Process for the enzymatic synthesis of n-methyl-beta-alanine

Abstract

The application discloses a new method for synthesizing N-methyl-beta-alanine by catalysis of aspartate ammonia-lyase, and comprises the following steps: taking acrylic acid and methylamine as substrates, and using aspartate ammonia-lyase or a mutant thereof with an amino acid sequence shown in SEQ ID NO:1 to catalyze a synthesis reaction to obtain N-methyl-beta-alanine. The application provides a biological catalysis path for the production of N-methyl-beta-alanine.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically, it relates to a method for the synthesis of N-methyl-β-alanine catalyzed by aspartate aminopyrate synthase. Background Technology

[0002] N-Methyl-β-alanine, also known as 3-methylaminopropionic acid, is a derivative of β-alanine. N-Methyl-β-alanine and its derivatives have various applications in multiple fields. For example, N-methyl-β-alanine can be used as a pharmaceutical intermediate; its derivative, ethyl 3-(methylamino)propionate, is a key intermediate in the antithrombotic drug dabigatran ester. The methylamino group in its structure may enhance drug activity by regulating the metabolic properties of the drug molecule or improving its stability. N-Methyl-β-alanine can also be used to synthesize hapten 3 (J. Agric. Food Chem. 1994, 42, 413-422), for highly sensitive and rapid enzyme-linked immunosorbent assay (ELISA) of pesticide residues. N-Methyl-β-alanine can be used as an intermediate in the synthesis of daily chemical products, such as N-acyl-N-methyl-β-alanine, hydroxy fatty acid acyl-N-methyl-β-alanine, N-myristoyl-N-methyl-β-alanine ester, N-cyclohexyl-N-methyl-β-alanine, and N-lauroyl-N-methyl-β-alanine. Currently, there is limited research on the direct functions of N-methyl-β-alanine, and existing preparation methods for this component are also relatively scarce.

[0003] The patent document with publication number CN112209844A discloses a method for preparing an N-methyl-β-alanine derivative. The method includes the following steps: reacting acrylic acid and N-methylbenzylamine in an ethanol solution to generate compound 1; and subjecting compound 1 to debenzylation by hydrogenation under atmospheric pressure with 10% palladium on carbon catalysis to generate the target compound.

[0004] Patent document CN113548977A discloses a method for preparing an N-methyl-β-alanine derivative, comprising the following steps: acylation reaction of compound RCOOH with oxalyl chloride to prepare a first reaction solution; addition of methylamine to the first reaction solution to carry out a first substitution reaction to prepare a second reaction solution; addition of sodium hydride to the second intermediate reaction solution, stirring, and then addition of chloropropionic acid or bromopropionic acid to carry out a second substitution reaction to prepare the N-methyl-β-alanine derivative.

[0005] Patent document JP3107426B2 discloses a method for preparing N-methyl-β-alanine. The method involves adding β-methylaminopropionitrile dropwise to an aqueous solution containing additives at a reaction temperature below 80°C, followed by hydrolysis. The additives include dilute hydrochloric acid, dilute sulfuric acid, sodium hydroxide, potassium hydroxide, hydrochloric acid, barium chloride, etc.

[0006] As mentioned above, N-methyl-β-alanine is currently mainly produced industrially through chemical synthesis. Chemical synthesis methods often require strong bases and acids, high temperatures, and high pressures, and involve numerous side reactions, cumbersome product purification, and environmental pollution. With the rapid development of biotechnology, the biosynthesis of N-methyl-β-alanine aligns with future development trends. Summary of the Invention

