Alpha-keto acid decarboxylase mutants, methods of making and using the same

Abstract

The application discloses an alpha-keto acid decarboxylase mutant, a preparation method and application thereof. The amino acid sequence of the alpha-keto acid decarboxylase mutant comprises any one of the following sequences: (1) a sequence which is subjected to R75S and / or I249T mutation on the basis of the sequence shown in SEQ ID NO: 3; or (2) a sequence which is obtained by substituting, deleting or adding one or at least two amino acid residues from the sequence in (1) and is functionally identical or similar to the sequence in (1); or (3) a sequence which has at least 90% sequence identity with the sequence in (1) or (2) and is functionally identical or similar to the sequence in (1). The application introduces mutations in the wild-type alpha-keto acid decarboxylase, improves the catalytic activity of the alpha-keto acid decarboxylase, and further improves the yield of malonic acid, reduces the separation cost, and has high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to α-keto acid decarboxylase mutants, their preparation methods, and applications. Background Technology

[0002] Malonic acid is a three-carbon dicarboxylic acid, a colorless and odorless crystal that is hygroscopic. It was first discovered as a product of the oxidation of malic acid, hence its other name, condensed malic acid. The methylene group at the intermediate position of malonic acid is highly reactive, easily substituted, and readily decarboxylated, making it widely used as an intermediate in pharmaceuticals, pesticides, fragrances, and vitamins. Currently, the main method for producing malonic acid is chemical synthesis from chloroacetic acid and sodium cyanide. This process generates significant amounts of waste, causing considerable pollution, and the byproducts are difficult to treat. Besides chemical methods, the production of malonic acid through bio-fermentation is noteworthy, as this process is environmentally friendly and produces less waste compared to other methods.

[0003] There is no natural biosynthetic pathway for malonic acid in nature. Most of the reported biosynthetic pathways are artificially designed. Although some reports have achieved gram-level production, they are far from meeting the requirements for industrial production. For example, CN113832087A discloses a method for the complete biosynthesis of malonic acid using Escherichia coli. Using Escherichia coli BL21(DE3) as the host, the method co-expresses the phosphoenolpyruvate carboxylase gene and aspartate transaminase gene of Escherichia coli, expresses the aspartate-α-dehydrogenase gene from Corynebacterium glutamicum alone, and co-expresses the succinate semialdehyde dehydrogenase gene of Escherichia coli and the heterologous gene β-alanine pyruvate transaminase gene from Pseudomonas aeruginosa. This constructs a complete biosynthetic pathway for malonic acid via oxaloacetate-aspartate, achieving a yield of 0.74 g / L. Although this is a breakthrough, the complete biosynthetic pathway of malonic acid via oxaloacetate-aspartate is relatively long, and the yield needs further improvement.

[0004] Furthermore, a novel biosynthetic pathway for malonic acid has been reported, which synthesizes malonic acid via the oxaloacetate-malonic acid half-aldehyde-malonic acid route. This biosynthetic pathway is shorter and has significant application potential. For current malonic acid synthesis pathways, increasing yield hinges on using highly active α-keto acid decarboxylases. Therefore, the discovery and design of highly active α-keto acid decarboxylases is of great importance. Summary of the Invention

[0005] In response to the shortcomings of existing technologies and practical needs, this invention provides α-keto acid decarboxylase mutants, their preparation methods, and applications. The invention designs and modifies α-keto acid decarboxylase mutants with higher activity in order to promote the production of malonic acid.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an α-keto acid decarboxylase mutant, wherein the amino acid sequence of the α-keto acid decarboxylase mutant comprises any one of the following sequences:

[0008] (1) A sequence that has undergone R75S and / or I249T mutations based on the sequence shown in SEQ ID NO:3; or,

[0009] (2) A sequence obtained by substituting, deleting, or adding one or at least two amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1); or,

[0010] (3) A sequence that has at least 90% sequence identity with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

[0011] In this invention, a wild-type α-keto acid decarboxylase derived from the genus Brettanomyces bruxellensis was designed and modified by introducing R75S and / or I249T mutations, which can improve catalytic activity and thus increase the yield of malonic acid.

