L-aspartate-alpha-decarboxylase mutants, nucleic acid molecules, recombinant expression vectors, vector cells and production methods and uses

Abstract

The application discloses an L-aspartate-alpha-decarboxylase mutant, a nucleic acid molecule, a recombinant expression vector, a carrier cell and a production method and application. The L-aspartate-alpha-decarboxylase mutant provided in the application comprises amino acid mutations in one or more of the following positions: 31st, 34th, 38th, 51st, 65th, 66th, 87th, 100th, 135th, 198th, 325th, 433rd, 453rd and 490th, compared with the amino acid sequence of the L-aspartate-alpha-decarboxylase shown in SEQ ID No. 1. The L-aspartate-alpha-decarboxylase mutant is obtained by site-directed mutagenesis based on an insect-derived L-aspartate-alpha-decarboxylase. The L-aspartate-alpha-decarboxylase mutant is applied to the preparation of beta-alanine and has high catalytic efficiency.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biocatalysis, and relates to an L-aspartate-α-decarboxylase mutant, nucleic acid molecule, recombinant expression vector, vector cell, production method and application. Background Technology

[0002] β-alanine is the only β-type non-protein amino acid found in nature. It is mainly used to synthesize pantothenic acid and calcium pantothenate, carnosine, pamidronate sodium, balsalazine, etc. It has a wide range of applications in the fields of medicine, feed, and food, and there is a very large market demand.

[0003] Currently, the main method for synthesizing β-alanine is chemical synthesis, which suffers from drawbacks such as high pollution, high energy consumption, and difficult post-processing. In recent years, biological synthesis of β-alanine has attracted attention. L-aspartate-α-decarboxylase (ADC) can specifically remove the α-carboxyl group from L-aspartate to generate β-alanine. However, currently discovered L-aspartate-α-decarboxylases generally suffer from low enzyme activity. Improving the enzyme activity of L-aspartate-α-decarboxylase, and thus its conversion efficiency, is of great significance for the industrial synthesis of β-alanine. Summary of the Invention

[0004] Therefore, it is necessary to provide an L-aspartate-α-decarboxylase mutant, nucleic acid molecule, recombinant expression vector, vector cell, production method, and application to meet the needs of industrial production.

[0005] In some embodiments, an L-aspartate-α-decarboxylase mutant is provided, said L-aspartate-α-decarboxylase mutant comprising, compared with the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1, an amino acid mutation at one or more of the following sites:

[0006] The 31st, 34th, 38th, 51st, 65th, 66th, 87th, 100th, 135th, 198th, 325th, 433rd, 453rd and 490th positions.

[0007] In some embodiments, the provided L-aspartate-α-decarboxylase mutant includes one or any suitable mutations as follows:

[0008] (1) Aspartic acid at position 31 is missing;

[0009] (2) Valine at position 34 is mutated to threonine;

[0010] (3) Serine at position 38 is mutated to methionine;

[0011] (4) The lysine at position 51 is mutated to glutamic acid;

[0012] (5) The lysine at position 65 is mutated to serine;

[0013] (6) Threonine at position 66 is mutated to glycine;

[0014] (7) Threonine at position 87 is mutated to glutamine;

[0015] (8) The 100th position of asparagine is mutated to glutamic acid;

[0016] (9) Leucine at position 135 is mutated to isoleucine;

[0017] (10) The lysine at position 198 is mutated to isoleucine;

[0018] (11) Glutamine at position 325 is mutated to alanine;

[0019] (12) Glutamine at position 325 is mutated to glutamic acid;

[0020] (13) Glutamine at position 325 is mutated to lysine;

[0021] (14) The phenylalanine at position 433 is mutated to tyrosine;

[0022] (15) Threonine at position 453 is mutated to leucine;

[0023] (16) Threonine at position 453 is mutated to asparagine; and

[0024] (17) Glutamine at position 490 is mutated to lysine.