[0007] Referring to patent document CN110791493B, the inventors discovered as early as 2018 that aspartate ammonia-lyase (Aspartate ammonia-lyase, abbreviated AspB, also known as aspartic acid lyase or aspartate ammonia lyase) derived from Bacillus sp. YM55-1 has the function of catalyzing the reaction of substrates acrylic acid or acrylonitrile with ammonia to produce β-alanine. Currently, an industrial aspartate ammonia-lyase (company number B001), which is a multi-site mutant of this aspartate ammonia-lyase (amino acid sequence shown in SEQ ID NO: 1), is being used for the industrial production of β-alanine. Inspired by the catalytic function of aspartate aminopyrase, we attempted to use this enzyme to catalyze the reaction of acrylic acid with methylamine to investigate whether N-methyl-β-alanine could be synthesized. The desired results were achieved, thus developing a biosynthetic route for N-methyl-β-alanine. Furthermore, we modified the initial aspartate enzyme B001 using site-directed mutagenesis, obtaining the aspartate enzyme mutant B027 with significantly enhanced catalytic activity, thereby improving its feasibility for industrial application. Specifically, this invention includes the following technical solutions.

[0008] The first aspect of this invention provides a method for the production of N-methyl-β-alanine catalyzed by aspartate aminopyruvate synthase, characterized by comprising the following steps: using acrylic acid and methylamine as substrates, and using aspartate aminopyruvate synthase (B001) with the amino acid sequence shown in SEQ ID NO: 1 or a conserved variant polypeptide thereof to catalyze a synthetic reaction to obtain N-methyl-β-alanine. The conserved variant polypeptide is a mutant with amino acid sequence exhibiting 90% or more homology to SEQ ID NO: 1, preferably 92% or more homology, preferably 95% or more homology, preferably 96% or more homology, preferably 97% or more homology, preferably 98% or more homology, and more preferably 99% or more homology, and with enhanced enzyme activity.

[0009]

[0010] MNTDVRIEKDFLGEKEIPKDAYYGVQTIRATENFPITGYRFVVELIKSLGIVKKSAALANMEVGLLDKEVGQYIVKAADEVIEGKWNDQFIVDPIQGGAGTSINMNANEVIANRALEL MGEEKGNYSKISPNSHVNMSQSTNDAFPTATHIAVLSLLNQLIETTKYMQQEFMEKADEFAGVIKMGRIHLQDAVPILLGQEFEAYAHVIARDIERIAYTRNNLYDINMGATAVGTGL NADPEYISIVTEHLAKFSGHPLRSAQHLVDATQNTDCYTEVSSALKVCMINMSKIANDLRLMASGPRAGLSEIVLPARQPGSSIMPGMVTPVMPEVMNQVAFQVFGNDLTITSASEAG QFELNVMEPVLFFNLIQSISIMTNVFKSFTENCLKGIKANEERMKEYVEKSIGIITAINPHVGYETATKLAREAYLTGESIRELCIKYGVLTEEQLNEILNPYEMTHPGIAGRK (SEQ ID NO: 1).

[0011] In one embodiment, the above-mentioned conserved variant polypeptide is a mutant of the amino acid sequence of aspartate amino acid synthase selected from the following sites in SEQ ID NO: 1: E14L, K68P, F145I, K252W, M321I and / or V360F.

[0012] Preferably, the above-mentioned conserved mutant polypeptide is an E14L, K68P, F145I, K252W, M321I, V360F mutant of aspartate amino acid lyase (named B027 in this document), and its amino acid sequence is shown in SEQ ID NO: 3.

[0013] MNTDVRIEKDFLGLKEIPKDAYYGVQTIRATENFPITGYRFVVELIKSLGIVKKSAALANMEVGLLDPEVGQYIVKAADEVIEGKWNDQFIVDPIQGGAGTSINMNANEVIANRALEL MGEEKGNYSKISPNSHVNMSQSTNDAIPTATHIAVLSLLNQLIETTKYMQQEFMEKADEFAGVIKMGRIHLQDAVPILLGQEFEAYAHVIARDIERIAYTRNNLYDINMGATAVGTGL NADPEYISIVTEHLAWFSGHPLRSAQHLVDATQNTDCYTEVSSALKVCMINMSKIANDLRLMASGPRAGLSEIVLPARQPGSSIIPGMVTPVMPEVMNQVAFQVFGNDLTITSASEAG QFELNFMEPVLFFNLIQSISIMTNVFKSFTENCLKGIKANEERMKEYVEKSIGIITAINPHVGYETATKLAREAYLTGESIRELCIKYGVLTEEQLNEILNPYEMTHPGIAGRK (SEQ ID NO: 3).

