Ppds mutant, its encoding gene, recombinant plasmid, recombinant bacteria and application in synthesis of protopanaxadiol

Abstract

The application belongs to the technical field of enzymes, and particularly relates to a PPDS mutant, a coding gene of the PPDS mutant, a recombinant plasmid, a recombinant bacterium and application of the PPDS mutant in synthesis of protopanaxadiol, wherein the amino acid sequence of the PPDS mutant is an amino acid sequence with at least one mutation of N178S and Y312N in the amino acid sequence shown in SEQ ID NO. 1. The mutant can significantly improve the enzyme activity of PPD, and the recombinant bacterium constructed by using the mutant can improve the synthesis efficiency of protopanaxadiol when the recombinant bacterium uses glycerol as the only carbon source.

Description

Technical Field

[0001] This invention belongs to the field of enzyme technology, specifically relating to the application of PPDS mutants and their encoding genes, recombinant plasmids, recombinant bacteria, and protopanaxadiol synthesis. Background Technology

[0002] Protopanaxadiol (PPD) is the core aglycone of ginsenosides and a key precursor for the synthesis of various ginsenoside compounds. It belongs to the dammarane-type tetracyclic triterpenoids and has significant pharmacological activities such as antitumor, antioxidant, antidepressant and cardiovascular protection. It has broad application prospects and extremely high market value in the fields of medicine, health products, and cosmetics.

[0003] With the development of synthetic biology technology, the efficient synthesis of protopanaxadiol using microbial cell factories can not only break through the production bottleneck of traditional plant extraction, but also provide sufficient and stable precursor raw materials for the large-scale synthesis of various ginsenoside compounds, becoming a research hotspot in related fields.

[0004] In the biosynthetic pathway of protopanaxadiol, protopanaxadiol synthase (PPDS) is the key rate-limiting enzyme, responsible for catalyzing the directed conversion of dammarene diol-II to protopanaxadiol. Its activity directly determines the synthesis efficiency and yield of protopanaxadiol, thus affecting the synthesis efficiency and large-scale production potential of subsequent ginsenoside compounds. However, wild-type PPDS suffers from prominent problems such as low catalytic activity, weak substrate affinity, and poor heterologous expression stability in Pichia pastoris. This results in consistently low protopanaxadiol yields from recombinant strains, failing to provide sufficient precursor raw materials for the synthesis of subsequent ginsenoside compounds. Ultimately, this hinders the industrial application of protopanaxadiol and downstream ginsenosides, and also limits the promotion and implementation of synthetic biology technology in the production of ginsenoside compounds.

[0005] Existing technologies also disclose some mutant enzymes and mutant enzyme-catalyzed synthesis strategies. For example, patent document CN119709712A discloses a dammarene diol synthase mutant and its application in the preparation of β-amyrin alcohol. The dammarene diol synthase undergoes the following amino acid mutation: T128L, while keeping other amino acid sequences unchanged; or, the dammarene diol synthase undergoes the following amino acid mutations: T128L, L242M, F253L, F255M, V327L, A377V, V485Q, and A493G, while keeping other amino acid sequences unchanged. As another example, publication WO2023039518A1 discloses an engineered microbial cell, an enzyme, and a composition and method related to the production of dammarene diol and compounds derived from dammarene diol; the method involves engineering microbial host cells to express a heterologous biosynthetic pathway for the production of dammarene diol or its derivatives. Host cells may optionally express heterouridine diphosphate-dependent glycosyltransferase (UGT) enzymes to produce dammarene glycol, protopanaxadiol, or protopanatriol in natural or non-natural glycosylated forms. For example, patent document CN111378681A discloses a method for producing dammarene glycol-II glycoside 3β-O-Glc-DM and a method for constructing recombinant bacteria, the recombinant bacteria obtained by the above method, and the use of the above recombinant bacteria in the preparation of 3β-O-Glc-DM.

[0006] In summary, while obtaining higher-performance mutants through mutation is a conventional approach, differences in the original sequence and mutation method can fundamentally affect the spatial structure of the mutant, leading to unpredictable impacts on its performance. The existing mutant enzymes of PPDS and their performance require further development. Summary of the Invention

[0007] To improve the catalytic activity of PPDS enzyme, the primary objective of this invention is to provide a novel PPDS mutant, which aims to provide a novel mutant with excellent catalytic activity and selectivity of ginsenoside PPD enzyme.

[0008] A second objective of this invention is to provide a gene capable of encoding the PPDS mutant, a recombinant plasmid, and a recombinant bacterium expressing the PPDS mutant.

[0009] A third objective of this invention is to provide the application of the PPDS mutant in the synthesis of PPD enzyme.

[0010] The PPDS mutant has an amino acid sequence containing at least one mutation in N178S or Y312N in the amino acid sequence shown in SEQ ID NO.1.

[0011] This invention provides a novel mutant, an enzyme mutant obtained by site-directed mutagenesis of the protopanaxadiol synthase gene (Uniprot accession number: H2DH16) derived from ginseng. Specifically, N178S refers to the mutation of asparagine (N) at position 178 to serine (S), and Y312N refers to the mutation of tyrosine (Y) at position 312 to asparagine (N) (Y312N). This mutant significantly improves the synthesis efficiency of protopanaxadiol in recombinant Pichia pastoris when glycerol is the primary carbon source.