[0025] In some embodiments, the provided L-aspartate-α-decarboxylase mutant includes one of the following amino acid mutant groups:

[0026] Group 1: Lysine at position 51 is mutated to glutamic acid;

[0027] Group 2: Lysine at position 65 is mutated to serine;

[0028] Group 3: Threonine at position 87 is mutated to glutamine;

[0029] Group 4: Lysine at position 198 is mutated to isoleucine;

[0030] Group 5: Leucine at position 135 is mutated to isoleucine;

[0031] Group 6: Serine at position 38 is mutated to methionine;

[0032] Group 7: Valine at position 34 is mutated to threonine;

[0033] Group 8: Asparagine at position 100 is mutated to glutamic acid;

[0034] Group 9: Phenylalanine at position 433 is mutated to tyrosine;

[0035] Group 10: Threonine at position 66 is mutated to glycine;

[0036] Group 11: Aspartic acid at position 31 is deleted, glutamine at position 325 is mutated to alanine, threonine at position 453 is mutated to leucine, and glutamine at position 490 is mutated to lysine.

[0037] Group 12: Aspartic acid at position 31 is deleted, glutamine at position 325 is mutated to lysine, threonine at position 453 is mutated to asparagine, and glutamine at position 490 is mutated to lysine.

[0038] Group 13: Aspartic acid at position 31 is deleted, glutamine at position 325 is mutated to glutamic acid, threonine at position 453 is mutated to asparagine, and glutamine at position 490 is mutated to lysine.

[0039] Group 14: Aspartic acid is deleted at position 31, glutamine at position 325 is mutated to glutamic acid, threonine at position 453 is mutated to leucine, and glutamine at position 490 is mutated to lysine.

[0040] In some embodiments, a nucleic acid molecule is provided, the nucleic acid molecule comprising encoding the L-aspartate-α-decarboxylase mutant.

[0041] In some embodiments, a recombinant expression vector is provided that contains the aforementioned nucleic acid molecule.

[0042] In some implementations, the provided recombinant expression vector includes a plasmid.

[0043] In some embodiments, a vector cell is provided that contains the nucleic acid molecule or the recombinant expression vector, optionally, the vector cell comprising Escherichia coli cells.

[0044] In some embodiments, a method for producing an L-aspartate-α-decarboxylase mutant is provided, comprising the steps of: culturing the vector cells; and isolating the L-aspartate-α-decarboxylase mutant from the resulting culture.

[0045] In some embodiments, the L-aspartate-α-decarboxylase mutant or the vector cell is provided for use in the preparation of β-alanine.

[0046] In some embodiments, a method for preparing β-alanine is provided, comprising the following steps: using the L-aspartate-α-decarboxylase mutant or the culture substrate of the carrier cell to prepare β-alanine.

[0047] The aforementioned L-aspartate-α-decarboxylase mutant was obtained through site-directed mutagenesis of insect-derived L-aspartate-α-decarboxylase. This L-aspartate-α-decarboxylase mutant exhibits high enzyme activity and can be used to prepare β-alanine with high catalytic efficiency. Detailed Implementation

[0048] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0050] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0051] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0052] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0053] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0054] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0055] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0056] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0057] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0058] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0059] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0060] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0061] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0062] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0063] In this application, the term "mutation" refers to the deletion, addition, or substitution of amino acid residues in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the deletion of an amino acid at a specific position in the protein sequence is indicated by annotations such as D31-, where D31- refers to the deletion of aspartic acid at position 31 of the reference protein's amino acid sequence; the substitution of an amino acid at a specific position in the protein sequence is indicated by annotations such as V34T, where V34T refers to the mutation of valine (V) residue at position 34 to threonine (T) residue; S38M refers to the mutation of serine (S) residue at position 38 to methionine (M) residue; K51G refers to the mutation of lysine (K) residue at position 51 to glutamic acid (G) residue; K65S refers to the mutation of lysine (K) residue at position 65 to serine (S) residue; T66G refers to the mutation of threonine (T) residue at position 66 to glycine (G) residue; T87Q refers to the mutation of threonine (T) residue at position 87 to glutamine (Q) residue; and N100E refers to the deletion of aspartic acid at position 100. The mutation of an amide (N) residue to a glutamic acid (E) residue is as follows: L135I refers to the mutation of the leucine (L) residue at position 135 to an isoleucine (I) residue; K198I refers to the mutation of the lysine (K) residue at position 198 to an isoleucine (I) residue; Q325A refers to the mutation of the glutamine (Q) residue at position 325 to an alanine (A) residue; Q325E refers to the mutation of the glutamine (Q) residue at position 325 to a glutamic acid (E) residue; and Q325K is... The mutation at position 325 (Q) is replaced by a lysine (K) residue; F433Y is replaced by a phenylalanine (F) residue at position 433 (Y) is replaced by a tyrosine (Y) residue; T453L is replaced by a threonine (T) residue at position 453 (L) is replaced by a leucine (L) residue; T453N is replaced by an asparagine (N) residue at position 453 (T) is replaced by a threonine (N) residue; and Q490K is replaced by a glutamine (Q) residue at position 490 (K) is replaced by a lysine (K) residue.