[0012] In this invention, a specific mutation is introduced into wild-type α-keto acid decarboxylase to obtain an α-keto acid decarboxylase mutant, which can improve its catalytic activity. It is understood that, based on the α-keto acid decarboxylase mutant, those skilled in the art can use common technical means in the art to substitute, delete or add one or at least two amino acid residues to obtain other sequences with the same or similar functions.

[0013] In some specific embodiments of the present invention, in addition to introducing the R75S and / or I249T mutations, conserved substitutions of amino acids can be further performed at other sites, resulting in the mutated α-keto acid decarboxylase exhibiting superior substrate specificity. Preferably, the conserved substitution of amino acids preserves the substrate specificity of the α-keto acid decarboxylase mutant of the present invention. It will be apparent to those skilled in the art that such substitutions can occur in regions other than those described above, while still retaining the corresponding activity. Preferably, the conserved substitution variant has at least one conserved substitution of an amino acid. Examples of conserved substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (such as glycine, alanine, serine, threonine, and methionine). The most common amino acid interchanges are G to A; A to G, S; V to I, L, A, T or S; I to V, L or M; L to I, M or V; M to L, I or V; P to A, S or N; F to Y, W or H; Y to F, W or H; W to Y, F or H; R to K, E or D; K to R, E or D; H to Q, N, S; D to N, E, K, R or Q; E to Q, D, K, R or N; S to T or A; T to S, V or A; C to S, T or A; N to D, Q, H or S; Q to E, N, H, K or R, as well as their opposite interchanges. An α-keto acid decarboxylase mutant that has a certain degree of amino acid homology with the above-mentioned α-keto acid decarboxylase mutant, preferably with a homology of 70%-99%, more preferably with a homology of 80%-99%, even more preferably with a homology of 90%-99%, and most preferably with a homology of 99%, should also fall within the scope of protection of this invention.

[0014] SEQ ID NO:3:

[0015] .

[0016] In a second aspect, the present invention provides a nucleic acid molecule that encodes the α-keto acid decarboxylase mutant described in the first aspect.

[0017] In this invention, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0018] Preferably, the nucleic acid molecule includes the sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, or a sequence that hybridizes with SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 and encodes the α-keto acid decarboxylase mutant, or a sequence that has more than 90% identity with SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 and encodes the α-keto acid decarboxylase mutant.

[0019] SEQ ID NO:7:

[0020]

[0021] SEQ ID NO:8:

[0022]

[0023] SEQ ID NO:9:

[0024]

[0025] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0026] In this invention, the recombinant vector includes a cloning vector and an expression vector. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be pRSFDuet-1.

[0027] In some embodiments, the recombinant vector is obtained by replacing the sequence between the NcoI and EcoRI restriction sites of pRSFDuet-1 with the nucleic acid molecule, while leaving the rest of the sequence unchanged.

[0028] Fourthly, the present invention provides recombinant cells containing the recombinant vector described in the third aspect.

[0029] In some embodiments, the recombinant cells are induced (e.g., by IPTG induction) to produce the α-keto acid decarboxylase mutant.

[0030] In some embodiments, the method for constructing the recombinant cells includes: converting the recombinant vector into host cells.

[0031] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., specifically Escherichia coli BL21(DE3), Rosetta(DE3), BL21(DE3)plysS, M15 or Top10f, etc., preferably Escherichia coli BL21(DE3).

[0032] Fifthly, the present invention provides a method for preparing the α-keto acid decarboxylase mutant described in the first aspect, the method comprising:

[0033] The nucleic acid molecule encoding the α-keto acid decarboxylase mutant described in the first aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and purified to obtain the α-keto acid decarboxylase mutant.

[0034] Preferably, the expression vector includes pRSFDuet-1 vector, pETDuet-1 vector, pACYCDuet-1 vector, pTrc99a vector, or pET28a vector, etc.

[0035] Preferably, the host cells include Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, or Yersinia lipolytica.

[0036] Preferably, the Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli Rosetta(DE3), Escherichia coli BL21(DE3)plysS, Escherichia coli M15, or Escherichia coli Top10f, etc.