[0012] Preferably, the PPDS mutant is the sequence shown in SEQ ID NO.2 (N178S single-point mutation), SEQ ID NO.3 (Y312N single-point mutation), or SEQ ID NO.4 (N178S and Y312N double-point mutant).

[0013] Studies have shown that the N178S and Y312N double-point mutants can further improve the synthesis efficiency of protopanaxadiol.

[0014] The present invention also provides an application of the PPDS mutant described above, using it as an enzyme catalyst for the enzymatic synthesis of protopanaxadiol.

[0015] The present invention also provides a gene that can encode the PPDS mutant described above.

[0016] The present invention also provides a recombinant plasmid loaded with the gene of the PPDS mutant.

[0017] The present invention also provides a recombinant bacterium capable of expressing the PPDS mutant described above.

[0018] The recombinant bacteria are recombinant bacteria inoculated with the recombinant plasmid. Further, the recombinant bacteria are Pichia pastoris inoculated with the recombinant plasmid.

[0019] Preferably, the recombinant bacteria further incorporate the genes for 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), mevalonate phosphate kinase (ERG8), acetyl-CoA thiolase (ERG10), squalene oxidase (ERG1), dammarene diol synthase (DDS), glycerol kinase (GUT1), and glycerol-3-phosphate dehydrogenase (GUT2).

[0020] The present invention also provides an application of the recombinant bacteria in the preparation of protopanaxadiol, wherein the recombinant bacteria are fermented and then protopanaxadiol is obtained by separation.

[0021] Introducing the aforementioned protopanaxadiol synthase mutant gene (PPDS) and its HMGR, FPPS, SQS, ERG8, ERG10, ERG1, DDS, GUT1, and GUT2 genes into Pichia pastoris GS115 can optimize the glycerol metabolism pathway, construct a recombinant Pichia pastoris strain, and enable it to grow efficiently and synthesize protopanaxadiol using only glycerol as the main carbon source.

[0022] In this invention, the fermentation method and means can be conventional.

[0023] Furthermore, the fermentation medium can be well-known, such as conventional YPG liquid medium.

[0024] Beneficial effects

[0025] This invention provides a novel PPDS mutant, the mutated coding gene, and the microorganism expressing it. The mutant and the recombinant bacteria described in this invention can synthesize PPD with high selectivity. Attached Figure Description

[0026] Figure 1 The yield data for PPD of the constructed Pichia pastoris strain are shown. Detailed Implementation

[0027] The process steps of this invention include:

[0028] (1) This invention provides a protopanaxadiol synthase mutant, the amino acid sequence of which is an amino acid sequence containing at least one mutation of N178S or Y312N in the amino acid sequence shown in SEQ ID NO.1. Among the mutants, the amino acid sequence of the mutant labeled PgPPDS-N178S is shown in SEQ ID NO.2, the amino acid sequence of the mutant PgPPDS-Y312N is shown in SEQ ID NO.3, and the amino acid sequence of the mutant PgPPDS-N178S-Y312N is shown in SEQ ID NO.4.

[0029] (2) This invention provides a method for constructing a recombinant Pichia pastoris strain that metabolizes glycerol to produce protopanaxadiol, comprising the following steps:

[0030] 1) A truncated expression cassette of the mevalonate pathway (MVA pathway) rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase HMGR (tHMG1), farnesyl pyrophosphate synthase (FPPS), and squalene synthase (SQS) was introduced into Pichia pastoris GS115 to increase the metabolic flux of squalene and obtain recombinant strain MQ1. The nucleotide sequence of the truncated expression cassette of the MVA pathway rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase HMGR (tHMG1) is shown in SEQ ID NO. 5; the nucleotide sequence of the farnesyl pyrophosphate synthase (FPPS) is shown in SEQ ID NO. 6; and the nucleotide sequence of the squalene synthase (SQS) is shown in SEQ ID NO. 7.

[0031] 2) The mevalonate kinase ERG8 gene and the acetyl-CoA thiolase ERG10 expression cassette were introduced into the recombinant strain MQ1 to increase the throughput of the MVA pathway and obtain the recombinant strain MQ2. The nucleotide sequence of the mevalonate kinase ERG8 is shown in SEQ ID NO.8; the nucleotide sequence of the acetyl-CoA thiolase ERG10 is shown in SEQ ID NO.9.

[0032] 3) The squalene oxidase ERG1 gene expression cassette and the dammarene diol synthase DDS gene expression cassette were introduced into the recombinant strain MQ2 to open the dammarene diol synthesis pathway, thus obtaining the recombinant strain MQ3. The nucleotide sequence of the squalene oxidase ERG1 is shown in SEQ ID NO.10; the nucleotide sequence of the dammarene diol synthase DDS gene is shown in SEQ ID NO.11.

[0033] 4) Introduce the key enzymes of glycerol metabolism, glycerol kinase GUT1 and glycerol-3-phosphate dehydrogenase GUT2, into the above recombinant strain MQ3 to enhance the absorption and metabolism of glycerol, ensuring that the recombinant strain can grow and synthesize products using only glycerol as the sole carbon source, thus constructing recombinant strain MQ4. The nucleotide sequence of glycerol kinase GUT1 is shown in SEQ ID NO.12; the nucleotide sequence of glycerol-3-phosphate dehydrogenase GUT2 is shown in SEQ ID NO.13.