[0014] Optionally, in the above reaction system, aspartate aminotransferase B001 or its conserved variant polypeptide / mutant such as B027 is in enzyme form or expressed in microbial cell form.

[0015] In one embodiment, the concentration of the substrate acrylic acid in the above reaction system is 100 g / L or more, more preferably 200 g / L or more.

[0016] Furthermore, the pH value of the reaction system is 6.5-9.0, preferably 7.0-8.5, preferably 7.2-8.0, and more preferably around 7.5.

[0017] The reaction temperature is 25℃~55℃, preferably 30℃~50℃, more preferably 35℃~45℃, and even more preferably around 40℃.

[0018] It should be understood that in this article, when describing numerical characteristics, the terms "around" or "approximately" mean that the expressed number may have an error range or fluctuation range of ±10%, ±9%, ±8%, ±7%, ±6%, or ±5%.

[0019] The above method can be further extended to include the following steps: using the product N-methyl-β-alanine as a raw material, an N-methyl-β-alanine derivative is obtained through a chemical synthesis reaction. The N-methyl-β-alanine derivative is N-acyl-N-methyl-β-alanine or hydroxy fatty acid acyl-N-methyl-β-alanine, for example, a compound selected from the group consisting of N-myristoyl-N-methyl-β-alanine ester, N-lauroyl-N-methyl-β-alanine, N-cyclohexyl-N-methyl-β-alanine, etc.

[0020] For example, the N-methyl-β-alanine derivative is N-lauroyl-N-methyl-β-alanine, which is prepared by acylation reaction using N-methyl-β-alanine and lauroyl chloride as raw materials.

[0021] A second aspect of this invention provides an aspartate amino acid lyase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3 and is named B027 herein.

[0022] A third aspect of the present invention provides a gene encoding the above-mentioned aspartate aminotransferase mutant SEQ ID NO: 3.

[0023] Preferably, the nucleotide sequence of the above-mentioned encoding gene is shown in SEQ ID NO: 4.

[0024] Accordingly, the present invention further provides a DNA molecule comprising the above-described coding gene and an upstream promoter such as a Lac promoter or a T7 promoter and / or a downstream terminator such as a T7 terminator, for example, an expression cassette / expression box of an aspartate aminotransferase mutant SEQ ID NO: 3.

[0025] A fourth aspect of the present invention provides a recombinant plasmid, which is an expression plasmid formed by cloning the above-mentioned coding gene on a plasmid vector.

[0026] The plasmid vectors mentioned above can be selected from the pET series (e.g., pET22b, pET24a, pET28a), pMAL series, pGEX series, pQE series, pBAD series, pCAI series, pSH series, pRSFDuet series, or other vectors.

[0027] A fifth aspect of the present invention provides a microbial engineered bacterium that expresses the above-described coding gene.

[0028] The aforementioned engineered microorganisms can be transformants that have been transformed with the aforementioned recombinant plasmids; or positive recombinant bacteria that have cloned the aforementioned encoding genes into the host genome through gene editing technology.

[0029] Preferably, the host bacteria of the above-mentioned engineered microorganisms are microorganisms with rapid proliferation rates and suitable for expressing exogenous recombinant proteins, such as Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Vibrio natriureticis, Candida magnolius, Yersinia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae. Escherichia coli is a preferred microorganism, and more preferably Escherichia coli BL21(DE3).