[0037] Preferably, the method for preparing the nucleic acid encoding the α-keto acid decarboxylase mutant includes: codon optimization of the wild-type α-keto acid decarboxylase gene (SEQ ID NO:1) to obtain the gene shown in SEQ ID NO:2, so as to improve the expression efficiency of the gene; inserting the gene shown in SEQ ID NO:2 into an expression vector; and introducing the mutations R75S and / or I249T into the gene shown in SEQ ID NO:2 using the inserted vector as a template.

[0038] Preferably, the method for introducing mutations can be the Mut Express MultiS Fast Mutagenesis KitV2.

[0039] In this invention, the methods for inducing and culturing recombinant cells and for isolating α-keto acid decarboxylase from the culture can both employ conventional methods in the art. The culture medium used when recombinant cells express α-keto acid decarboxylase can be any culture medium in the art that can enable the recombinant cells to grow and produce the α-keto acid decarboxylase mutant of this invention, such as LB medium.

[0040] In this invention, there are no special requirements for the culture method and culture conditions, as long as the recombinant cells can grow normally and express the α-keto acid decarboxylase mutant.

[0041] SEQ ID NO:1:

[0042]

[0043] SEQ ID NO:2:

[0044]

[0045] In a sixth aspect, the present invention provides the application of the α-keto acid decarboxylase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the recombinant cell described in the fourth aspect, or the method for preparing the α-keto acid decarboxylase mutant described in the fifth aspect in the preparation of malonic acid.

[0046] In a seventh aspect, the present invention provides a method for preparing malonic acid, the method comprising: using the α-keto acid decarboxylase mutant described in the first aspect to catalyze the substrate glucose to obtain malonic acid.

[0047] Preferably, the method for preparing malonic acid specifically includes:

[0048] The nucleic acid molecule encoding the α-keto acid decarboxylase mutant described in the first aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell for fermentation and malonic acid separation and purification to obtain malonic acid.

[0049] Preferably, the fermentation includes an aerobic fermentation stage and an acid production stage.

[0050] Preferably, the fermentation conditions for the aerobic fermentation stage include: dissolved oxygen (DO) greater than 30%, temperature of 35-42℃ (e.g., 36℃, 37℃, 38℃, 39℃, 40℃ or 41℃, etc.), and pH of 6.0-7.2, including but not limited to 6.2, 6.4, 6.6, 6.8, 7 or 7.1, etc.

[0051] In some implementations, initially, the rotation speed is 100 r / min, the aeration rate is 0.5 VVM, the temperature is 37°C, and the pH is 7.0. During fermentation, ammonia is used to maintain the pH at 7.0. As the cells continue to grow, dissolved oxygen will continuously decrease. During this period, the aeration rate and rotation speed are correlated with dissolved oxygen (DO) to maintain DO above 30%. The maximum aeration rate is 3 VVM, and the maximum rotation speed is 1000 r / min.

[0052] Preferably, the fermentation conditions for the acid-producing stage include: OD 600When the concentration reaches 10–20 g / L (e.g., 11, 12, 13, 15, 16, 17, 18, or 19), IPTG is added to induce protein expression. The glucose concentration in the culture medium is 20–30 g / L (e.g., 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, or 29 g / L), the dissolved oxygen value is greater than 15%, the temperature is 35–42℃ (e.g., 36℃, 37℃, 38℃, 39℃, 40℃, or 41℃), and the pH is 6.0–7.2, including but not limited to 6.2, 6.4, 6.6, 6.8, 7, or 7.1.

[0053] For example, OD 600 After reaching a concentration of 15, add IPTG to a final concentration of 0.5 mM, replace the pH adjuster ammonia with sodium hydroxide, and add glucose solution to the fermenter to maintain the glucose concentration between 20-30 g / L, DO above 20%, temperature at 35-42℃, and pH at 6.0-7.2.