[0034] 5) Introduce the protopanaxadiol synthase PPDS (or mutant) and its reductase fusion protein PPDS-ATR1 gene expression cassette into the above recombinant strain MQ4 to open the protopanaxadiol synthesis pathway and obtain MQ5, MQ6, MQ7, MQ8, and MQ9 (MQ5 is a recombinant strain with the original PPDS enzyme gene sequence introduced; MQ6, MQ7, MQ8, and MQ9 are recombinant strains with the PPDS enzyme mutant gene sequence introduced); the nucleotide sequence of the PPDS and its reductase ATR1 fusion gene is shown in SEQ ID NO.14; the nucleotide sequence of the PPDS mutant and its reductase ATR1 fusion gene PgPPDS-GGGGS-ATR1 is shown in SEQ ID NO.15-18;

[0035] This invention constructed a recombinant strain of Pichia pastoris GS115 capable of synthesizing protopanaxadiol using glycerol as the main carbon source. Codon optimization and site-directed mutagenesis were performed on the key rate-limiting enzyme, protopanaxadiol synthase PPDS, with at least one of the following mutation sites: PgPPDS-N178S, PgPPDS-K235F, PgPPDS-Y312N, and PgPPDS-N178S-Y312N. Compared to the wild-type PgPPDS, the mutant strain MQ9 significantly improved protopanaxadiol production, providing a more stable and efficient chassis cell for the subsequent biosynthesis of ginsenosides.

[0036] Example 1: Construction of the CRISPR-Cas9 plasmid used for the insertion sites of the above strains:

[0037] According to the Pichia pastoris gene editing tool plasmid BB3cK (also known as...) provided on the ADDGENE website,... After purchasing, according to the literature (Gassler, T., Heistinger, L., Mattanovich, D., Gasser, B. & Prielhofer, R. CRISPR / Cas9-mediated homology-directed genome editing in...), Pichia pastoris . Methods Mol. Biol. (1923, 211–225 (2019)) The sequence of its gRNA expression cassette was obtained, synthesized, and seamlessly cloned into... A complete, editable CRISPR plasmid MK01 was constructed on the backbone.

[0038] After selecting N20, PCR amplification was performed using plasmid MK01 as a template. The obtained PCR product was digested with DpnI and then purified by gel extraction. 100 ng of the recovered fragment was homologously recombinated using a seamless cloning kit and transformed into 50 μL of commercial Trans5α competent cells, which were then plated on LB solid medium containing kanamycin sulfate and cultured overnight. Two transformants were randomly selected and sent to Qingke Sequencing. Plasmids containing the correct N20 were extracted to obtain PNS4-2 CRISPR, PNS2-8 CRISPR, PNS4-10 CRISPR, HRD1 CRISPR, and PAH1 CRISPR plasmids.

[0039] The names of the sites used in this invention and N20 are shown in Table 1:

[0040] Table 1. Genomic insertion site information used in this invention.

[0041] Example 2: A method for constructing recombinant bacteria MQ1, comprising the following steps:

[0042] Based on the protein sequences of 3-hydroxy-3-methylglutaryl-CoA reductase HMGR (accession number: P12683) and farnesyl pyrophosphate synthase (FPPS, accession number: P08524; squalene synthase SQS, accession number: P29704) from *Saccharomyces cerevisiae* provided by UniProt, Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the proteins artificially and optimize the sequences according to the codon bias of *Pichia pastoris*. The sequences were constructed on the plasmid pPGAP, with the truncated HMGR, i.e., tHMG1, representing amino acids 552-1054 of HMGR. Expression plasmids pPGAP-SctHMG1, pPGAP-ScFPPS, and pPGAP-ScSQS were obtained. The nucleotide sequence of the truncated form tHMG1 of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase HMGR in the MVA pathway is shown in SEQ ID NO.5; the nucleotide sequence of the farnesyl pyrophosphate synthase FPPS is shown in SEQ ID NO.6; and the nucleotide sequence of the squalene synthase SQS is shown in SEQ ID NO.7.

[0043] (1) Expression box construction

[0044] Using the primer pairs and templates in Table 2, the following fragments were amplified by PCR: pGAP-SctHMG1-tAOX1 (the truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 gene expression cassette, also known as module M1), pGAP-ScSQS-tAOX1 (the squalene synthase SQS gene expression cassette, also known as module M2), tTEF1, and pGAP-S.cFPPS. The amplified tTEF1 and pGAP-ScFPPS fragments were then fused to obtain the pGAP-ScFPPS-tTEF1 gene expression cassette (the farnesyl pyrophosphate synthase FPPS gene expression cassette, also known as module M3).

