A 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant, and a construction method and application thereof

Abstract

The application discloses a 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant, a construction method and application thereof, and belongs to the technical field of biology. Eight 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutants which are significantly superior to wild-type enzymes in catalytic efficiency are obtained through rational design and screening, and a recombinant Escherichia coli based on the mutants is constructed, so that the yield of vitamin K2 produced by the whole cell catalysis of the constructed recombinant bacteria is more than 30% higher than that of a wild-type strain. The excellent catalytic performance of the menA mutant provided by the application provides strong technical support for the industrialized production of vitamin K2, and has wide application prospect and market value.

Description

Technical Field

[0001] This invention relates to a 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant, its construction method, and its application, belonging to the field of biotechnology. Background Technology

[0002] 1,4-Dihydroxy-2-naphthylcarboxylic acid-polyisoprenetransferase (menA, EC 2.5.1.74) is a key enzyme in the bacterial vitamin K2 biosynthesis pathway, responsible for catalyzing the rate-limiting step of this pathway—the isoprenylation of polyisoprene diphosphate with 1,4-dihydroxy-2-naphthylcarboxylic acid (DHNA) to generate isoprenoidized naphthoquinone derivatives, which are direct precursors for the synthesis of vitamin K2 with different side chain lengths. Vitamin K2 not only serves as an electron carrier in microbial anaerobic respiration but also plays an important role in human bone and cardiovascular health. Therefore, the menA enzyme, as a core target for vitamin K2 synthesis in microbial cell factories, has significant research and application value, as its catalytic efficiency directly determines the yield of the final product.

[0003] However, wild-type menA enzymes have significant limitations in industrial applications: low catalytic efficiency, strong specificity to substrate side chain length, poor stability, and susceptibility to product inhibition, resulting in low efficiency and limited product diversity throughout the synthetic pathway, making it difficult to meet the demands of large-scale production. Therefore, there is an urgent need to construct novel enzyme mutants with higher catalytic activity, wider substrate adaptability, enhanced stability, and resistance to feedback inhibition, in order to improve the production efficiency of vitamin K2, reduce costs, and achieve diversified product development. Summary of the Invention

[0004] The technical problem to be solved by this invention is to improve the ability of existing 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase to synthesize vitamin K2.

[0005] This invention provides a 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant, which, based on the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase parent, has at least one of the following mutations: N294W, S157Q, G131W, P281W, P281L, G131F, P281C, or G110M.

[0006] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating glycine at position 110 of the amino acid sequence shown in SEQ ID NO.2 to methionine, and the mutant is named G110M.

[0007] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating glycine at position 131 of the amino acid sequence shown in SEQ ID NO.2 to tryptophan, and the resulting mutant is named G131W.

[0008] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating glycine at position 131 to phenylalanine, and the resulting mutant is named G131F.

[0009] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating serine at position 157 to glutamine, and the resulting mutant is named S157Q.

[0010] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating proline at position 281 to tryptophan, and the resulting mutant is named P281W.

[0011] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating proline at position 281 to leucine, and the resulting mutant is named P281L.

[0012] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating proline at position 281 to cysteine, and the resulting mutant is named P281C.

[0013] In one embodiment, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant is constructed by mutating asparagine at position 294 to tryptophan, and the resulting mutant is named N294W.

[0014] In one embodiment, the amino acid sequence of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant G131F is shown in SEQ ID NO.3.

[0015] The present invention also provides an enzyme preparation containing the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant.

[0016] In one embodiment, the enzyme preparation contains the protein of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant.

[0017] In one embodiment, the enzyme preparation is an immobilized enzyme of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant.

[0018] The present invention also provides a gene encoding the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant.

[0019] In one embodiment, the nucleotide sequence of the gene encoding the mutant G131F is shown in SEQ ID NO.4.

[0020] The present invention also provides a recombinant plasmid containing the said gene.

[0021] The present invention also provides a recombinant microbial cell expressing a 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant.

[0022] In one embodiment, the recombinant microorganism uses Escherichia coli BL21(DE3) as the host and pSB1a3-AD011 as the expression vector to express the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant.

[0023] The present invention also provides a method for preparing vitamin K2, wherein the recombinant microorganism is fermented in a culture medium, and then vitamin K2 is synthesized by whole-cell catalysis in a culture medium with glucose as the carbon source; arabinose is used to induce the fermentation process.