[0054] In some implementations, the fermentation medium used during fermentation consists of: 5-60 g / L glucose (digested), 15-16 g / L Na₂HPO₄·12H₂O, 2.5-3.5 g / L KH₂PO₄, 0.8-1.2 g / L NH₄Cl, and 0.4-0.6 g / L NaCl. Before fermentation begins, 0.01-0.1% (v / v) of MgSO₄ (1 mol / L) and 0.01-0.1% (v / v) of trace elements are added. For example, it can be: 40 g / L glucose (digested), 15.11 g / L Na₂HPO₄·12H₂O, 3 g / L KH₂PO₄, 1 g / L NH₄Cl, and 0.5 g / L NaCl. Sterilize at 121°C for 20 min, and add 0.1% (v / v) of MgSO₄ (1 mol / L) and 0.1% (v / v) of trace elements before fermentation begins.

[0055] The trace elements may be: FeCl3·6H2O 2-3 g / L, CoCl2·6H2O 0.2-0.4 g / L, CuCl2·2H2O 0.1-0.2 g / L, ZnCl2 0.2-0.4 g / L, Na2MO4·2H2O 0.2-0.4 g / L, H3BO3 0.07-0.08 g / L, MnCl2·4H2O 0.49-0.5 g / L; for example, it may be: FeCl3·6H2O 2.4 g / L, CoCl2·6H2O 0.3 g / L, CuCl2·2H2O 0.15 g / L, ZnCl2 0.3 g / L, Na2MO4·2H2O 0.3 g / L, H3BO3 0.075 g / L. g / L, MnCl2·4H2O 0.495 g / L.

[0056] In some implementations, the inoculum size for fermentation is 5% (v / v), with an initial OD... 600 It is 0.5.

[0057] In some embodiments, the culture medium used for seed culture is LB medium.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] This invention, through rational design, introduces R75S and / or I249T mutations into wild-type α-keto acid decarboxylase derived from the genus *Saccharomyces*, thereby improving the substrate specificity and catalytic activity of α-keto acid decarboxylase, increasing the yield of malonic acid, reducing separation costs, and possessing high industrial application value. Attached Figure Description

[0060] Figure 1 This is the liquid chromatogram of malonic acid standard;

[0061] Figure 2 The liquid chromatogram for malonic acid detection in recombinant bacteria containing wild-type α-keto acid decarboxylase;

[0062] Figure 3 The liquid chromatogram for malonic acid detection in recombinant bacteria containing the R75S mutation;

[0063] Figure 4 The liquid chromatogram shows the detection of malonic acid in recombinant bacteria containing the I249T mutation.

[0064] Figure 5 The liquid chromatogram shows the malonic acid detection of recombinant bacteria containing R75S and I249T mutations. Detailed Implementation

[0065] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0066] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0067] In this invention, "identity" can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0068] Example 1

[0069] This embodiment constructs an α-keto acid decarboxylase plasmid.

[0070] Codon optimization and total synthesis of the wild-type α-keto acid decarboxylase gene PDC1 from *Brettanomyces bruxellensis*: Based on the codon preference of *E. coli*, the codons of the wild-type α-keto acid decarboxylase gene PDC1 (SEQ ID NO:1) from *Brettanomyces bruxellensis* were optimized, and the DNA fragment shown in SEQ ID NO:2 was synthesized. The DNA fragment shown in SEQ ID NO:2 was used to replace the sequence between the NcoI and EcoRI restriction sites of pRSFDuet-1 (Novagen, catalog number 71341-3), while the remaining sequences remained unchanged, resulting in the recombinant plasmid pRSF-PDC1. Sequencing of the recombinant plasmid pRSF-PDC1 yielded results consistent with expectations. The yneI gene from *E. coli* was inserted between the NdeI and KpnI restriction sites of plasmid pRSF-PDC1, while the remaining sequences remained unchanged, resulting in the recombinant plasmid pRSF-PDC1-yneI. The recombinant plasmid pRSF-PDC1-yneI was sequenced, and the results were consistent with expectations.

[0071] Example 2

[0072] This embodiment involves α-keto acid decarboxylase mutation.

[0073] Using pRSF-PDC1-yneI as a template, site-directed mutations were introduced into the PDC1 gene shown in SEQ ID NO:2 synthesized in Example 1 using the Mut Express MultiS Fast Mutagenesis Kit V2 (Novazan, catalog number C215-01). These mutations resulted in single-point mutations of R75S or I249T, as well as combinations of both, in the amino acid sequence of wild-type α-keto acid decarboxylase (SEQ ID NO:3).

