L-aspartic acid-alpha-decarboxylase mutant, nucleic acid molecule, recombinant expression vector, vector cell, production method and application

Abstract

The invention discloses an L-aspartic acid-alpha-decarboxylase mutant, a nucleic acid molecule, a recombinant expression vector, a vector cell, a production method and application. Compared with the amino acid sequence of the L-aspartic acid-alpha-decarboxylase as shown in SEQ ID No.1, the provided L-aspartic acid-alpha-decarboxylase mutant comprises amino acid mutation of one or more of the following sites: the 31st site, the 34th site, the 38th site, the 51st site, the 65th site, the 66th site, the 87th site, the 100th site, the 135th site, the 198th site, the 325th site, the 433th site, the 453th site and the 490th site. The L-aspartic acid-alpha-decarboxylase mutant provided by the invention is obtained by carrying out site-specific mutagenesis on the basis of insect source L-aspartic acid-alpha-decarboxylase. The L-aspartic acid-alpha-decarboxylase mutant provided by the invention is applied to preparation of beta-alanine, and has relatively high catalytic efficiency.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering technology and biocatalysis, and relates to an L-aspartate-alpha-decarboxylase mutant, a nucleic acid molecule, a recombinant expression vector, a vector cell, a production method and an application. Background Art

[0002] β-Alanine is the only β-type non-protein amino acid existing in nature. It is mainly used to synthesize pantothenic acid and calcium pantothenate, carnosine, sodium pamidronate, balsalazide, etc. It is widely used in medicine, feed, food and other fields and has a very large market demand.

[0003] At present, the synthesis method of β-alanine mainly adopts chemical synthesis, which has the defects of high pollution, high energy consumption and difficult post-processing. In recent years, the biological synthesis of β-alanine has attracted people's attention. L-aspartate-α-decarboxylase (ADC) can specifically remove the α-carboxyl group of L-aspartate to generate β-alanine. However, the L-aspartate-α-decarboxylase discovered so far generally has the problem of low enzyme activity. Improving the enzyme activity of L-aspartate-α-decarboxylase and then improving its conversion efficiency is of great significance for the industrial synthesis of β-alanine. Summary of the invention

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

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

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

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

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

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

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

[0011] (4) Lysine 51 mutated to glutamic acid;

[0012] (5) Lysine 65 mutated to serine;

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

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

[0015] (8) Asparagine at position 100 mutated to glutamic acid;

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

[0017] (10) Lysine 198 mutated to isoleucine;

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

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

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

[0021] (14) Phenylalanine at position 433 mutated to tyrosine;

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

[0023] (16) mutation of Thr453 to Asparagine; and

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

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

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

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

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

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

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

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

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

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

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

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

[0036] Group 11: Aspartic acid at position 31 is missing, 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 missing, 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 missing, 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 at position 31 is deleted, 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, wherein the nucleic acid molecule comprises a protein encoding the L-aspartate-α-decarboxylase mutant.

[0041] In some embodiments, a recombinant expression vector is provided, which comprises the nucleic acid molecule.

[0042] In some embodiments, a recombinant expression vector is provided that includes a plasmid.

[0043] In some embodiments, a vector cell is provided, which comprises the nucleic acid molecule or the recombinant expression vector. Optionally, the vector cell comprises an Escherichia coli cell.

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

[0045] In some embodiments, there is provided use of the L-aspartate-α-decarboxylase mutant or the vector cell 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 of the carrier cell to catalyze a substrate to prepare β-alanine.

[0047] The L-aspartate-α-decarboxylase mutant provided above is obtained by site-directed mutagenesis of insect-derived L-aspartate-α-decarboxylase. The provided L-aspartate-α-decarboxylase mutant has high enzyme activity, can be used to prepare β-alanine, and has high catalytic efficiency. DETAILED DESCRIPTION

[0048] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0050] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0051] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

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

[0053] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0054] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0055] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0056] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

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

[0058] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0059] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0060] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

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

[0062] The "room temperature" in this application generally refers to 5°C to 30°C, preferably 25±5°C.