[0045] (2) Pichia pastoris transformation

[0046] A single colony of Pichia pastoris GS115 was inoculated into 3 mL of YPD liquid medium and cultured for 12 h at 30 °C and 220 rpm in a shaker. The cultured yeast seed culture was then transferred to a fresh 3 mL of YPD liquid medium and cultured for 5 h at 30 °C and 220 rpm. 1.5 mL of the culture was transferred to a pre-sterilized 1.5 mL centrifuge tube and centrifuged at 4000 rpm for 1 min. The supernatant was discarded, and the bacterial cells were collected. This process was repeated to collect the remaining 1.5 mL of bacterial cells. The cells were washed once with 1 mL of sterile water, centrifuged at 4000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were collected. Then, 1 mL of 1 M sorbitol aqueous solution was added to the bacterial cells, followed by washing. The cells were centrifuged at 4000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were collected. This washing process was repeated once. Resuspend competent cells in 50 μL of 1M sorbitol aqueous solution in a centrifuge tube containing bacterial pellet. Add 300 ng of PNS4-2 CRISPR plasmid and 300 ng each of expression cassette modules M1, M2, and M3, mix well, and aspirate into a 2 mm electroporation cuvette. Incubate on ice for 5 min. Then, electroporate using the Pichia pastoris electroporation program preset on the BIO-rad electroporator system. Gently mix with 1 mL of 1M sorbitol aqueous solution using a pipette and aspirate into a clean, sterile 1.5 mL centrifuge tube. Incubate at 30°C and 220 rpm for 2 h. Spread the culture onto YPD plates containing 1 mg / mL G418 and incubate at 30°C for 3 days. Once single colonies have grown, screen to obtain recombinant bacteria MQ1.

[0047] Table 2 Primers and templates for constructing recombinant bacterial MQ1

[0048] Example 3: A method for constructing recombinant bacteria MQ2, comprising the following steps:

[0049] Based on the endogenous Pichia pastoris ERG8 (PAS_chr2-2_0318) and ERG10 (PAS_chr1-4_0304) provided by GenBank, primers were directly designed to construct the relevant expression cassette using the Pichia pastoris GS115 genome as a template for PCR amplification. The nucleotide sequence of the mevalonate kinase ERG8 is shown in SEQ ID NO.8; the nucleotide sequence of the acetyl-CoA thiolase ERG10 is shown in SEQ ID NO.9.

[0050] (1) Expression box construction

[0051] Using the primer pairs and templates in Table 3, the fragments pGAP, ERG8, tDAS1, tERG10-ERG10, and pTEF1 were amplified by PCR. The pGAP, ERG8, and tDAS1 fragments were fused to obtain the pGAP-ERG8-tDAS1 gene expression cassette (mevaleric acid kinase ERG8 gene expression cassette, also known as module M4); the tERG10-ERG10 and pTEF1 fragments were fused to obtain the pGAP-ERG10-tERG10 gene expression cassette (acetyl-CoA thiolase ERG10 gene expression cassette, also known as module M5).

[0052] (2) Pichia pastoris transformation

[0053] Following the Pichia pastoris transformation method in step (2) of Example 1, the 300ng module M4, 300ng module M5, and 300ng site PNS2-8 CRISPR plasmid obtained in step (1) were transformed into the recombinant MQ1 competent cells, plated on YPD plates containing 1mg / mLG418, and placed in a 30℃ constant temperature incubator for 3 days. After single colonies grew, the recombinant MQ2 was obtained by screening.

[0054] Table 3 Primers and templates for constructing recombinant bacterial MQ2

[0055] Example 4: A method for constructing recombinant bacteria MQ3, comprising the following steps:

[0056] Based on ERG1 (accession number: P32476) from *Saccharomyces cerevisiae* and DDS (accession number: Q08IT1) from ginseng provided by UniProt, Beijing Qingke Biotechnology Co., Ltd. was commissioned to use artificial synthesis methods and optimize the sequences according to the codon bias of *Pichia pastoris* to construct the plasmid pPGAP, obtaining pPGAP-ScERG1, pPGAP-PgDDS, and expression plasmids. The nucleotide sequence of the squalene oxidase ERG1 is shown in SEQ ID NO. 10; the nucleotide sequence of the dammarene diol synthase DDS gene is shown in SEQ ID NO. 11.

[0057] (1) Expression box construction

[0058] Using the primer pairs and templates in Table 4, pGAP-ERG1-tAOX1 (squalene oxidase ERG1 gene expression cassette, also known as module M6) was amplified by PCR. The tTEF1 and pGAP-PgDDS fragments were fused to obtain the pGAP-PgDDS-tTEF1 gene expression cassette (dammarene diol synthase DDS gene expression cassette, also known as module M7).

[0059] (2) Pichia pastoris transformation

[0060] Following the Pichia pastoris transformation method in step (2) of Example 1, the 300ng module M6, 300ng module M7, and 300ng site PNS4-10 CRISPR plasmid obtained in step (1) were transformed into the recombinant MQ2 competent cells, plated on YPD plates containing 1mg / mLG418, and placed in a 30℃ constant temperature incubator for 3 days. After single colonies grew, the recombinant MQ3 was obtained by screening.