[0024] In one embodiment, the method involves culturing the recombinant microorganisms to OD200. 600 When the concentration reaches 0.8-1, add arabinose to a final concentration of 2 g / L as an inducer and induce culture at 28-30℃ for 15-20 h.

[0025] In one embodiment, the fermentation is carried out in LB and / or TB medium.

[0026] In one embodiment, the method involves culturing the recombinant Escherichia coli in TB medium at 37°C until OD reaches [a certain value]. 600 To achieve a final concentration of 0.8-1, induce at 28-30°C for 16-20 h using an arabinose inducer at a final concentration of 2 g / L.

[0027] In one embodiment, the method involves culturing the bacterial cells obtained from fermentation in M9 medium at 35-37°C for at least 10 hours.

[0028] In one embodiment, the M9 culture medium contains Na2HPO4, KH2PO4, NaCl, NH4Cl, glucose, MgSO4, and CaCl2.

[0029] The present invention also provides the use of the mutant, the enzyme preparation, or the recombinant microbial cell in the preparation of vitamin K2 or products containing vitamin K2.

[0030] Beneficial effects: 1. This invention innovatively employs a comprehensive rational design strategy, which includes three methods: (1) enhancing structural rigidity through folding energy optimization; (2) increasing surface charge to reduce aggregation; and (3) using consensus design to eliminate non-conserved residues. Information that can improve enzyme activity is analyzed from the perspective of enzyme structure and evolution. The sequences of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase family are integrated and analyzed. Combined with bioinformatics and crystallography methods, novel 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutants with high activity are obtained.

[0031] 2. The 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant with enhanced activity provided by this invention has excellent catalytic activity. After expression in recombinant Escherichia coli, the yield of vitamin K2 increased by 33.78% compared with wild-type recombinant Escherichia coli, showing good application prospects. Attached Figure Description

[0032] Figure 1 This is a standard curve for the high-performance liquid chromatography (HPLC) detection of vitamin K2 (MK-7) according to the present invention.

[0033] Figure 2 This is a gene map of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase provided in a specific embodiment of the present invention.

[0034] Figure 3 This is a schematic diagram of the simulated crystal structure of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein provided in a specific embodiment of the present invention.

[0035] Figure 4 This is a schematic diagram of a comprehensive rational design strategy for 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase based on AlphaFold structure modeling, provided in a specific embodiment of the present invention.

[0036] Figure 5 This is a protein gel electrophoresis image of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant with enhanced activity prepared in this invention. Detailed Implementation

[0037] (a) Culture medium LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. (ThermoScientific™ Oxoid) TB medium: Tryptone 12 g / L, Yeast Extract 24 g / L, Glycerol 4 mL / L, and a phosphate buffer system (KH₂PO₄ 2.31 g / L and K₂HPO₄ 12.54 g / L). Oxoid (a brand under Thermo Fisher Scientific) was commonly used for tryptone and yeast extract. Glycerol and phosphate were purchased from Sigma-Aldrich. M9 medium: Na2HPO4·7H2O 6.78 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L, glucose 2 g / L, MgSO4 1 mM, CaCl2 0.1 mM.

[0038] The inorganic salts (Na2HPO4, KH2PO4, NH4Cl, NaCl, MgSO4 and CaCl2) involved in this application were purchased from Sigma-Aldrich, and the glucose was purchased from Sinopharm.

[0039] (II) Detection Methods Vitamin K2 content detection: Take 2 mL of bacterial fermentation broth, centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend and disperse in 500 μL isopropanol, then add 500 μL of n-hexane, incubate in the dark at 1000 rpm and 37℃ for 3 h, and perform high-performance liquid chromatography (HPLC) analysis. The detection conditions were: sample volume 10 μL; flow rate 1 mL / min; mobile phase 100% methanol; detection wavelength 254 nm; column temperature 30℃; C18 column; detection time 35 min. The standard curve is shown below. Figure 1 As shown.

[0040] Example 1: Construction of recombinant bacteria expressing 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase The wild-type 1,4-dihydroxy-2-naphthylcarboxylic acid-polyisoprene transferase gene was codon-optimized using *E. coli* as the host cell. The gene fragment with the nucleotide sequence shown in SEQ ID NO.1 was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and amplified using upstream and downstream amplification primers. The nucleic acid sequences of the upstream and downstream primers are as follows: The upstream primer nucleic acid sequence is: 5'-ACTGCTATGACTGAACAACAAATTAGCCGAACTCAGG-3'; The downstream primer nucleic acid sequence is: 5'-TCAGCTTGCTGCCCACTGGCTTAGGAATATCC-3' The amplification conditions were as follows: amplification at 98℃ for 30 seconds, then amplification at 98℃ for 30 seconds, amplification at 60℃ for 10 seconds, amplification at 72℃ for 70 seconds, for a total of 35 cycles, and finally amplification at 72℃ for 5 minutes.