[0074] The primers used are shown in Table 1.

[0075] Table 1

[0076] R75S-F TAGCAAGagtAGCGGCAAACTGGGTGTTCTGG 10 R75S-R TGCCGCTactCTTGCTATAACCGTCCGCCGCG 11 I249T-F TCTGCGTactTCTCACGAAATCCAGGAGTTTGT 12 I249T-R CGTGAGAagtACGCAGACGATCTACCAGGCAG 13

[0077] pRSF-PDC1-yneI and the recombinant plasmid with a single point mutation were transformed into the Top 10 strains, plated, and transformants were picked. After confirmation, the transformants were cultured and the plasmid was extracted and transformed into BL21(DE3) strain. The transformed strains were plated on plates containing 50 μg / mL Kans. After growth, four colonies were picked for sequencing to screen for the correct mutation. Recombinant bacteria containing two or more mutation sites can be obtained through multiple rounds of mutation.

[0078] The amino acid sequence shown in SEQ ID NO:4 is the sequence obtained by R75S mutation of wild-type α-keto acid decarboxylase, and its corresponding nucleotide sequence is shown in SEQ ID NO:7.

[0079] The amino acid sequence shown in SEQ ID NO:5 is the sequence obtained by I249T mutation of wild-type α-keto acid decarboxylase, and its corresponding nucleotide sequence is shown in SEQ ID NO:8.

[0080] The amino acid sequence shown in SEQ ID NO:6 is the sequence obtained by R75S and I249T mutation of wild-type α-keto acid decarboxylase, and its corresponding nucleotide sequence is shown in SEQ ID NO:9.

[0081] SEQ ID NO:4:

[0082] MAPIALSSSKEENRISLSEYVFRRIASLGVHSVFGVPGDFNLEFLDFIYNVPELKWYGTCNELNGAYAADGYSKSSGKLGVLVTTMGVGELSAMNGISGSYAEYVPILSIVGTTPTTAKMQGLPSHHLITQLNPLEKNDHYVYQKMAASISCNVTSIDDPFEAPDMVDNLIRDILKEKKPGYLYIPCNLASVPVSDLNLRTTSGKFFFEKELCCDQVMVQNLAAKILDLIYMSHKPFVLSDCLVDRLRISHEIQEFVDKAKLPNSCTQMGKSTLNEQSSYYIGDYSGDETSIKTMNYVSSCDLMIHMGNFDNETNSGHFSIHKEFDDKKSGKTLIILNHKYIKIGSELFYGYSILDVLPEMLRMLDTSKLPQVPAPCIQNIIPAHKSSTPISETDVLKGIQTLLKPNDTLIVDTGSILYGISDIRLPKGCKVLTQPFYLSIGMGLPCSFGASVANRELGNKGRIILVEGDGAAQMTIQEFSNFNREGLNPLILLLNNEGYTVERAIKGPTRGYNDIRPNWKWTQLFDTFGMKDYKCQRVDTPDELSKTLKEFGSDNSCSRMIEIGLAKLDVPWRINGMVSHKK。

[0083] SEQ ID NO:5:

[0084] MAPIALSSSKEENRISLSEYVFRRIASLGVHSVFGVPGDFNLEFLDFIYNVPELKWYGTCNELNGAYAADGYSKRSGKLGVLVTTMGVGELSAMNGISGSYAEYVPILSIVGTTPTTAKMQGLPSHHLITQLNPLEKNDHYVYQKMAASISCNVTSIDDPFEAPDMVDNLIRDILKEKKPGYLYIPCNLASVPVSDLNLRTTSGKFFFEKELCCDQVMVQNLAAKILDLIYMSHKPFVLSDCLVDRLRTSHEIQEFVDKAKLPNSCTQMGKSTLNEQSSYYIGDYSGDETSIKTMNYVSSCDLMIHMGNFDNETNSGHFSIHKEFDDKKSGKTLIILNHKYIKIGSELFYGYSILDVLPEMLRMLDTSKLPQVPAPCIQNIIPAHKSSTPISETDVLKGIQTLLKPNDTLIVDTGSILYGISDIRLPKGCKVLTQPFYLSIGMGLPCSFGASVANRELGNKGRIILVEGDGAAQMTIQEFSNFNREGLNPLILLLNNEGYTVERAIKGPTRGYNDIRPNWKWTQLFDTFGMKDYKCQRVDTPDELSKTLKEFGSDNSCSRMIEIGLAKLDVPWRINGMVSHKK。

[0085] SEQ ID NO:6:

[0086] .