[0063] In the present 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 with the amino acid sequence of a reference protein or polypeptide. Throughout the specification and claims, the missing amino acid at a specific position in the protein sequence is annotated with, for example, D31-, which means that the aspartic acid at position 31 of the amino acid sequence of the reference protein is missing; the substituted amino acid at a specific position in the protein sequence is annotated with, for example, V34T, which means that the valine (V) residue at position 34 of the amino acid sequence of the reference protein is mutated to a threonine (T) residue, S38M means that the serine (S) residue at position 38 is mutated to a methionine (M) residue, K51G means that the lysine (K) residue at position 51 is mutated to a glutamic acid (G) residue, K65S means that the lysine (K) residue at position 65 is mutated to a serine (S) residue, T66G means that the threonine (T) residue at position 66 is mutated to a glycine (G) residue, T87Q means that the threonine (T) residue at position 87 is mutated to a glutamine (Q) residue, N100E means that the aspartic acid at position 100 is mutated to a glutamine (Q) residue, The amide (N) residue was mutated to a glutamic acid (E) residue, 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 refers to the mutation of the glutamine (Q) residue at position 325 to a glutamic acid (E) residue. Refers to the mutation of the glutamine (Q) residue at position 325 to a lysine (K) residue, F433Y refers to the mutation of the phenylalanine (F) residue at position 433 to a tyrosine (Y) residue, T453L refers to the mutation of the threonine (T) residue at position 453 to a leucine (L) residue, T453N refers to the mutation of the threonine (T) residue at position 453 to an asparagine (N) residue, and Q490K refers to the mutation of the glutamine (Q) residue at position 490 to a lysine (K) residue.

[0064] In this application, the term "mutant" refers to a polynucleotide or polypeptide that contains changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a wild-type or compared polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of 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 an external nucleic acid molecule. The term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector capable of expressing an insert in a target cell and generally containing control sequences (such as enhancers, promoters, and terminator sequences) that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. External nucleic acid molecules are referred to as "inserts" or "transgenics". Vectors are generally composed of inserts and larger sequences used as the backbone of the vector. Based on the structure or source of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus vectors), and artificial chromosomes.

[0066] In the present application, the term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the core metabolic machinery of the cell, and is typically in the form of a circular double-stranded DNA molecule. These elements can be autonomous replication sequences, genome integration sequences, phage or nucleotide sequences from any source, linear, circular or supercoiled single-stranded or double-stranded DNA or RNA. Typically, a plasmid contains a functional origin of replication in a host cell (e.g., Escherichia coli), and a selectable marker for detecting host cells containing the plasmid. In some embodiments, a plasmid is a closed-loop DNA molecule. A "co-expression plasmid" refers to different types of plasmids that can be expressed in the same host bacteria.

[0067] In this application, the term "nucleic acid" refers to any linear or sequentially arranged nucleotides and nucleosides, for example, cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. Nucleic acids may include bacterial plasmid vectors, including expression, cloning, cosmid and transformation vectors, and nucleic acids 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 represents a 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 polynucleotides or one or more nucleic acid sequences that are transcribed and translated into polypeptides in vitro or in vivo when placed under the control of appropriate regulatory or control sequences; control sequences, for example, 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, DNA sequences to which one or more transcription factors bind and positively (induce) or negatively (inhibit) alter the promoter activity of a gene, etc.

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

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

[0071] In this application, the terms "DNA", "RNA" or "nucleic acid" or "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 can be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form, as well as in double-stranded or single-stranded form. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been separated from its natural environment. For example, in the context of the present invention, a 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, etc.

[0072] In the present application, "gene" refers to a polynucleotide comprising nucleotides encoding functional molecules, including functional molecules (biologically active RNA) produced only by transcription or functional molecules (such as polypeptides) produced by transcription and translation. 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, which contains regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. "Natural gene" refers to any gene found in nature with its own regulatory sequence. "Chimeric gene" refers to any gene that is a non-natural gene and contains non-natural co-existing regulatory and / or coding sequences. "Endogenous gene" refers to a natural gene located in its natural position in the genome of an organism. "Foreign gene" or "heterologous gene" refers to a gene that is not usually in the host and is introduced into the host by gene transfer.

[0073] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0074] In this application, the term "recombinant" nucleic acid refers to non-natural nucleic acid, for example, by artificial intervention to connect originally separate nucleic acid fragments together to produce a combined nucleic acid fragment with a desired function. Artificial combination is often accomplished by chemical synthesis or by artificial manipulation of nucleic acid segments (e.g., by genetic engineering technology).

[0075] In the present application, the term "introduced" or "introduced into" a cell refers to the transformation of a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into a cell by various methods including but not limited to Agrobacterium transformation, gene gun bombardment, electroporation, PEG transformation, etc., so that the nucleic acid or protein can function in the cell.