[0030] In one embodiment, the transformation of the recombinant plasmid can be performed by conventional chemical transformation or electrotransformation into competent cells; the gene editing technology is selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0031] This invention develops a novel biotransformation process for the enzymatic synthesis of N-methyl-β-alanine. Aspartate enzyme and its mutants can catalyze the reaction of acrylic acid and methylamine to obtain N-methyl-β-alanine, overcoming many drawbacks of chemical synthesis methods and solving the environmental pollution problem of chemical synthesis methods. Moreover, the purity of the product is greater than 99%, and it can be used to prepare its derivatives or downstream products. Therefore, the new process developed in this invention has promising prospects for industrial application. Attached Figure Description

[0032] Figure 1 The structural map of the initial aspartate aminotransferase gene expression plasmid pET28a-B001 constructed in this invention is shown.

[0033] Figure 2 The diagram shows the working principle of high-throughput screening of mutants in Example 3.

[0034] Figure 3 The HPLC chromatogram of the sample after 6 h of reaction of acrylic acid and methylamine catalyzed by mutant B027 is shown. Detailed Implementation

[0035] The use of biosynthesis technology with mild reaction conditions to replace traditional organic synthesis methods for producing high-value-added fine chemical products represents an industrial trend and development direction.

[0036] Inspired by the function of aspartate aminopyrase catalyzing the synthesis of β-alanine (see CN110791493B) developed many years ago, we changed the reaction substrate of aspartate aminopyrase B001 and tried using acrylic acid and methylamine as substrates. The results confirmed the production of N-methyl-β-alanine, indicating that the biosynthesis of N-methyl-β-alanine can be achieved.

[0037] The aspartate aminotransferase B001 used in the experiment is a mutant of the Bacillus YM55-1-derived aspartate aminotransferase (Uniport ID accession number Q9LCC6.1) reported in CN110791493B, which has been industrially applied.

[0038] Considering that the initial aspartate amino acid lyase B001 exhibits low enzyme activity in the reaction of acrylic acid and methylamine to synthesize N-methyl-β-alanine, we further modified its amino acid sequence to obtain mutant enzymes with significantly enhanced catalytic activity in order to improve the feasibility of industrial production. To this end, we modified the initial aspartate enzyme using site-directed mutagenesis, obtaining several mutants with increased enzyme activity. Among them, the mutants E14L, K68P, F145I, K252W, M321I, and V360F (SEQ ID NO: 3) showed the highest enzyme activity.

[0039] In this document, the terms “initial (type) aspartate (amino lyase)” and “starting enzyme” have the same meaning and refer to aspartate amino lyase SEQ ID NO: 1 (i.e. B001).

[0040] Correspondingly, the terms "aspartate aminolysinase mutant," "mutant aspartate aminolysinase," "mutant," and "mutant enzyme" have the same meaning, all referring to mutants with increased activity compared to aspartate aminolysinase B001, such as polypeptide SEQ ID NO: 3. For the sake of brevity and convenience, the original aspartate aminolysinase and its mutants may be collectively referred to as "aspartate aminolysinase" in this invention, as long as it is not confused with the original enzyme B001.

[0041] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated into another aliphatic residue or another nonpolar residue.

[0042] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0043] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of engineered aspartate aminotransferase (AspB). Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0044] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, modified engineered aspartate amino acid synthase (AspB) includes inserting one or more amino acids into naturally occurring aspartate amino acid synthase (AspB) and into other modified aspartate amino acid synthase (AspB) polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl terminus or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0045] The aspartate amino acid lyase mutant of the present invention has 468 amino acids and a well-defined sequence. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassette (DNA molecule) containing these genes and plasmid, as well as transformants containing the plasmid.

[0046] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0047] In order to optimally express aspartate aminotransferase SEQ ID NO: 1 and its mutant SEQ ID NO: 3 in microbial hosts, such as Escherichia coli, which is most commonly used in genetic engineering, the present invention has optimized the codons of its expression gene.

[0048] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0049] After codon optimization, the encoding gene of the initial aspartate aminolysin SEQ ID NO: 1 can be the nucleotide sequence SEQ ID NO: 2, while the encoding gene of the aspartate aminolysin mutant SEQ ID NO: 3 can be the nucleotide sequence SEQ ID NO: 4.