[0087] Example 3

[0088] This embodiment involves catalytic activity testing.

[0089] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 15 g / L, pH 7.0. Sterilize at 121℃ for 20 min.

[0090] LB medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0. Sterilize at 121℃ for 20 min.

[0091] Trace elements: FeCl3·6H2O 2.4 g / L, CoCl2·6H2O 0.3 g / L, CuCl2·2H2O 0.15 g / L, ZnCl2 0.3 g / L, Na2MO4·2H2O 0.3 g / L, H3BO3 0.075 g / L, MnCl2·4H2O 0.495 g / L. Sterilize at 121℃ for 30 min.

[0092] Fermentation medium: glucose 40 g / L (digested), Na2HPO4·12H2O 15.11 g / L, KH2PO4 3 g / L, NH4Cl 1 g / L, NaCl 0.5 g / L, sterilized at 121℃ for 20 min. Before fermentation, add 1 / 1000 volume of MgSO4 (1 mol / L) and 1 / 1000 volume of trace elements.

[0093] 1. Activation of bacterial strains: Each recombinant strain constructed in Example 2, which was stored at -80℃, was streaked on LB solid medium and then placed in an incubator at 37℃ for 12 hours for activation. Single colonies were then picked and inoculated into seed medium.

[0094] 2. Seed culture: The seed culture medium was LB medium. After inoculation, the seed culture medium was cultured in a shaker at 220 r / min and 37℃ for 10 h to allow the OD of the seed culture medium to reach its maximum. 600 The level reaches 2 or higher. At this point, the seed culture can be inoculated into the fermentation medium.

[0095] 3. 3L Fermenter Cultivation: The 3L fermenter contains 1.5L of culture medium, and the pH of the sterilized fermentation medium is approximately 7. Before fermentation, the DO value in the fermenter is calibrated to 100%. The seed culture is then inoculated into the fermentation medium at a rate of 5% (v / v), with an initial OD value of... 600 The initial conditions were: rotation speed 100 rpm, aeration rate 0.5 VVM, temperature 37°C, and pH 7.0. Ammonia was used to maintain the pH at 7.0 during fermentation. As the cells grew, dissolved oxygen continuously decreased. During this period, aeration rate and rotation speed were correlated with dissolved oxygen (DO) to maintain DO above 30%. The maximum aeration rate was 3 VVM, and the maximum rotation speed was 1000 rpm. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 600 Once the pH reaches 15, add IPTG to a final concentration of 0.5 mM, replace the pH adjuster ammonia with sodium hydroxide, and add 600 g / L of glucose to the fermenter to maintain a glucose concentration between 20-30 g / L and DO above 20%. Fermentation then enters the acid-producing stage, starting malonic acid fermentation for 30 hours.

[0096] Glucose measurement: After centrifuging the fermentation broth, take the supernatant, dilute it to the range of 0-1 g / L, and measure it using a biosensor SBA-40D.

[0097] OD measurement: During the aerobic growth stage, the fermentation broth is diluted with pure water to a suitable concentration and measured using a UV spectrophotometer at a wavelength of 600 nm. During the anaerobic stage, the fermentation broth is diluted with dilute hydrochloric acid to a suitable concentration and measured using a UV spectrophotometer at a wavelength of 600 nm.

[0098] Malonic acid measurement: Take 1 mL of fermentation broth, centrifuge at 13000 r / min for 10 min, and pass the supernatant through a 0.22 μm filter membrane. Analysis was performed using a UV detector with the following parameters: Aminex HPX-87H organic acid analysis column, 5 mM sulfuric acid mobile phase, flow rate 1 mL / min, column temperature 40℃, and run time 1 h.