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

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

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

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

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

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

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

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

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

[0085] (3) mutation of serine at position 38 to methionine (S38M);

[0086] (4) Lysine 51 mutated to glutamic acid (K51G);

[0087] (5) Lysine 65 mutated to serine (K65S);

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

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

[0090] (8) Asparagine at position 100 mutated to glutamic acid (N100E);

[0091] (9) Leucine 135 mutated to isoleucine (L135I);

[0092] (10) Lysine 198 mutated to isoleucine (K198I);

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

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

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

[0096] (14) Phenylalanine at position 433 mutated to tyrosine (F433Y);

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

[0098] (16) mutation of threonine at position 453 to asparagine (T453N); and

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

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

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

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

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

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

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

[0106] Group 6: serine 38 mutated to methionine (S38M);

[0107] Group 7: valine at position 34 mutated to threonine (V34T);

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

[0109] Group 9: phenylalanine at position 433 mutated to tyrosine (F433Y);

[0110] Group 10: threonine at position 66 was mutated to glycine (T66G);

[0111] Group 11: Aspartate at position 31 is missing (D31-), glutamine at position 325 is mutated to alanine (Q325A), threonine at position 453 is mutated to leucine (T453L), and glutamine at position 490 is mutated to lysine (Q490K);

[0112] Group 12: Aspartate at position 31 is missing (D31-), glutamine at position 325 is mutated to lysine (Q325K), threonine at position 453 is mutated to asparagine (T453N), and glutamine at position 490 is mutated to lysine (Q490K);

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

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

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

[0116] In some embodiments, a recombinant expression vector is provided, which comprises the nucleic acid molecule.

[0117] In some embodiments, a recombinant expression vector is provided that includes a plasmid.

[0118] In some embodiments, a vector cell is provided, which comprises the nucleic acid molecule or the recombinant expression vector. Optionally, the vector cell comprises an Escherichia coli cell.

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

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

[0121] In some embodiments, there is provided use of the L-aspartate-α-decarboxylase mutant or the vector cell 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 of the carrier cell to catalyze a substrate to prepare β-alanine.

[0123] In a non-limiting manner, the gene encoding the L-aspartate-α-decarboxylase shown in SEQ ID No. 1 is modified by site-directed mutagenesis, thereby improving the enzymatic activity of the L-aspartate-α-decarboxylase encoded thereby, and significantly improving the catalytic performance of the genetically engineered bacteria expressing the ADC mutation, thereby having a higher industrial application value.

[0124] The present invention obtains a series of ADC enzyme mutants by performing site-directed mutagenesis on insect-derived L-aspartate-α-decarboxylase (ADC). The mutant recombinant plasmid is transformed into Escherichia coli, and the ADC enzyme activity of the strain obtained after shake flask fermentation is increased by 5%-43%. The mutant strain is applied to the preparation of β-alanine, and the catalytic efficiency is increased by 33%, which is more suitable for industrial production needs.

[0125] The molecular biology experiments in the examples include preparation of host competent cells, construction of plasmids, restriction digestion and ligation of genes, plasmid transformation, PCR gene amplification and other experimental techniques, which are mainly carried out with reference to the Molecular Cloning Experiment Guide (3rd edition).

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

[0127] The amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1 was subjected to random mutation at the site, and the gene sequence encoding the mutant of L-aspartate-α-decarboxylase after the site mutation was connected to the pET28a plasmid to prepare a recombinant plasmid. The obtained recombinant plasmid was transformed into Escherichia coli DH5α competent cells, and a plate containing kanamycin was coated for screening. The next day, positive clones were picked, plasmids were extracted after test tube culture, and these plasmids were re-electrotransformed into E.coli BL21 (DE3) competent cells to obtain a mutant cell library that can be used for expression, and the mutant cells obtained by culture were used to express L-aspartate-α-decarboxylase mutants, and L-aspartate-α-decarboxylase mutants with higher enzyme activity were screened, and mutant strains with significantly improved enzyme activity were sequenced to determine the mutation site, and the amino acid sequence of the L-aspartate-α-decarboxylase mutant was determined according to the mutation site of the gene to obtain the L-aspartate-α-decarboxylase mutant shown in Table 1 below.

[0128] The enzyme activity detection method of L-aspartate-α-decarboxylase mutants is as follows: first, single clone colonies are selected and grown in LB medium containing kanamycin and 0.2% isopropyl β-d-1-thiogalactopyranoside (IPTG) at 30°C for 20-24 hours, then the supernatant is removed by centrifugation, the cells are resuspended in buffer (KPB, pH7.0, 50mM) at a concentration of 10% (w / v) and ultrasonically disrupted to obtain the supernatant containing cell lysate.

[0129] Under the conditions of pH 6.5 and temperature 37°C, 50 μL of the above cell lysate was incubated in 50 mL of a 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, with mechanical stirring 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 the ADC activity, and the volume of sulfuric acid aqueous solution used in the reaction was recorded (recorded as "the volume of sulfuric acid aqueous solution used corresponding to ADC enzyme activity" in Table 1). The larger the volume, the higher the ADC enzyme activity. The results of enzyme activity detection can be found in Table 1.