[0041] After the reaction was complete, the PCR amplification product was detected by 1% agarose gel electrophoresis, yielding a 1kb band, the length of which met the expected result. The PCR product was purified using the Novizan PCR product purification kit to remove primers, dNTPs, enzymes, and other impurities. Following the kit's standard operating procedures, the target fragment was recovered and purified. The purified target gene fragment was recombined with the vector pSB1a3-AD011 (Addgene: Plasmid #116852). The resulting ligation product was transformed into *E. coli* BL21(DE3) competent cells. The transformed cells were plated on LB agar plates containing 50 μg / ml ampicillin, and positive clone plasmids were extracted and sequenced. The results showed that the cloned 1,4-dihydroxy-2-naphthyl-polyisoprene transferase gene sequence was correct and correctly inserted into the pSB1a3-AD011 plasmid, yielding the recombinant plasmid pSB1a3-AD011-menA, as shown in the image. Figure 2 .

[0042] Example 2 Expression and purification of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase The engineered bacteria from the glycerol tube were inoculated at a volume ratio of 1% into a 3 mL LB medium tube containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 12 h. 800 μL of the bacterial suspension was then transferred to a 50 mL TB medium shake flask containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 2 h until the OD600 reached approximately 1. Then, 1% arabinose inducer was added, and the culture was induced at 30℃ and 200 rpm for 18 h. The harvested *E. coli* bacterial suspension was ultrasonically disrupted and then subjected to a one-step Ni-NTA affinity chromatography to obtain 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein with a purity >95%, the amino acid sequence of which is SEQ ID NO.2.

[0043] Example 3: Rational Design of Mutation Sites for 1,4-Dihydroxy-2-naphthoic acid-polyisoprene transferase Based on AlphaFold structural modeling, a high-confidence model (pLDDT>90, such as...) was obtained. Figure 3Using RosettaCartesian_ddg and a custom Python script, all 308 amino acid residues (shown in SEQ ID NO.2) were replaced one by one with 20 standard amino acids, and the ΔΔG values ​​were calculated using energy functions (including fa_atr, fa_rep, hbond_sc, etc.). Fold energy calculations showed that 8.1% of single-point mutations predicted ΔΔG < 0 kcal / mol, indicating a potential for enhanced stiffness, while 0.9% of mutations showed ΔΔG < -5 kcal / mol, demonstrating a strong stabilizing effect. Figure 4 A) Preliminary screening identified 30 potential stabilization mutation sites, including: N294W, N198I, S157Q, D208L, A137F, G131W, G169W, S60W, G131I, G110L, G149L, G131F, G131V, P281V, S166W, G169F, P281W, P281L, G131Y, G110M, P281Y, G169Y, P281C, P281I, G126F, A137D, N198L, G131C, N294L, and S166Y.

[0044] Furthermore, in surface charge engineering, aiming to reduce the net charge from -1 to -50, 20 mutation sites were ultimately screened that could adjust the net charge to -37. Figure 4 B). To avoid disrupting protein stability, mutations with ΔΔG > 0 kcal / mol were excluded, ultimately resulting in 15 potential activity-related mutation sites, including: N294W, N198I, S157Q, D208L, A137F, G131W, G169W, S60W, G131I, G110L, G149L, G131F, G131V, P281V, and S166W; Furthermore, by searching the Pfam and NCBI databases for the amino acid sequence shown in SEQ ID NO.2, removing redundant amino acid sequences, and selecting amino acid sequences with greater than 50% identity to the amino acid sequence shown in SEQ ID NO.2, consensus design based on analysis of 676 homologous sequences identified 18 non-conserved residues, of which 10 mutations resulted in a conservation score exceeding 50% after the site mutation. Figure 4 C) Organize the remaining amino acid sequences into fasta. format, input them into Clustalx 1.83 software for multiple sequence alignment, and output the alignment results in aln. and fasta. formats, where aln. and fasta. files are sequence files in different formats; Furthermore, the aforementioned fasta. file is uploaded to the online server Consensus Maker v2.0.0 (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html). After modifying the settings parameters as needed, the online software will generate a consensus sequence that can be edited later.