[0061] Table 4 Primers and templates for constructing recombinant bacterial MQ3

[0062] Example 5: A method for constructing recombinant bacteria MQ4, comprising the following steps:

[0063] Based on the key endogenous glycerol metabolism enzymes GUT1 (accession number: F2QY70) and GUT2 (accession number: F2QW06) in Pichia pastoris provided by UniProt, the absorption and metabolism of glycerol were enhanced to ensure that the recombinant strain could grow and synthesize products using glycerol as the sole carbon source. GUT1 and GUT2 were transformed into the HRD1 site of strain MQ3. The HRD1 gene encodes an E3 ubiquitin-protein ligase involved in endoplasmic reticulum-related degradation processes. Inactivation of this gene leads to increased levels of triglycerides and sterol esters, thereby exacerbating lipid droplet (LD) formation, thus constructing the recombinant strain MQ4. The nucleotide sequence of the glycerol kinase GUT1 is shown in SEQ ID NO.12; the nucleotide sequence of the glycerol-3-phosphate dehydrogenase GUT2 is shown in SEQ ID NO.13.

[0064] (1) Expression box construction

[0065] Using the primer pairs and templates in Table 5, pGAP, GUT1, tDAS1, tTEF1, pGAP, and GUT2 were amplified by PCR. The pGAP, GUT1, and tDAS1 fragments were fused to obtain the pGAP-GUT1-tDAS1 gene expression cassette (glycerol kinase GUT1 gene expression cassette, also known as module M8); the tTEF1, pGAP, and GUT2 fragments were fused to obtain the pGAP-PgGUT2-tTEF1 gene expression cassette (glycerol-3-phosphate dehydrogenase GUT2 gene expression cassette, also known as module M9).

[0066] (2) Pichia pastoris transformation

[0067] Following the Pichia pastoris transformation method in step (2) of Example 1, the 300ng module M8, 300ng module M9, and 300ng site HRD1 CRISPR plasmid obtained in step (1) were transformed into recombinant MQ3 competent cells, plated on YPD plates containing 1mg / mLG418, and placed in a 30℃ constant temperature incubator for 3 days. After single colonies grew, recombinant MQ4 was obtained by screening.

[0068] Table 5 Primers and templates for constructing recombinant bacterial MQ4

[0069] Example 6: A method for constructing recombinant bacterial MQ5, comprising the following steps:

[0070] Based on PgPPDS (accession number: H2DH16) from ginseng and ATR1 (accession number: QqSB48) from Arabidopsis thaliana provided by UniProt, Beijing Qingke Biotechnology Co., Ltd. was commissioned to use artificial synthesis methods and optimize the sequences according to the codon preference of Pichia pastoris. The two genes were constructed into the plasmid pPGAP using LinkerGGGGS, resulting in the pPGAP-PgPPDS-GGGGS-ATR1 fusion plasmid. The gene expression cassette of the protopanaxadiol synthase PPDS and its reductase fusion protein PPDS-ATR1 was transferred into the PAH1 site of MQ4. The PAH1 gene encodes a key phosphatidylphosphatase, which participates in the competitive branching pathway of phospholipid synthesis. Due to the increased phospholipid supply, the disruption of the PAH1 gene can lead to an increase in the volume of the endoplasmic reticulum (ER), thereby opening up the protopanaxadiol synthesis pathway and increasing the spatial structure of the ER, resulting in strain MQ5. The nucleotide sequence of the fusion gene of the PPD synthase PPDS and its reductase ATR1 is shown in SEQ ID NO.14 (the nucleic acid sequence corresponding to the wild-type amino acid sequence of SEQ ID NO.1);

[0071] (1) Expression box construction

[0072] Using the primer pairs and templates in Table 6, pGAP-PgPPDS-GGGGS-ATR1 (the gene expression cassette of the fusion protein PPDS-ATR1 of ginsenoside diol synthase PPDS and its reductase, also known as module M10) was amplified by PCR.

[0073] (2) Pichia pastoris transformation

[0074] Following the Pichia pastoris transformation method in step (2) of Example 1, the 300ng module M10 and 300ng site PAH1 CRISPR plasmid obtained in step (1) were transformed into recombinant MQ4 competent cells, plated on YPD plates containing 1mg / mL G418, and placed in a constant temperature incubator at 30℃ for 3 days. After single colonies grew, recombinant MQ5 was obtained by screening.

[0075] Table 6 Primers and templates for constructing recombinant bacterial MQ5

[0076] Example 7: Method for constructing recombinant bacteria MQ6, 7, 8, and 9, comprising the following steps:

[0077] The mutant plasmids pPGAP-PgPPDS(N178S)-GGGGS-ATR1, pPGAP-PgPPDS(K235F)-GGGGS-ATR1, pPGAP-PgPPDS(Y312N)-GGGGS-ATR1, and pPGAP-PgPPDS(N178S-Y312N)-GGGGS-ATR1 were constructed using site-directed mutagenesis. Primers were designed as shown in Table 7 to embed the target mutation sites. PCR was used to amplify the fragment containing the mutated amino acid site using pPGAP-PgPPDS-GGGGS-ATR1 as a template. The PCR product was purified using a DNA purification kit and transformed into *E. coli* DH5α competent cells. The cells were plated on LB (50 μg / mL zeocin) plates. Transformants were picked and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the results showed that the mutant plasmids were obtained. The mutant PgPPDS-GGGGS-ATR1 gene expression cassette was constructed and transformed into MQ4 to construct mutant strains MQ6-MQ9. The nucleotide sequences of the fusion gene PgPPDS-GGGGS-ATR1, which is a fusion of the mutant PPD synthase PPDS and its reductase ATR1, are as follows:

[0078] Positive mutation group: pPGAP-PgPPDS(N178S)-GGGGS-ATR1 (the nucleic acid sequence corresponding to the N178S mutant amino acid sequence shown in SEQ ID NO.2) see SEQ ID NO.15;

[0079] Negative mutation group: pPGAP-PgPPDS(K235F)-GGGGS-ATR1 (comparative mutation, i.e., the nucleic acid sequence corresponding to the amino acid sequence of K235F in the original amino acid sequence) see SEQ ID NO.16;

[0080] Positive mutation group: pPGAP-PgPPDS(Y312N)-GGGGS-ATR1 (the nucleic acid sequence corresponding to the Y312N mutant amino acid sequence shown in SEQ ID NO.3) see SEQ ID NO.17;

[0081] Positive mutation group: pPGAP-PgPPDS(N178S-Y312N)-GGGGS-ATR1 (the nucleic acid sequence corresponding to the N178S-Y312N mutant amino acid sequence shown in SEQ ID NO.4) see SEQ ID NO.18;

[0082] (1) Expression box construction

[0083] Using the primer pairs and templates in Table 7, the gene expression cassettes of the mutated protopanaxadiol synthase PPDS and its reductase fusion protein PPDS-ATR1 were amplified by PCR: pGAP-PgPPDS(N178S)-GGGGS-ATR1 (also known as module M11); pGAP-PgPPDS(K235F)-GGGGS-ATR1 (also known as module M12); pGAP-PgPPDS(Y312N)-GGGGS-ATR1 (also known as module M13); and pPGAP-PgPPDS(N178S-Y312N)-GGGGS-ATR1 (also known as module M14).

[0084] (2) Pichia pastoris transformation

[0085] Following the Pichia pastoris transformation method in step (2) of Example 1, modules M11, M12, M13, and M14 obtained in step (1) were transformed into recombinant MQ4 competent cells with 300ng PAH1 CRISPR plasmids, respectively. The cells were then plated on YPD plates containing 1 mg / mL G418 and incubated at 30°C for 3 days. Once single colonies grew, recombinant MQ6~MQ9 were obtained through screening.

[0086] Table 7 Primers and templates for constructing recombinant bacteria MQ6-MQ9

[0087] Example 8: Fermentation process and extraction and determination of metabolites of recombinant bacteria:

[0088] (1) Fermentation process and metabolite extraction of recombinant bacteria

[0089] The recombinant bacteria containing complete protopanaxadiol, namely MQ5 (original gene recombinant bacteria), MQ6 (N178S mutant recombinant bacteria), MQ7 (K235F mutant recombinant bacteria, and unfavorable mutant control recombinant bacteria), MQ8 (Y312N mutant recombinant bacteria), and MQ9 (N178S-Y312N mutant recombinant bacteria), were constructed above and subjected to shake-flask fermentation to detect the yield of protopanaxadiol (PPD) in the metabolites.

[0090] Single colonies of recombinant bacteria MQ5, MQ6, MQ7, MQ8, and MQ9 were inoculated into 3 mL of YPG liquid medium and cultured overnight at 30°C and 220 rpm. An appropriate amount of seed culture was inoculated into a shake flask containing 30 mL of YPG liquid medium to make the initial OD of the fermentation broth 0.1, and fermentation was carried out for 5 days.

[0091] After fermentation, 1 mL of fermentation broth was taken, centrifuged and the supernatant was discarded. 1 mL of methanol:acetone (1:1 v / v) and quartz sand of equal volume to the cell volume were added to the precipitate. The mixture was vortexed for 30 minutes, sonicated for 30 minutes, centrifuged at 12000 rpm for 5 minutes, and the upper organic phase was collected. After filtration through a 0.22 μm filter membrane, the mixture was used for HPLC analysis.

[0092] (2) HPLC-MS determination of metabolites Chromatographic conditions: DAD detector, Waters Symmetry® C18 column (250 mm × 4.6 mm, 5 μm), mobile phase A is 10% methanol, mobile phase B is acetonitrile, isocratic elution, A:B is 10:90, flow rate is 1.0 mL / min, detection time is 25 min, column temperature is 30℃, detection wavelength is 203 nm; a standard curve of protopanaxadiol (PPD) concentration was prepared for quantitative analysis of the yield of protopanaxadiol (PPD) in the metabolites of the strain.

[0093] (3) The results showed that the PPD yields of ginsenoside diol in recombinant bacteria MQ5, MQ6, MQ7, MQ8 and MQ9 were 243.07 mg / L, 269.77 mg / L, 178.30 mg / L, 279.47 mg / L and 292.43 mg / L, respectively.

[0094] (4) YPD liquid culture medium used in the examples: final concentration of glucose 20 g / L, final concentration of yeast extract 10 g / L, final concentration of peptone 20 g / L, prepared with distilled water; YPD solid culture medium was prepared by adding 20 g / L of agar powder to YPD liquid culture medium; YPG liquid culture medium used: final concentration of glycerol 20 g / L, final concentration of yeast extract 10 g / L, final concentration of peptone 20 g / L, prepared with distilled water; LB liquid culture medium used: final concentration of yeast extract 50 g / L, final concentration of peptone 10 g / L, final concentration of sodium chloride 10 g / L, prepared with distilled water; LB solid culture medium was prepared by adding 20 g / L of agar powder to LB liquid culture medium.