[0099] The above-mentioned recombinant bacteria were used to ferment α-keto acid decarboxylase before and after mutation, and the malonic acid in the fermentation broth was measured. The liquid chromatogram of malonic acid standard is shown in the figure. Figure 1 As shown, the liquid chromatograms for malonic acid detection in each recombinant strain are as follows: Figures 2-5 As shown in the figure, the results are shown in Table 2.

[0100] Table 2

[0101] Recombinant bacteria containing pRSF-PDC1-yneI 1.2 Recombinant bacteria containing the R75S mutation 1.6 Recombinant bacteria containing the I249T mutation 1.5 Recombinant bacteria containing R75S and I249T mutations 2.0

[0102] As shown in Table 2, mutating wild-type α-keto acid decarboxylase with R75S or I249T can increase the yield of malonic acid. Furthermore, combining R75S and I249T mutations can further increase the yield of malonic acid, reduce the cost of downstream separation, and thus lower the production cost of malonic acid.

[0103] In summary, this invention optimizes the codons of the wild-type α-keto acid decarboxylase gene from the genus *Escherichia coli* based on codon bias to improve the gene's expression efficiency. Furthermore, by introducing single-point or multi-point mutations into the gene through gene mutation, the modified α-keto acid decarboxylase acquires catalytic activity, increasing malonic acid production, facilitating downstream malonic acid separation, and promoting the production and application of malonic acid.

[0104] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An α-keto acid decarboxylase mutant, characterized in that, The amino acid sequence of the α-keto acid decarboxylase mutant is selected from any one of the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the α-keto acid decarboxylase mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule includes the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2 or 3.

5. Recombinant cells, characterized in that, The recombinant cells contain the recombinant vector as described in claim 4.

6. The method for preparing the α-keto acid decarboxylase mutant according to claim 1, characterized in that, The preparation method includes: The nucleic acid molecule encoding the α-keto acid decarboxylase mutant of claim 1 is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and purified to obtain the α-keto acid decarboxylase mutant.

7. The method for preparing the α-keto acid decarboxylase mutant according to claim 6, characterized in that, The expression vectors include pRSFDuet-1, pETDuet-1, pACYCDuet-1, pTrc99a, or pET28a.

8. The method for preparing the α-keto acid decarboxylase mutant according to claim 6, characterized in that, The host cells include Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, or Yeast lipolytica.

9. The method for preparing the α-keto acid decarboxylase mutant according to claim 8, characterized in that, The Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli Rosetta(DE3), Escherichia coli BL21(DE3)plysS, Escherichia coli M15, or Escherichia coli Top10f.

10. The application of the α-keto acid decarboxylase mutant of claim 1, the nucleic acid molecule of claim 2 or 3, the recombinant vector of claim 4, the recombinant cell of claim 5, or the method for preparing the α-keto acid decarboxylase mutant of any one of claims 6 to 9 in the preparation of malonic acid.

11. A method for preparing malonic acid, characterized in that, The method for preparing malonic acid includes: using the α-keto acid decarboxylase mutant of claim 1 to catalyze the substrate glucose to obtain malonic acid.

12. The method for preparing malonic acid according to claim 11, characterized in that, The method for preparing malonic acid specifically includes: The nucleic acid molecule encoding the α-keto acid decarboxylase mutant of claim 1 is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell for fermentation and malonic acid separation and purification to obtain malonic acid.

13. The method for preparing malonic acid according to claim 12, characterized in that, The fermentation includes an aerobic fermentation stage and an acid production stage.

14. The method for preparing malonic acid according to claim 13, characterized in that, The fermentation conditions for the aerobic fermentation stage include: dissolved oxygen value greater than 30%, temperature of 35~42℃, and pH of 6.0~7.

2.

15. The method for preparing malonic acid according to claim 13, characterized in that, The fermentation conditions for the acid-producing stage include: OD 600 When the concentration reaches 10-20, IPTG is added to induce protein expression. The glucose concentration in the culture medium is 20-30 g / L, the dissolved oxygen value is greater than 15%, the temperature is 35-42℃, and the pH is 6.0-7.2.

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