[0045] Furthermore, the amino acid sequence (SEQ ID NO.2) of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein was compared with the consensus sequence of this family and the amino acid abundance map at each point.

[0046] Furthermore, the three-dimensional structure of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein (amino acid sequence SEQ ID NO.2) was predicted using the AlphaFold online tool, and structural modeling was performed. The crystal structure of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase (amino acid sequence SEQ ID NO.2) was observed using PyMOL. Based on the structural information, the above-mentioned candidate mutation sites and mutation modes were reviewed, and the mutant sites most likely to improve the activity of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase were screened out. The screening criteria are as follows: (1) The criteria for determining a site as a candidate site are: ①Most proteins in this family have a generally high amino acid abundance at this site; ②The amino acid at this site is conserved; ③ The amino acids that appear frequently at this site have significant differences in physicochemical properties compared to the amino acids at this site in 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase, such as differences in charge, polarity, and steric hindrance.

[0047] (2) Remove amino acid residues near the active site, i.e., within 10 Å of the catalytic residues, and remove amino acid residues that are in an embedded or semi-embedded state.

[0048] After the above two screening steps, a total of 15 differential sites remain, most of which are located on the surface of the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein molecule.

[0049] (3) Based on the crystal structure of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein, each structure was analyzed in detail to screen out mutants that may improve the activity of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein.

[0050] The main criteria for judgment are: ① Mutations should eliminate existing force forms that are detrimental to activity, such as electrostatic repulsion and charge accumulation; ② Mutations should not destroy existing force forms that are beneficial to activity and stable protein structures; ③ Mutations should introduce new force forms that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0051] A total of 25 single-point mutants were designed, with the following mutation sites: N294W, N198I, S157Q, D208L, A137F, G131W, G169W, S60W, G131I, G110L, G149L, G131F, G131V, P281V, S166W, G169F, P281W, P281L, G131Y, G110M, P281Y, G169Y, P281C, P281I, and G126F.

[0052] Example 4 Construction, expression and purification of a single-point mutant of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase (1) Construction of recombinant bacteria expressing 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant Using the recombinant plasmid pSB1a3-AD011-menA constructed in Example 1 as a template, and a pair of complementary oligonucleotides with mutation sites as amplification primers, the whole plasmid was amplified by PCR using the Vazyme high-fidelity PCR kit to obtain the recombinant plasmid with specific mutation sites. The amplification primer pairs used are shown in Tables 1 and 2.

[0053] Table 1. Primer sequences for amplification of single-point mutations

[0054] Table 2. Primer sequences for amplification of single-point mutations

[0055] The reaction mixture (50 μL) consisted of: 1 μL template DNA (TAPC), 2 μL forward primer (F), 2 μL reverse primer (R), 25 μL 2×Phanta Max Buffer, 2 μL dNTP Mix, 1 μL Phanta Max Super-Fidelity DNA Polymerase, and ddH2O to a final volume of 50 μL. The PCR program was set as follows: 98℃ pre-denaturation for 30 s; 35 cycles of amplification (98℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 70 s, adjusting the extension time by 1 kb / 10 s); and a final extension at 72℃ for 300 s. After amplification, 4 μL of PCR product was mixed with 1 μL of 5× Loading buffer and electrophoresed on a 1% agarose gel (containing 0.5 μg / mL GoldView™ dye) (120 V, 50 min). The gel was then imaged using the GelDoc XR+ system. The PCR product was recovered from the gel and digested with DpnI enzyme at 37°C for 1 h to degrade the initial template. The digested product was recombined and transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Positive clones were screened and sequenced to verify the results, yielding recombinant bacteria containing a single-point mutant of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase. The PCR amplification was performed using the Vazyme high-fidelity PCR kit; the DpnI enzyme was provided by Fermentas.

[0056] (2) Expression and purification of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase single-point mutant The recombinant bacteria constructed in step (1) were inoculated into a 3 mL LB medium tube containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 12 h. 800 μL of the bacterial suspension was transferred to a 50 mL TB medium shake flask containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 2 h until the OD600 reached 0.8-1. Then, arabinose inducer was added to a final concentration of 2 g / L, and the culture was induced at 30℃ and 200 rpm for 18 h. The *E. coli* bacterial suspension was collected, sonicated, and then subjected to a Ni-NTA affinity chromatography step to obtain 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase protein with a purity >95%. The protein gel image is shown below. Figure 5 As shown.