[0064] In this application, the term "mutant" refers to a polynucleotide or polypeptide that, relative to the wild type or comparison, contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0065] In this application, the term "vector" refers to a nucleic acid molecule capable of transporting or transferring foreign nucleic acid molecules. This term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector capable of expressing an insert in target cells and typically contains control sequences (such as enhancer, promoter, and terminator sequences) that drive the expression of the insert. The term "transcription vector" refers to a vector capable of being transcribed but not translated. Transcription vectors are used to amplify their inserts. Foreign nucleic acid molecules are referred to as "insertions" or "transgenic molecules." Vectors typically consist of an insert and a larger sequence that serves as the vector's backbone. Based on their structure or origin, major types of vectors include plasmid vectors, granular vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0066] In this application, the term "plasmid" refers to an extrachromosomal element that typically carries a gene that is not part of the core metabolic mechanisms of the cell and is usually in the form of a circular double-stranded DNA molecule. These elements can be autonomously replicating sequences, genome-integrated sequences, bacteriophage or nucleotide sequences from any source, and linear, circular or supercoiled single-stranded or double-stranded DNA or RNA. Typically, plasmids contain a functional origin of replication in the host cell (e.g., *E. coli*) and selection markers for detecting host cells containing the plasmid. In some embodiments, the plasmid is a closed circular DNA molecule. "Co-expression plasmid" refers to a plasmid of different types that can be expressed in the same host bacterium.

[0067] In this application, the term "nucleic acid" refers to any linearly or sequentially arranged nucleotides and nucleosides, such as cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides, and their derivatives. Nucleic acids may include bacterial plasmid vectors, including expression, cloning, granulation, and transformation vectors, and may include modified or derived nucleotides and nucleosides.

[0068] In this application, the term "nucleotide sequence" refers to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, and refers to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, and denotes the sense or antisense strand. The terms "polynucleotide," "oligonucleotide," "nucleotide sequence," and "nucleic acid" are used interchangeably herein and include, but are not limited to, coding sequences. That is, one or more polynucleotide or nucleic acid sequences that, when placed under the control of appropriate regulatory or control sequences, are transcribed and translated into polypeptides in vitro or in vivo; control sequences, such as translation start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, and DNA sequences to which one or more transcription factors bind and positively (inducing) or negatively (inhibiting) alter the promoter activity of a gene, etc.

[0069] In this application, the term "expression" refers to the transformation of sequence information into a corresponding expression product, including direct transcription products (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, structural RNA, or any other type of RNA) or proteins produced by translation of mRNA.

[0070] In this application, the term "coding" refers to a DNA polynucleotide sequence that can be transcribed into RNA (mRNA) that translates into proteins; or that can be transcribed into RNA that does not translate into proteins (tRNA, rRNA, or other non-coding RNA); or an RNA polynucleotide sequence that can be translated into proteins.