[0130] Table 1

[0131]

[0132] From the results in Table 1, it can be seen that the enzyme activity of the provided L-aspartate-α-decarboxylase mutant is increased by 5%-43% compared with the enzyme activity of the non-mutated L-aspartate-α-decarboxylase.

[0133] The amino acid sequence of SEQ ID No.1 is as follows:

[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 mutants

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

[0166] The high performance liquid chromatograph was Waters 2695-PDA996, ​​the chromatographic column was Phenomenex Luna C18 (250 mm × 4.6 mm × 5 μm), and 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; the flow rate was 0.8 mL / min; the detection wavelength was 360 nm; the column temperature was 30 °C; 2,4-dinitrofluorobenzene (AR) was pre-column derivatized, 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 ADC12 mutant of 8% is similar to that of the initial ADC enzyme of 12%, and the catalytic efficiency of the mutated ADC enzyme is increased by 33%.

[0173] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. An L-aspartate-α-decarboxylase mutant, characterized in that: Compared with the amino acid sequence of L-aspartate-α-decarboxylase shown in SEQ ID No. 1, the L-aspartate-α-decarboxylase mutant comprises amino acid mutations at one or more of the following sites: 31st, 34th, 38th, 51st, 65th, 66th, 87th, 100th, 135th, 198th, 325th, 433rd, 453rd and 490th.

2. The L-aspartate-α-decarboxylase mutant according to claim 1, characterized in that The L-aspartate-α-decarboxylase mutant includes the following mutations or any suitable multiple mutations: (1) Aspartic acid at position 31 is missing; (2) Valine at position 34 mutated to threonine; (3) Serine 38 mutated to methionine; (4) Lysine 51 mutated to glutamic acid; (5) Lysine 65 mutated to serine; (6) Threonine at position 66 mutated to glycine; (7) Threonine at position 87 mutated to glutamine; (8) Asparagine at position 100 mutated to glutamic acid; (9) Leucine at position 135 mutated to isoleucine; (10) Lysine 198 mutated to isoleucine; (11) Glutamine at position 325 was mutated to alanine; (12) Glutamine at position 325 mutated to glutamic acid; (13) Glutamine at position 325 mutated to lysine; (14) Phenylalanine at position 433 mutated to tyrosine; (15) Threonine at position 453 was mutated to leucine; (16) mutation of Thr453 to Asparagine; and (17) Glutamine at position 490 was mutated to lysine.

3. The L-aspartate-α-decarboxylase mutant according to claim 1 or 2, characterized in that The L-aspartate-α-decarboxylase mutant includes one of the following amino acid mutation groups: Group 1: Lysine 51 mutated to glutamic acid; Group 2: Lysine 65 mutated to serine; Group 3: Threonine at position 87 was mutated to glutamine; Group 4: Lysine 198 mutated to isoleucine; Group 5: leucine at position 135 mutated to isoleucine; Group 6: Serine 38 mutated to methionine; Group 7: Valine at position 34 mutated to threonine; Group 8: Asparagine at position 100 mutated to glutamic acid; Group 9: phenylalanine at position 433 mutated to tyrosine; Group 10: Threonine at position 66 was mutated to glycine; Group 11: Aspartic acid at position 31 is missing, 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 missing, 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 missing, 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 at position 31 is deleted, 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.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a gene encoding the L-aspartate-α-decarboxylase mutant according to any one of claims 1 to 3. A recombinant expression vector comprising the nucleic acid molecule according to claim 4 .

6. The recombinant expression vector according to claim 5, characterized in that The recombinant expression vector includes a plasmid.

7. A vector cell comprising the nucleic acid molecule of claim 4 or the recombinant expression vector of claim 5 or 6, optionally, the vector cell comprises an Escherichia coli cell.

8. A method for producing an L-aspartate-α-decarboxylase mutant, comprising the steps of: culturing the vector cell according to claim 7; and isolating the L-aspartate-α-decarboxylase mutant from the obtained culture.

9. Use of the L-aspartate-α-decarboxylase mutant according to any one of claims 1 to 3 or the vector cell according to claim 7 in the preparation of β-alanine.

10. A method for preparing β-alanine, characterized in that: The method comprises the following steps: using the L-aspartate-α-decarboxylase mutant according to any one of claims 1 to 3 or the culture of the carrier cell according to claim 7 to catalyze the substrate to prepare β-alanine.

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