[0057] Example 5: Whole-cell catalysis of vitamin K2 by a 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant The recombinant bacteria expressing wild-type 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase prepared in Example 1 and the recombinant bacteria expressing a mutant of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase prepared in Example 4 were used for whole-cell catalytic synthesis of vitamin K2. The specific steps included: The recombinant *E. coli* expressing wild-type 1,4-dihydroxy-2-naphthyl-polyisoprene transferase constructed in Example 1 and the recombinant *E. coli* expressing the mutant constructed in Example 4 were streaked onto LB agar plates containing 100 μg / mL ampicillin and cultured overnight at 37°C. Three single colonies from each culture were picked and transferred to 3 mL LB medium tubes containing 100 μg / mL ampicillin and cultured at 37°C and 220 rpm for 12 h. 800 μL of the bacterial culture was then transferred to a shake flask containing 50 mL TB medium containing 100 μg / mL ampicillin and cultured at 37°C and 220 rpm for 2 h to allow OD to develop. 600 When the bacterial culture reaches a concentration of 0.8-1, add arabinose inducer to a final concentration of 2 g / L and induce culture for 18 h at 30℃ and 200 rpm. Transfer the bacterial culture to 50 mL centrifuge tubes in a clean bench, centrifuge at 5000 rpm and 4℃ for 5 min, and dilute with M9 medium to an OD value of 0.8-1. 600 The result was 10, and the mixture was cultured for another 10 h at 37℃ and 220 rpm. The fermentation broth was collected, and the vitamin K2 yield in the fermentation broth was detected. The results are shown in Table 3.

[0058] The results showed that the recombinant bacteria expressing the mutants had a significantly higher vitamin K2 production rate during whole-cell transformation compared to the wild-type enzyme. Specifically, the recombinant bacteria expressing the mutants N294W, S157Q, G131W, P281W, P281L, G110M, P281C, and G131F had production rates of 71.53 mg / L, 71.57 mg / L, 69.80 mg / L, 70.13 mg / L, 70.81 mg / L, 84.71 mg / L, 68.87 mg / L, and 69.54 mg / L, respectively.

[0059] Table 3. Whole-cell catalytic synthesis of vitamin K2 by wild-type 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase and mutants

[0060] Note: Relative activity is the percentage of vitamin K2 production catalyzed by the mutant relative to the wild-type enzyme. It is calculated as: vitamin K2 production of recombinant bacteria expressing the mutant ÷ vitamin K2 production of recombinant bacteria expressing the wild-type enzyme.

[0061] As shown in Table 2, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutants provided by this invention have excellent catalytic activity. There are 8 mutants with enhanced activity. Among them, G131F increases the production of vitamin K2 by 33.78% compared with wild-type 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase, which has broad application prospects and market value.

[0062] Comparative example: Referring to the methods in Examples 3-5, mutants as shown in Table 3 were also constructed. Recombinant cells expressing mutants were used for whole-cell catalytic preparation of vitamin K2 according to the method in Example 5. The results are shown in Table 4. Some mutants did not achieve the effect of increasing vitamin K2 production.

[0063] Table 4. Whole-cell catalytic synthesis of vitamin K2 by wild-type 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase and mutants

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. 1,4-Dihydroxy-2-naphthoic acid-polyisoprene transferase mutant, characterized in that, It has a G131F mutation based on the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase parent.

2. The 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant according to claim 1, characterized in that, The mutant is based on the parent shown in SEQ ID NO.2, with glycine at position 131 mutated to phenylalanine.

3. An enzyme preparation containing the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant as described in claim 1 or 2.

4. The gene encoding the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant of claim 1 or 2.

5. A recombinant plasmid containing the gene of claim 4.

6. Recombinant microbial cells expressing the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant of claim 1 or 2.

7. Recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pSB1a3-AD011 as the expression vector, the 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant of claim 2 was expressed.

8. A method for preparing vitamin K2, characterized in that, The recombinant Escherichia coli of claim 7 is fermented in a culture medium, and arabinose is used to induce fermentation during the process.

9. The method according to claim 8, characterized in that, The fermentation is carried out in LB medium and / or TB medium.

10. The use of the mutant of claim 1 or 2, or the enzyme preparation of claim 3, or the recombinant microbial cell of claim 6, or the recombinant Escherichia coli of claim 7, or the method of any one of claims 8 to 9 in the preparation of vitamin K2 or products containing vitamin K2.

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