[0071] In this application, the terms “DNA,” “RNA,” “nucleic acid,” “nucleic acid fragment,” or “polynucleotide” refer to any one or more nucleic acid segments present in a polynucleotide or construct. Nucleic acids or fragments thereof may be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form, and in double-stranded or single-stranded form. “Isolated” nucleic acid or polynucleotide means a nucleic acid molecule, DNA, or RNA that has been isolated from its natural environment. For example, in the context of this invention, the recombinant polynucleotide contained in the vector is isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides in solution.

[0072] In this application, "gene" refers to a polynucleotide containing nucleotides encoding a functional molecule, including functional molecules produced solely by transcription (biologically active RNA) or functional molecules produced through transcription and translation (e.g., polypeptides). The term "gene" includes cDNA and genomic DNA nucleic acids, and also refers to a nucleic acid fragment expressing a specific RNA, protein, or polypeptide, containing regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding sequence. "Natural gene" refers to any gene found in nature that has its own regulatory sequences. "Chimeric gene" refers to any non-natural gene containing non-naturally co-occurring regulatory and / or coding sequences. "Endogenous gene" refers to a naturally occurring gene located at its natural position in an organism's genome. "Exogenous gene" or "heterologous gene" refers to a gene that is not normally present in the host but is introduced into the host through gene transfer.

[0073] In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acid residues.

[0074] In this application, the term "recombinant" nucleic acid refers to non-naturally occurring nucleic acids, such as those created by artificially linking previously separate nucleic acid fragments together to produce a combined nucleic acid fragment with a desired function. Artificial combination is often accomplished through chemical synthesis or by artificially manipulating segments of nucleic acids (e.g., through genetic engineering techniques).

[0075] In this application, the term "import" or "introduction" to cells refers to the conversion of nucleic acid molecules (e.g., plasmids, linear nucleic acid fragments, RNA, etc.) or proteins into cells through various methods including but not limited to Agrobacterium-mediated transformation, gene gun bombardment, electroporation, and PEG transformation, so that the nucleic acid or protein can function in the cells.

[0076] In this application, "ADC enzyme mutant" refers to L-aspartate-α-decarboxylase mutant.

[0077] In this application, "initial ADC enzyme" refers to the L-aspartate-α-decarboxylase shown in SEQ ID No. 1.

[0078] In some embodiments, an L-aspartate-α-decarboxylase mutant is provided, which, compared to the L-aspartate-α-decarboxylase shown in SEQ ID No. 1, includes amino acid mutations at one or more sites.

[0079] The amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is derived from insects, and the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1 has been disclosed in prior art CN115261364 A. In that prior art CN115261364 A, the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1 involved in this invention is named CtADC.

[0080] In some embodiments, an L-aspartate-α-decarboxylase mutant is provided, wherein the provided L-aspartate-α-decarboxylase mutant, compared with the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1, includes amino acid mutations at one or more of the following sites:

[0081] The 31st, 34th, 38th, 51st, 65th, 66th, 87th, 100th, 135th, 198th, 325th, 433rd, 453rd and 490th positions.

[0082] In some embodiments, the provided L-aspartate-α-decarboxylase mutant, compared to the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1, includes amino acid mutations at one or more of the following sites:

[0083] (1) Deletion of aspartic acid at position 31 (D31-);

[0084] (2) Valine at position 34 is mutated to threonine (V34T);

[0085] (3) The serine at position 38 is mutated to methionine (S38M).

[0086] (4) The lysine at position 51 is mutated to glutamic acid (K51G).

[0087] (5) The lysine at position 65 is mutated to serine (K65S);

[0088] (6) Threonine at position 66 is mutated to glycine (T66G).

[0089] (7) Threonine at position 87 is mutated to glutamine (T87Q);

[0090] (8) The 100th position of asparagine is mutated to glutamic acid (N100E);

[0091] (9) The leucine at position 135 is mutated to isoleucine (L135I);

[0092] (10) The lysine at position 198 is mutated to isoleucine (K198I);

[0093] (11) Glutamine at position 325 is mutated to alanine (Q325A);

[0094] (12) Glutamine at position 325 is mutated to glutamic acid (Q325E);

[0095] (13) Glutamine at position 325 is mutated to lysine (Q325K);

[0096] (14) The phenylalanine at position 433 is mutated to tyrosine (F433Y).