[0050] As used herein, “DNA molecule” and “expression cassette” refer to a gene expression system containing all the necessary elements required to express the target aspartate aminotransferase SEQ ID NO: 1 or 3, typically including the following elements: a promoter, a gene sequence encoding aspartate aminotransferase such as SEQ ID NO: 1 or 3 SEQ ID NO: 2 or 4, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc.; these biological elements are operatively linked.

[0051] As used herein, “operable linking” refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO: 2 or 4, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operable linking” the promoter region to the nucleic acid sequence.

[0052] Preferably, the recombinant plasmid (or nucleic acid construct) includes one or more copies, two or more copies, preferably four or more copies, six or more copies, or eight or more copies of the aspartate aminotransferase, for example, the gene encoding SEQ ID NO: 3.

[0053] When used as a biocatalyst for the preparation of N-methyl-β-alanine, the aspartate aminotransferase of the present invention can be in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on a carrier; the bacterial cell form includes live cells and dead cells.

[0054] In the field of biocatalysis, it is well known that compared with free enzyme methods, the application of immobilized enzyme technology has advantages such as simplified production processes and improved production efficiency. Furthermore, because the enzyme can be used multiple times and its stability is improved, the productivity per unit enzyme is effectively increased. Secondly, immobilized enzymes are easily separated from substrates and products, simplifying purification processes, resulting in higher yields and better product quality.

[0055] Those skilled in the art will readily understand that bacterial cells themselves are a natural form of enzyme immobilization, and can be used as an enzyme preparation for catalytic reactions without the need for disruption or even extraction and purification. Since the reaction substrates and products can easily cross the bacterial cell membrane—the biological barrier—disruption of the cells is unnecessary, which is economically advantageous.

[0056] On the other hand, compared with the catalysis of isolated enzymes, the present invention can provide a continuous and inexhaustible supply of enzymes or other substances through simple microbial fermentation, without the need for further extraction, purification, or enzyme separation. The economic benefits are obvious, creating conditions for industrial application.

[0057] Since the product N-methyl-β-alanine, which is produced by the reaction of aspartic acid and its mutants with acrylic acid and methylamine, has a purity of more than 99%, it can be directly used as a raw material to synthesize high-value-added derivatives such as N-myristoyl-N-methyl-β-alanine ester, N-cyclohexyl-N-methyl-β-alanine, and N-lauroyl-N-methyl-β-alanine through chemical reactions.

[0058] In one embodiment, the derivative of N-methyl-β-alanine is N-lauroyl-N-methyl-β-alanine, which can be prepared by acylation of N-methyl-β-alanine and lauroyl chloride.

[0059] In one exemplary embodiment, the preparation of N-lauroyl-N-methyl-β-alanine includes the following steps:

[0060] (1) Raw material pretreatment

[0061] Dissolve N-methyl-β-alanine in a suitable organic solvent (such as dichloromethane) and stir until dissolved. Add a suitable amount of base (such as triethylamine, molar ratio of approximately 2.2:1) to neutralize the carboxylic acid group and activate the amine group.

[0062] (2) Acylation reaction

[0063] Under ice-water bath conditions, lauroyl chloride (molar ratio approximately 1.1:1) was slowly added dropwise while maintaining the reaction temperature at 0–10°C to avoid the formation of byproducts. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 6–7 hours (the reaction progress was monitored by TLC).

[0064] (3) Post-processing

[0065] After the reaction was complete, the mixture was poured into ice water to quench excess acyl chloride, and the organic phase was collected in layers. The aqueous phase was extracted 2-3 times with an organic solvent (such as dichloromethane), and the organic phases were combined. The organic phase was washed with 5% NaHCO3 solution to remove residual acid, then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain the crude product.

[0066] (4) Purification

[0067] The crude product was eluted by column chromatography (silica gel column, eluent: petroleum ether / ethyl acetate = 4:1 → 2:1) to obtain the N-lauroyl-N-methyl-β-alanine.