[0095] In summary, single-point or double-point mutations of the amino acid sequence shown in SEQ ID NO.1 at N178S and Y312N can improve enzyme activity and enhance the synthesis efficiency and selectivity of protopanaxadiol PPD.

[0096] sequence: SEQ ID NO.1 Pg PPDS MAAAMVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVKYRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVE KMFPRSHGESNADNFSKVRGKMMFLLKVDGMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTMSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPINIPGTAMNRAIKTVKLLTRE VEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYTTLNGTITFVLNYLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPG VGTFREAITDFTYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVVTNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN; SEQ ID NO.2 Pg PPDS-N178S MAAAMVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVKYRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVE KMFPRSHGESNADNFSKVRGKMMFLLKVDGMKKYVGLMDRVMKQFLETDWNRQQQISVHNTVKKYTVTMSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPINIPGTAMNRAIKTVKLLTRE VEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYTTLNGTITFVLNYLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPG VGTFREAITDFTYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVVTNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN; SEQ ID NO. 3 Pg PPDS-Y312N MAAAMVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVKYRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVE KMFPRSHGESNADNFSKVRGKMMFLLKVDGMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTMSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPINIPGTAMNRAIKTVKLLTRE VEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYTTLNGTITFVLNNLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPG VGTFREAITDFTYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVVTNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN; SEQ ID NO. 4 Pg PPDS -N178S-Y312N MAAAMVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVKYRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVEKMFPRSHGESNADNFSKVRGKMMFLLKVDGMKKYVGLMDRVMKQFLETDWNRQQQISVHNTVKKYTVTMSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPINIPGTAMNRAIKTVKLLTREVEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYTTLNGTITFVLNNLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPGVGTFREAITDFTYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVVTNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN; SEQ ID NO.5 SC THMG1 SEQ ID NO.6 SC FPPS SEQ ID NO.7 SC SQS SEQ ID NO.8 ERG8 SEQ ID NO.9 ERG10 SEQ ID NO.10 SC ERG1 SEQ ID NO.11 PG DDS SEQ ID NO.12 PP GUT1 SEQ ID NO.13 GUT2 SEQ ID NO. 14 PG PPDS-GGGGS-ATR1 SEQ ID NO.15 PG PPDS(N178S)-GGGGS-ATR1 SEQ ID NO. 16 PG PPDS(K235F)-GGGGS-ATR1 SEQ ID NO.17 PG PPDS(Y312N)-GGGGS-ATR1 SEQ ID NO.18 PG PPDS(N178S-Y312N)-GGGGS-ATR1