[0097] (15) Threonine at position 453 is mutated to leucine (T453L);

[0098] (16) The threonine at position 453 is mutated to asparagine (T453N); and

[0099] (17) Glutamine at position 490 is mutated to lysine (Q490K).

[0100] In some embodiments, the provided L-aspartate-α-decarboxylase mutant includes one of the following amino acid mutant groups:

[0101] Group 1: Lysine at position 51 is mutated to glutamic acid (K51G);

[0102] Group 2: Lysine at position 65 is mutated to serine (K65S);

[0103] Group 3: Threonine at position 87 is mutated to glutamine (T87Q);

[0104] Group 4: Lysine at position 198 is mutated to isoleucine (K198I);

[0105] Group 5: Leucine at position 135 is mutated to isoleucine (L135I);

[0106] Group 6: Serine at position 38 is mutated to methionine (S38M).

[0107] Group 7: Valine at position 34 is mutated to threonine (V34T).

[0108] Group 8: Asparagine at position 100 is mutated to glutamic acid (N100E);

[0109] Group 9: Phenylalanine at position 433 is mutated to tyrosine (F433Y).

[0110] Group 10: Threonine at position 66 is mutated to glycine (T66G).

[0111] Group 11: Aspartic acid deletion at position 31 (D31-), glutamine at position 325 mutated to alanine (Q325A), threonine at position 453 mutated to leucine (T453L), and glutamine at position 490 mutated to lysine (Q490K).

[0112] Group 12: Aspartic acid deletion at position 31 (D31-), glutamine at position 325 mutated to lysine (Q325K), threonine at position 453 mutated to asparagine (T453N), and glutamine at position 490 mutated to lysine (Q490K).

[0113] Group 13: Aspartic acid deletion at position 31 (D31-), glutamine at position 325 mutated to glutamic acid (Q325E), threonine at position 453 mutated to asparagine (T453N), and glutamine at position 490 mutated to lysine (Q490K).

[0114] Group 14: Aspartic acid deletion at position 31 (D31-), glutamine at position 325 mutated to glutamic acid (Q325E), threonine at position 453 mutated to leucine (T453L), and glutamine at position 490 mutated to lysine (Q490K).

[0115] In some embodiments, a nucleic acid molecule is provided, the nucleic acid molecule comprising encoding the L-aspartate-α-decarboxylase mutant.

[0116] In some embodiments, a recombinant expression vector is provided that contains the aforementioned nucleic acid molecule.

[0117] In some implementations, the provided recombinant expression vector includes a plasmid.

[0118] In some embodiments, a vector cell is provided that contains the nucleic acid molecule or the recombinant expression vector, optionally, the vector cell comprising Escherichia coli cells.

[0119] In some embodiments, a recombinant genetically engineered bacterium is provided. The host cell used for the genetically engineered bacterium is Escherichia coli, preferably E. coli BL21 (DE3), but not limited to the aforementioned E. coli host.

[0120] In some embodiments, a method for producing an L-aspartate-α-decarboxylase mutant is provided, comprising the steps of: culturing the vector cells; and isolating the L-aspartate-α-decarboxylase mutant from the resulting culture.

[0121] In some embodiments, the L-aspartate-α-decarboxylase mutant or the vector cell is provided for use in the preparation of β-alanine.

[0122] In some embodiments, a method for preparing β-alanine is provided, comprising the following steps: using the L-aspartate-α-decarboxylase mutant or the culture substrate of the carrier cell to prepare β-alanine.

[0123] Non-limitingly, by modifying the gene encoding the L-aspartate-α-decarboxylase shown in SEQ ID No. 1 through site-directed mutagenesis, the enzyme activity of the encoded L-aspartate-α-decarboxylase was increased, resulting in a significant improvement in the catalytic performance of the genetically engineered bacteria expressing the ADC mutation, thus having higher industrial application value.