[0068] The N-lauroyl-N-methyl-β-alanine prepared according to the above method can be used as a surfactant in the field of cosmetics or skin care products.

[0069] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0070] Example

[0071] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0072] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0073] Materials and methods

[0074] In the embodiments, the whole gene synthesis, primer synthesis, and sequencing were all performed by Nanjing GenScript Biotech Co., Ltd. To optimally express aspartate aminolysase and its mutants in microbial hosts, such as *E. coli*, which is the most commonly used host in genetic engineering, the codons of the expression gene were optimized in this invention.

[0075] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0076] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0077] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0078] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K₂HPO₄•3H₂O, 2.31 g / L KH₂PO₄, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium is supplemented with 20 g / L agar powder.)

[0079] The HPLC detection conditions for the substrate acrylic acid and the product N-methyl-β-alanine are as follows:

[0080] Detection instrument and chromatographic column: Agilent (Shanghai Yuexu OAA chromatographic column)

[0081] Mobile phase A (100%): 10 mM, pH 2.5 potassium dihydrogen phosphate

[0082] Flow rate: 1 mL / min

[0083] Column oven: 20℃

[0084] Detection time: 20 min

[0085] Detection wavelength: 210nm

[0086] Injection volume: 50 μL

[0087] N-methyl-β-alanine RT: 3.994 min

[0088] For ease of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number can share the same number, that is, the same number can refer to different biological forms in different descriptive scenarios. For example, B001 can represent the initial enzyme SEQ ID NO: 1 number, the initial enzyme-encoding gene SEQ ID NO: 2 number, and the initial enzyme SEQ ID NO: 1 expression strain (WT).

[0089] Example 1: Construction of recombinant Escherichia coli expressing initial aspartate aminopyrease

[0090] 1.1 For the initial aspartate amino acid lyase B001, based on its amino acid sequence SEQ ID NO: 1, codon optimization according to E. coli preference was performed to obtain the coding gene sequence SEQ ID NO: 2. The entire gene was synthesized into this nucleotide sequence, and restriction endonuclease sites Nde I and Xho I were designed at both ends of the gene. The gene was subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-B001. The constructed plasmid map is shown below. Figure 1 .

[0091] 1.2 The recombinant plasmid pET24a-B001 was transformed into host Escherichia coli BL21(DE3) competent cells (Invitrogen) by electroporation to obtain recombinant Escherichia coli expressing initial aspartate aminolysis enzyme, designated B001.

[0092] It should be understood that the recombinant plasmid pET24a-B001 can also be transformed into other host bacteria such as Bacillus subtilis and Pichia pastoris to express the initial aspartate aminotransferase.

[0093] Example 2: Construction of the B001 mutant library

[0094] Based on the stereochemical model of the initial aspartate amino acid synthase B001, molecular docking was performed with the substrate acrylic acid. The amino acids within the 8 Å range for enzyme-substrate binding were rationally designed, and the mutation point with higher affinity for enzyme-substrate binding after mutation was determined by molecular dynamics simulation. We designed and selected amino acids at positions 26, 98, 99, 100, 101, 104, 105, 108, 132, 136, 140, 142, 145, 146, 187, 188, 190, 192, 231, 316, 317, 318, 319, 320, 321, 324, 325, 326, 331, 357, 358, 360, and 362 of aspartate amino acid synthase for saturation mutagenesis.

[0095] Simultaneously, by rationally analyzing the surface charge of aspartate aminotransferase, its optimal pH value was modified to improve its catalytic activity under neutral or weakly alkaline conditions. The optimal pH values ​​were then adjusted, and the following positions of aspartate aminotransferase were selected: positions 4, 7, 14, 15, 19, 40, 54, 68, 76, 85, 87, 125, 128, 132, 158, 165, and 16... A total of 38 amino acid positions, including positions 9, 173, 190, 217, 220, 252, 259, 262, 303, 314, 315, 386, 389, 392, 397, 399, 426, 449, 450, 451, 455, and 461, were subjected to saturation mutations.