[0097] Primer sequences: PNS4-2: GTACAATTCACTCGATACGT; PNS2-8: GTTACTGGATTAGAACGACG; PNS4-10: TACCAAGTTAATCAGCAGCG; HRD1: TGGAATAGACCAGGAAGAAT; PAH1: ATTACTAAATCTGATGCCTT; (4-2)-PGAP-F: AGTAGTGTAGTGGGAGACCATACGCGAAACTCAGGTGCTGCAATTTTCATTTTTTGTAGAAATGTCTTGGTGTCC; (PGAP)-TAOX1-R: CAGAGATGGCTACCTGATTGGACGAGGACACCAAGACATTTCTACAAAAATCTCACTTAATCTTCTGTACTCTGAAG; (TAOX1)-PGAP-F: TTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGATTTTTGTAGAAATGTCTTGGTGTCC; (TTEF1)-TAOX1-R: TAATCTATTGACTAGCAGCGACCTCTTCAACCGAAGGGCAAGTACACAGCTCTCACTTAATCTTCTGTACTCTGAAG; (TAOX1)-TTEF1-F: TTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAGCTGTGTACTTGCCCTTCGGTTGAAGAG; (FPPS)-TTEF1-R: TAAGGTTTACAAGAGAAGTAAATGAATTGCTTGAAGCTTTAATTTATTTTATTAAC; FPPS-F: TCATTTACTTCTCTTGTAAACCTTATTCAAG; (4-2)-PGAP-R: GGAGCGCAAATAAGAGATCAGGTCCAGCCAACTATGACCTTTCGTAGTTGTTTTTGTAGAAATGTCTTGGTGTCC; (2-8)-PGAP-F: AACACTGAGCTTTTCCCGCCGCCTATGTTACGGGAATCAACATCCGTGATTTTTTGTAGAAATGTCTTGGTGTCC; PGAP-R: ATAGTTGTTCAATTGATTGAAATAG; (PGAP)-ERG8-F: CTATTTCAATCAATTGAACAACTATATGAAAGCTTTCAGCGCTCCGAGCA; (TDAS1)-ERG8-R: ACTAAAACTGTAAAGACTTCCCGTTCTATTCTGCAAATAAATAGCTCTGGAC; TDAS1-F: AACGGGAAGTCTTTACAGTTTTAGTTAG; (TERG10)-TDAS1-R: TCAGGATAATCTTTCAATGATTTCCAAGAGTGCACTGTTGGCGTACCTAGTGACCCTTGTGACTGACACTTTG; (TDAS1)-TERG10-F: TATGAGACTAAGTAGAATAGGGACTCCCAAAGTGTCAGTCACAAGGGTCACTAGGTACGCCAACAGTGCAC; (PTEF1)-ERG10-R: AGAAAGCATAGCAATCTAATCTAAGATGAGTGAACCTGTTTACATTGTTA; PTEF1-F: CTTAGATTAGATTGCTATGCTTTCTTTC; (2-8)-PTEF1-R: CGTCGGCTGTTTCAGATTAAAGCAAGCAAGCAATCTGAAACTTGCGAATTGATCCCCCACACACCATAG; (4-10)-PGAP-F: ATTAATTATCGTTTTCGGGGCTGTTGACATGAATGAAATAGTGAGTTGATTTTTTGTAGAAATGTCTTGGTGTCC; (TTEF1)-TAOX1-R: TAATCTATTGACTAGCAGCGACCTCTTCAACCGAAGGGCAAGTACACAGCTCTCACTTAATCTTCTGTACTCTGAAG; (TAOX1)-TTEF1-F: TTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAGCTGTGTACTTGCCCTTCGGTTGAAGAG; (DDS)-TTEF1-R: AAAGCATCAACAACTGAAAATTTAGATTGCTTGAAGCTTTAATTTATTTTATTAAC; DDS-F: CTAAATTTTCAGTTGTTGATGCTTTG; (2-8)-PGAP-R: GCAGTTTTAATTTACTGATCATGACCCTATTATGTCTTTTAAACCCCGCTTTTTTGTAGAAATGTCTTGGTGTCC; (HRD1)-PGAP-F: CAACAGGACAGCAACTCCTCAAATAATACCTCATACCCTTCACTAAGTGTTTTTTGTAGAAATGTCTTGGTGTCC; (PGAP)-GUT1-F: CTATTTCAATCAATTGAACAACTATATGGGAAAAGACTATACACCATTGG; (TDAS1)-GUT1-R: ACTAAAACTGTAAAGACTTCCCGTTTTAAGCAGTGTCCTTGAGCCAG; (TTEF1)-TDAS1-F: TAATCTATTGACTAGCAGCGACCTCTTCAACCGAAGGGCAAGTACACAGCTGACCCTTGTGACTGACACTTTG; (TDAS1)-TTEF1-F: TATGAGACTAAGTAGAATAGGGACTCCCAAAGTGTCAGTCACAAGGGTCAGCTGTGTACTTGCCCTTCGGTTGAAGAG; (GUT2)-TTEF1-R: AAGTTTCTTGGAAAACGAGGTGTAGATTGCTTGAAGCTTTAATTTATTTTA; GUT2-F: CTACACCTCGTTTTCCAAGAAACTT; GUT2-R: ATGTTTCGTCAGCAGATTAGAAGAT; (GUT2)-PGAP-F: ATCTTCTAATCTGCTGACGAAACATATAGTTGTTCAATTGATTGAAATAGGGAC; (HRD1)-PGAP-R: TGTAAGATATTATTTTTGTTCCGTGCGTTGTGTATATGCTGTGGTAGTCCTTTTTGTAGAAATGTCTTGGTGTCC; (PAH1)-PGAP-F: TCAACATCTCTTCCTCTTCTCTGCTTCCTTTTTCCTCTTGTTTGTTATCATTTTTGTAGAAATGTCTTGGTGTCC; (PAH1)-PGAP-R: AATAATGTATATAAATAGAAAATGCAGATTTAACTAATACTTATTTACCCTCTCACTTAATCTTCTGTACTCTGA; N178-F: AGCGTACACAATACGGTCAAGAAATATAC; N178-R: TTCTTGACCGTATTGTGTACGCTGATTTGCTGCTGTCGGTTCC; K235-F: TTCACAGTTAAGTTGCTCACAAGGG; K235-R : TGTGAGCAACTTAACTGTGAAGATTGCTCTGTTCATAGCTGTTC; Y312-F: AACCTAGCCGAATTTCCTGACGTT; Y312-R: CGTCAGGAAATTCGGCTAGGTTATTCAAAACGAAAGTTATCGTCCC。

Claims

1. A PPDS mutant, characterized in that, The sequence of the PPDS mutant is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

2. An application of the PPDS mutant according to claim 1, characterized in that, It was used as an enzyme catalyst for the enzymatic synthesis of protopanaxadiol.

3. A gene capable of encoding the PPDS mutant of claim 1.

4. A recombinant plasmid loaded with the gene of the PPDS mutant of claim 3.

5. A recombinant bacterium capable of expressing the PPDS mutant of claim 1.

6. The recombinant bacteria as described in claim 5, characterized in that, To introduce recombinant bacteria containing the recombinant plasmid of claim 4.

7. The recombinant bacteria as described in claim 6, characterized in that, For introducing Pichia pastoris containing the recombinant plasmid of claim 4.

8. The recombinant bacteria according to any one of claims 5 to 7, characterized in that, The recombinant bacteria also contained the genes for 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, mevalonate phosphate kinase, acetyl-CoA thiolase, squalene oxidase, dammarene diol synthase, glycerol kinase (a key enzyme in glycerol metabolism), and glycerol-3-phosphate dehydrogenase.

9. The application of the recombinant bacteria according to claim 8 in the preparation of protopanaxadiol, characterized in that, The recombinant bacteria were fermented, and then protopanaxadiol was isolated.

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