[0124] This invention obtains a series of ADC enzyme mutants by site-directed mutagenesis of insect-derived L-aspartate-α-decarboxylase (ADC). Transforming the recombinant plasmids of these mutants into *E. coli* and fermenting them in shake flasks resulted in strains with ADC enzyme activity increased by 5%-43%. Applying these mutant strains to the preparation of β-alanine improved catalytic efficiency by 33%, making them more suitable for industrial production.

[0125] The molecular biology experiments in the examples include the preparation of host competent cells, plasmid construction, gene digestion and ligation, plasmid transformation, PCR gene amplification, and other experimental techniques, mainly referring to "Molecular Cloning: A Laboratory Manual" (3rd edition).

[0126] Example 1: Expression of L-aspartate-α-decarboxylase mutant

[0127] The amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 was randomly mutated at specific sites. The gene sequence of the L-aspartate-α-decarboxylase mutant encoding the mutated site was ligated into the pET28a plasmid to prepare a recombinant plasmid. The obtained recombinant plasmid was transformed into E. coli DH5α competent cells and plated on kanamycin-containing plates for screening. Positive clones were picked the next day, and after in vitro culture, the plasmid was extracted. These plasmids were then re-electroporated into E. coli BL21(DE3) competent cells to obtain a mutant cell library that could be used for expression. The mutant cells were cultured to express the L-aspartate-α-decarboxylase mutant. L-aspartate-α-decarboxylase mutants with high enzyme activity were screened. The mutant strains with significantly increased enzyme activity were sequenced to determine the mutation site. Based on the mutation site of the gene, the amino acid sequence of the L-aspartate-α-decarboxylase mutant was determined, resulting in the L-aspartate-α-decarboxylase mutants shown in Table 1 below.

[0128] The enzyme activity detection method for L-aspartate-α-decarboxylase mutant is as follows: First, select single colonies and grow them at 30℃ in LB medium containing kanamycin and 0.2% isopropyl β-d-1-thiogalactopyranoside (IPTG) for 20-24 hours. Then, centrifuge to remove the supernatant, resuspend the cells in buffer (KPB, pH 7.0, 50mM) at a concentration of 10% (w / v), and sonicate to obtain the supernatant containing cell lysates.

[0129] At pH 6.5 and 37°C, 50 μL of the above cell lysate was incubated in a 50 mL solution containing L-aspartic acid at a final concentration of 60 g / L and pyridoxal phosphate (PLP) at a final concentration of 0.2 g / L. The solution was mechanically stirred at 200 rpm, and then 1 M sulfuric acid aqueous solution was titrated into the reaction solution to maintain pH. After half an hour, the amount of sulfuric acid used in the reaction was determined to directly measure ADC activity, and the volume of sulfuric acid aqueous solution used in the reaction was recorded (recorded in Table 1 as "volume of sulfuric acid aqueous solution used corresponding to ADC enzyme activity"). The larger the volume, the higher the ADC enzyme activity. The enzyme activity detection results can be found in Table 1.

[0130] Table 1

[0131]

[0132] As shown in Table 1, the enzyme activity of the provided L-aspartate-α-decarboxylase mutant was increased by 5%-43% compared with that of the unmutated L-aspartate-α-decarboxylase.

[0133] The amino acid sequence of SEQ ID No. 1 is shown below:

[0134] MPTNGMLDVALQVIEDANLSSGSDSAGVSEDEDVQLFSTTGNIVSSKPLKKPALKPATKDEDQNKTKANAKRYASLPNREQHQRFLTDFLSEVLNGAIFNATDRSNKVLNWVDPEELKRSIDLSLKDEPDSDEKLLELARATIDHSVKTGHPYFMNQLFSSVDPYGFAGQVLTDALNPSVYTFEVSPVFVLMEEVVLKEMRTIVGFPGGVGDGIFCPGGSMANGYAISCARFKHMPDVKTKGLHSLPRLVIFTSEDAHYSIKKLASFMGIGSDNVYPIRTDAVGKIQPDHLEAEILRAKSEGAVPFMVSATAGTTVIGAFDPLEQIADLCQKYNLWMHVDAAWGGGALMSKKYRTLLKGVERADSVTWNPHKLLAAPQQCSTFLTRHEGILSGCHSTNATYLFQKDKFYDTQYDTGDKHIQCGRRADVLKFWFMWRAKGTSGLEQHIDKVFETAEYFTNSIKARPGFEMVIENPECTNVCFWYVPPGLRQVPRDSAEFGERLHKVAPKVKERMMREGSMMITYQPIHDKPNFFRLVLQNSALDKSDMNYIIDEIERLASDL。