[0096] Finally, using DLKcat prediction, the following sites were selected for site-directed mutagenesis: V265M, E276M, S278F, T410W, S278W, L264D, L264R, Q269M, L264M, V416M, S279W, V265Y, N287W, T275I, V277H, M137T, G354D, S279Q, L264H, and G97Y.

[0097] For example, taking the E14 mutation as an example, the PCR primer pair is designed as follows:

[0098] Forward primer E14-F:

[0099]

[0100] Reverse primer E14-R: TCCTAAAAAGTCTTTCTCAATACGAACATC.

[0101] The 50 μL PCR reaction system includes: 100 ng plasmid template pET28a-B001, 0.4 μM primer pair E14-F and E14-R, 25 μL PrimeSTAR Max Premix (2X), and the remainder is made up with ddH2O.

[0102] The PCR reaction conditions were: 98℃ for 2 min; 23 cycles (98℃ for 10 s, 60℃ for 15 s, 72℃ for 30 s / kb); 72℃ for 10 min. After PCR, 5 μL of the PCR product was run on a gel to confirm the band size. If the band size was correct, the plasmid template was digested with DpnI to obtain the mutant plasmid pET24a-E14, which was then electrotransformed into E. coli BL21(DE3) to obtain a saturated mutant library.

[0103] Example 3: High-throughput screening of mutant libraries

[0104] 3.1 Culture using 96-well plates

[0105] (1) Take a 96-well plate and add self-induction medium ZYM (400 μL) (containing kanamycin 50 μg / mL); pick single colonies from LB kan agar plates into the 96-well plate with a toothpick and incubate at 30℃ and 280 rpm for 16 h;

[0106] (2) Take 100 μL of culture medium and add an equal volume of sterile 50% glycerol, then freeze at -20℃ for short-term storage. Centrifuge the remaining culture medium and collect the bacterial cells for screening.

[0107] 3.2 Screening with a 96-well plate

[0108] (1) Take out the 96-well plate, resuspend the frozen-thawed bacterial body in 200 μL of water, add 200 μL of reaction solution (360 g / L acrylic acid, pH adjusted to 7.5 with methylamine), place the plate on a shaker, react at 40℃ and 280 rpm for 18 h, centrifuge and take 15 μL of supernatant, add 150 μL of colorimetric solution and incubate at 37℃ for 3 h for color development; then detect OD. 438 value.

[0109] (2) The formula of the colorimetric solution is shown in the table below:

[0110]

[0111] (3) Screening principle

[0112] like Figure 2As shown, using acrylic acid and methylamine as substrates, N-methylβ-alanine is catalyzed by AspB (aspartate aminotransferase) to form N-methylβ-alanine. N-methylβ-alanine is then catalyzed by BAPAT (β-alanine pyruvate transaminase) to form N-methyl-L-alanine. N-methyl-L-alanine is further catalyzed by AlaDH (alanine dehydrogenase) to form pyruvate and methylamine. In this reaction process, NAD... + The WST-1 is reduced to NADH, and the generated NADH, under the action of GsDI (flavodiase), reduces WST-1 to form yellow formazan. The maximum absorption wavelength corresponding to yellow is at OD438. Therefore, the larger the OD438 value, the more complete the reaction is, which also indirectly indicates that the catalytic effect of Aspartate aminotransferase (ASTB) is better.

[0113] (3) Screening results

[0114] Through screening of 72 saturated mutant libraries and 20 site-directed mutants, the mutant with the highest enzyme activity, designated B027, was finally selected based on colorimetric results and HPLC data. Compared with the naïve aspartate aminotransferase B001, this mutant contains the following mutations: E14L, K68P, F145I, K252W, M321I, and V360F.

