significantly higher than that of experimental group 1-2, proving that the multi-enzyme system provided by this invention can effectively drive the conversion of acetophenone to S-phenylethylamine.

[0158] Example 5: Preparation of R-phenylethylamine

[0159] Three experimental groups were set up. Experimental group 2-1 was supplemented with 2 mL of crude enzyme solution R01 and 2 mL of crude enzyme solution CO2. Experimental group 2-2 was supplemented with 2 mL of crude enzyme solution R01. The remaining components were obtained by adding mother liquor to achieve the final concentration of the reaction solution, with a total volume of 10 mL. The reaction was carried out at 30 °C for 24 h. After the reaction, 1 mL of the reaction solution was taken, centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The concentration of R-phenylethylamine was determined using the HPLC method described above. Three replicate experiments were conducted, with three parallel experiments set up for each experimental group in each replicate. The results showed that the R-phenylethylamine yield of experimental group 2-1 reached 15.60 ± 1.85 g / L, which was significantly higher than that of experimental group 2-2, proving that the multi-enzyme system provided by this invention can effectively drive the conversion of acetophenone to R-phenylethylamine.

[0160] Table 4 Experimental Results

[0161]

Claims

1. An enzyme composition for the production of S-phenylethylamine, comprising a protein or polypeptide having the following functions: A) S-transaminase, B) alanine aminotransferase, C) glutamate dehydrogenase, D) formate dehydrogenase.

2. The enzyme composition according to claim 1; characterized in that, The A) S-transaminase was derived from Ochrobactrum anthropi, the B) alanine aminotransferase from barley (Hordeum vulgare), the C) glutamate dehydrogenase from Pyrobaculum calidifontis, and the D) formate dehydrogenase from Candida boidinii.

3. The enzyme composition according to claims 1 to 2; characterized in that, The amino acid sequence of A) S-transaminase includes the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of B) alanine aminotransferase includes the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:

8.

4. A method for synthesizing S-phenylethylamine using acetophenone and formate as raw materials, comprising: Using acetophenone and formate as substrates, water, L-alanine, L-glutamic acid, and NAD+ were added. + The enzyme composition as described in any one of claims 1 to 3, or at least one enzyme in the enzyme composition, is reacted, and the supernatant collected by centrifugation after the reaction is completed is the S-phenylethylamine solution.

5. A group of recombinant engineered bacteria for the production of S-phenylethylamine, including: I) Recombinant Escherichia coli S01, containing an introduced or enhanced gene encoding A) S-transaminase; II) Recombinant Escherichia coli CO2, containing the introduced or enhanced expression of genes encoding B) alanine aminotransferase, C) glutamate dehydrogenase, and D) formate dehydrogenase.

6. The group of recombinant engineered bacteria according to claim 5; characterized in that, The A) S-transaminase was derived from Ochrobactrum anthropi, the B) alanine aminotransferase from barley (Hordeum vulgare), the C) glutamate dehydrogenase from Pyrobaculum calidifontis, and the D) formate dehydrogenase from Candida boidinii.

7. The group of recombinant engineered bacteria according to claims 5 to 6; characterized in that, The amino acid sequence of A) S-transaminase includes the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of B) alanine aminotransferase includes the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:

8.

8. A method for synthesizing S-phenylethylamine using acetophenone and formate as raw materials, comprising: Using acetophenone and formate as substrates, water, L-alanine, L-glutamic acid, and NAD+ were added. + The engineered bacterial cells or cell fragments as described in any one of claims 5 to 7 are reacted, and the supernatant collected by centrifugation after the reaction is completed is the S-phenylethylamine solution.

9. An enzyme composition for the production of R-phenylethylamine, comprising a protein or polypeptide having the following functions: F) R-transaminase, G) alanine racemic enzyme, B) alanine aminotransferase, C) glutamate dehydrogenase, and D) formate dehydrogenase.

10. The enzyme composition according to claim 9; characterized in that, The F) R-transaminase was derived from Fusarium graminearum, the G) alanine racemic enzyme from Bacillus amyloliquefaciens, the B) alanine aminotransferase from barley (Hordeum vulgare), the C) glutamate dehydrogenase from Pyrobaculum calidifontis, and the D) formate dehydrogenase from Candida boidinii.

11. The enzyme composition according to claims 9 to 10; characterized in that, The amino acid sequence of F) R-transaminase includes the amino acid sequence shown in SEQ ID NO:10, the amino acid sequence of G) alanine racemic enzyme includes the amino acid sequence shown in SEQ ID NO:12, the amino acid sequence of B) alanine aminotransferase includes the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:

8.

12. A method for synthesizing R-phenylethylamine using acetophenone and formate as raw materials, comprising: Using acetophenone and formate as substrates, water, L-alanine, L-glutamic acid, and NAD+ were added. + The enzyme composition as described in any one of claims 9 to 11, or at least one enzyme in the enzyme composition, is reacted, and the supernatant collected by centrifugation after the reaction is completed is the R-phenylethylamine solution.

13. A group of recombinant engineered bacteria for the production of R-phenylethylamine, including: III) Recombinant Escherichia coli R01, containing introduced or enhanced expression of genes encoding F) R-transaminase and genes encoding G) alanine racemase; II) Recombinant Escherichia coli CO2, containing the introduced or enhanced expression of genes encoding B) alanine aminotransferase, C) glutamate dehydrogenase, and D) formate dehydrogenase.

14. The group of recombinant engineered bacteria according to claim 13; characterized in that, The F) R-transaminase was derived from Fusarium graminearum, the G) alanine racemic enzyme from Bacillus amyloliquefaciens, the B) alanine aminotransferase from barley (Hordeum vulgare), the C) glutamate dehydrogenase from Pyrobaculum calidifontis, and the D) formate dehydrogenase from Candida boidinii.

15. The group of recombinant engineered bacteria according to claims 13 to 14; characterized in that, The amino acid sequence of F) R-transaminase includes the amino acid sequence shown in SEQ ID NO:10, the amino acid sequence of G) alanine racemic enzyme includes the amino acid sequence shown in SEQ ID NO:12, the amino acid sequence of B) alanine aminotransferase includes the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence of C) glutamate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of D) formate dehydrogenase includes the amino acid sequence shown in SEQ ID NO:

8.

16. A method for synthesizing R-phenylethylamine using acetophenone and formate as raw materials, comprising: Using acetophenone and formate as substrates, water, L-alanine, L-glutamic acid, and NAD+ were added. + The engineered bacterial cells or cell fragments as described in any one of claims 13 to 15 are reacted, and the supernatant collected by centrifugation after the reaction is completed is the R-phenylethylamine solution.

17. The method according to claim 4, 8, 12, or 16, wherein, In the reaction system, the final concentrations of formate, acetophenone, L-alanine, L-glutamic acid, and NAD+ were 50 mM-1 mM, 1-5 mM, 2-10 mM, and 0.5-1 mM, respectively.

18. The use of the enzyme composition or recombinant engineered bacteria according to any one of claims 1 to 16 in the production of S-phenylethanol or R-phenylethanol.

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