[0135] The amino acid sequences of the obtained L-aspartate-α-decarboxylase mutants are shown in SEQ ID No.2 to SEQ ID No.15 respectively:

[0136] SEQ ID No.2

[0137] 。

[0138] SEQ ID No. 3

[0139] 。

[0140] SEQ ID No.4

[0141] 。

[0142] SEQ ID No.5

[0143] 。

[0144] SEQ ID No.6

[0145] 。

[0146] SEQ ID No.7

[0147] 。

[0148] SEQ ID No.8

[0149] 。

[0150] SEQ ID No.9

[0151] 。

[0152] SEQ ID No.10

[0153] 。

[0154] SEQ ID No.11

[0155] MPTNGMLDVALQVIEDANLSSGSDSAGVSEDEDVQLFSTTGNIVSSKPLKKPALKPATKDEDQNKGKANAKRYASLPNREQHQRFLTDFLSEVLNGAIFNATDRSNKVLNWVDPEELKRSIDLSLKDEPDSDEKLLELARATIDHSVKTGHPYFMNQLFSSVDPYGFAGQVLTDALNPSVYTFEVSPVFVLMEEVVLKEMRTIVGFPGGVGDGIFCPGGSMANGYAISCARFKHMPDVKTKGLHSLPRLVIFTSEDAHYSIKKLASFMGIGSDNVYPIRTDAVGKIQPDHLEAEILRAKSEGAVPFMVSATAGTTVIGAFDPLEQIADLCQKYNLWMHVDAAWGGGALMSKKYRTLLKGVERADSVTWNPHKLLAAPQQCSTFLTRHEGILSGCHSTNATYLFQKDKFYDTQYDTGDKHIQCGRRADVLKFWFMWRAKGTSGLEQHIDKVFETAEYFTNSIKARPGFEMVIENPECTNVCFWYVPPGLRQVPRDSAEFGERLHKVAPKVKERMMREGSMMITYQPIHDKPNFFRLVLQNSALDKSDMNYIIDEIERLASDL。

[0156] SEQ ID No.12

[0157] 。

[0158] SEQ ID No.13

[0159] 。

[0160] SEQ ID No.14

[0161] MPTNGMLDVALQVIEDANLSSGSDSAGVSEEDVQLFSTTGNIVSSKPLKKPALKPATKDEDQNKTKANAKRYASLPNREQHQRFLTDFLSEVLNGAIFNATDRSNKVLNWVDPEELKRSIDLSLKDEPDSDEKLLELARATIDHSVKTGHPYFMNQLFSSVDPYGFAGQVLTDALNPSVYTFEVSPVFVLMEEVVLKEMRTIVGFPGGVGDGIFCPGGSMANGYAISCARFKHMPDVKTKGLHSLPRLVIFTSEDAHYSIKKLASFMGIGSDNVYPIRTDAVGKIQPDHLEAEILRAKSEGAVPFMVSATAGTTVIGAFDPLEEIADLCQKYNLWMHVDAAWGGGALMSKKYRTLLKGVERADSVTWNPHKLLAAPQQCSTFLTRHEGILSGCHSTNATYLFQKDKFYDTQYDTGDKHIQCGRRADVLKFWFMWRAKGTSGLEQHIDKVFENAEYFTNSIKARPGFEMVIENPECTNVCFWYVPPGLRKVPRDSAEFGERLHKVAPKVKERMMREGSMMITYQPIHDKPNFFRLVLQNSALDKSDMNYIIDEIERLASDL。

[0162] SEQ ID No.15

[0163] .