[0115] Example 4: Catalytic performance test of aspartate amino acid lyase

[0116] The initial aspartate aminotransferase B001 and mutant strain B027 were inoculated into LB medium (test tubes) at a 1% v / v inoculation rate and cultured at 37℃ and 220 rpm for 16 h to obtain seed culture. The seed culture was inoculated into 400 mL LB medium at a 1% v / v inoculation rate and cultured at 37℃ and 220 rpm until the logarithmic growth phase was reached. 0.5 mM IPTG inducer was added and cultured at 18℃ and 120 rpm for 16 h to induce enzyme production. The cells were then centrifuged at 4℃ and 4000 rpm for 10 min to collect the wet cells.

[0117] 5 mL reaction system: 200 g / L acrylic acid, pH adjusted to 7.5 with methylamine; 20 g / L wet bacterial cells, reacted at 40℃ and 220 rpm for 6 h, and then samples were taken for HPLC detection of N-methylβ-alanine content.

[0118] The test results are shown in Table 1:

[0119] Table 1. Catalytic results of aspartate aminopyrase mutant

[0120]

[0121] The results showed that, compared with the initial aspartate aminotransferase B001, the mutant aspartate aminotransferase B027 significantly increased the concentration of the product N-methyl-β-alanine. The HPLC chromatogram of the sample taken after 6 hours of reaction catalyzed by the mutant B027 is shown below. Figure 3 As shown.

[0122] Furthermore, after separation and purification, the purity of N-methyl-β-alanine fermentation products from different strains was all above 99%, indicating that the N-methyl-β-alanine prepared by the method of the present invention has extremely high purity, no by-products, and can be directly used to prepare its derivatives or other downstream products, such as N-acyl-N-methyl-β-alanine.

[0123] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for producing N-methyl-β-alanine catalyzed by aspartate aminolyase, characterized in that, The method includes the following steps: using acrylic acid and methylamine as substrates, an aspartate amino acid lyase with the amino acid sequence shown in SEQ ID NO: 1 or its conserved variant polypeptide is used to catalyze a synthesis reaction to obtain N-methyl-β-alanine, wherein the conserved variant polypeptide is a mutant with more than 90% homology to SEQ ID NO: 1 in amino acid sequence and enhanced enzyme activity.

2. The method as described in claim 1, characterized in that, The conserved variant polypeptide is a mutant of the amino acid sequence of aspartate amino acid lyase selected from the following sites in SEQ ID NO: 1: E14L, K68P, F145I, K252W, M321I and / or V360F.

3. The method as described in claim 1, characterized in that, The conserved mutant polypeptide is an E14L, K68P, F145I, K252W, M321I, V360F mutant of aspartate amino acid lyase, and its amino acid sequence is shown in SEQ ID NO:

3.

4. The method as described in claim 1, characterized in that, In the reaction system, the aspartate aminotransferase or its conserved variant polypeptide / mutant is in enzyme form or expressed in microbial cell form.

5. The method as described in claim 1, characterized in that, The concentration of acrylic acid substrate in the reaction system is above 100 g / L to above 200 g / L; the pH value of the reaction system is 6.5-9.0; and the reaction temperature is 25℃~55℃.

6. The method as described in claim 1, characterized in that, The process further includes the following steps: using the product N-methyl-β-alanine as a raw material, an N-methyl-β-alanine derivative is obtained through a chemical synthesis reaction, wherein the N-methyl-β-alanine derivative is N-acyl-N-methyl-β-alanine or hydroxy fatty acid acyl-N-methyl-β-alanine.

7. An aspartate aminolysin mutant, characterized in that, The amino acid sequence of the aspartate aminopyrase mutant is shown in SEQ ID NO:

3.

8. The gene encoding the aspartate aminolysin mutant SEQ ID NO: 3 as described in claim 7.

9. A recombinant plasmid, characterized in that, It is an expression plasmid formed by cloning the encoding gene as described in claim 8 on a plasmid vector.

10. A type of engineered microbial bacterium, characterized in that, Its expression is the encoding gene as described in claim 8.

Images