[0164] Example 2: Catalytic performance test of L-aspartate-α-decarboxylase mutant

[0165] After fermentation and centrifugation, strains expressing the initial ADC enzyme and the ADC enzyme mutant ADC12 obtained in Example 1 were added to 0.4 L of reaction solution at concentrations of 12% (w / w) and 8% (w / w) (as a percentage of the substrate L-aspartic acid). The substrate L-aspartic acid concentration in the reaction solution was 200 g / L. Pyridoxal phosphate (PLP) was added to a final concentration of 0.02 g / L. The reaction was catalyzed at 37 °C and 300 rpm. A 2 M sulfuric acid aqueous solution was added to the reaction solution to maintain pH 6.5. Samples were taken periodically, and the amounts of substrate and product β-alanine produced were analyzed using HPLC. The HPLC method is as follows:

[0166] The high-performance liquid chromatograph (HPLC) was a Waters 2695-PDA996, ​​and the column was a Phenomenex Luna C18 (250 mm × 4.6 mm × 5 μm). The HPLC conditions were as follows: mobile phase A was water + 0.1% (v / v) acetic acid + 0.1% (v / v) triethylamine; mobile phase B was acetonitrile; flow rate was 0.8 mL / min; detection wavelength was 360 nm; column temperature was 30 °C; 2,4-dinitrofluorobenzene (AR) was used for pre-column derivatization, and the elution gradient can be found in Table 2.

[0167] Table 2

[0168]

[0169] The results are shown in Table 3 below:

[0170] Table 3

[0171]

[0172] As shown in Table 3, the catalytic efficiency of the 8% ADC12 mutant is similar to that of the initial 12% ADC enzyme, and the catalytic efficiency of the mutated ADC enzyme is increased by 33%.

[0173] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0174] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An L-aspartate-α-decarboxylase mutant, characterized in that, The L-aspartate-α-decarboxylase mutant, compared with the amino acid sequence of the L-aspartate-α-decarboxylase shown in SEQ ID No. 1, includes only the following amino acid mutations: The 31st, 325th, 453rd, and 490th positions; The L-aspartate-α-decarboxylase mutant includes only one of the following amino acid mutant groups: Group 11: Aspartic acid at position 31 is deleted, glutamine at position 325 is mutated to alanine, threonine at position 453 is mutated to leucine, and glutamine at position 490 is mutated to lysine. Group 12: Aspartic acid at position 31 is deleted, glutamine at position 325 is mutated to lysine, threonine at position 453 is mutated to asparagine, and glutamine at position 490 is mutated to lysine. Group 13: Aspartic acid at position 31 is deleted, glutamine at position 325 is mutated to glutamic acid, threonine at position 453 is mutated to asparagine, and glutamine at position 490 is mutated to lysine. Group 14: Aspartic acid is deleted at position 31, glutamine at position 325 is mutated to glutamic acid, threonine at position 453 is mutated to leucine, and glutamine at position 490 is mutated to lysine.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes the L-aspartate-α-decarboxylase mutant of claim 1.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector includes a plasmid.

5. A carrier cell, characterized in that, It comprises the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3 or 4; The carrier cells are not animal species.

6. The carrier cell according to claim 5, characterized in that, The carrier cells include Escherichia coli cells.

7. A method for producing an L-aspartate-α-decarboxylase mutant, comprising the following steps: culturing the vector cells of claim 5; and isolating the L-aspartate-α-decarboxylase mutant from the resulting culture.

8. The use of the L-aspartate-α-decarboxylase mutant of claim 1 or the carrier cell of claim 5 or 6 in the preparation of β-alanine.

9. A method for preparing β-alanine, characterized in that, The method includes the following steps: preparing β-alanine using the L-aspartate-α-decarboxylase mutant of claim 1 or the culture substrate of the carrier cell of claim 5 or 6.

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