Kaurene-like synthetase mutant, coding gene, genetically engineered bacterium and application thereof
By performing site-directed mutations and heterologous expression of similar kauriene synthetases, the problem of low catalytic efficiency was solved, and the efficient biosynthesis of setanshinone diene was achieved, which significantly improved yield and broke through the synthesis bottleneck.
Patent Information
- Application Number
- CN202510595226.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-08-08
AI Technical Summary
The existing kauridine synthases similar to kauridine are low catalytic efficiency, poor substrate specificity, and poor stereo/regional selectivity, resulting in insufficient biosynthesis yield of setanshinone diene and its downstream products.
By performing site-directed mutations on the amino acid sequence similar to kaurilene synthetase, specifically asparagine at 504 is muted to threonine or tyrosine at 518 is muted to cysteine, genetically engineered strains are constructed and heterologously expressed, improving the catalytic efficiency of the enzyme.
The efficient production of subtanshinone diene in Saccharomyces cerevisiae was achieved, and the fermentation yield was significantly improved, breaking through the bottleneck problem of heterologous synthesis, with the output reaching 13.9 mg/L and 16.2 mg/L, which was 31.3% and 53.0% higher than that of wild type, respectively.
Smart Images
Figure CN120442609A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of enzyme genetic engineering and enzyme engineering, and specifically relates to a kaurene synthase-like mutant, an encoding gene, a genetically engineered bacterium and applications thereof. Background Art
[0002] Plant-derived diterpenoids are a vast resource pool waiting to be developed and utilized. Abietane-type diterpenoids such as carnosic acid, tanshinone and triptolide have excellent biological activity and a wide range of clinical application value. Miltiradiene is an important intermediate in the biosynthesis of this type of diterpenoids. Establishing an efficient miltiradiene synthesis platform is crucial for the synthesis of downstream products. Currently, the sources of these diterpenoids rely on chemical synthesis and natural product extraction. However, the chemical synthesis pathway has complex steps and low conversion rates, while natural product extraction is easily limited by raw materials and difficult to produce on a large scale. The development of synthetic biology technology has provided a new biosynthetic model for these diterpenoids. The construction of miltiradiene-producing engineered strains has made it possible to prepare these diterpenoids on a large scale using biofermentation.
[0003] Kaurene-like synthase is a key, rate-limiting enzyme in the biosynthesis of hypotanshinone dienes. Natural kaurene-like synthases suffer from low catalytic efficiency, poor substrate specificity, poor stereo / regioselectivity, and instability, severely impacting the biosynthesis yield of hypotanshinone dienes and their downstream products. Therefore, increasing the activity of kaurene-like synthase is crucial for ensuring the efficient synthesis of hypotanshinone dienes and their downstream products. Summary of the Invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a mutant of a kaurene synthase.
[0005] The second object of the present invention is to provide a gene encoding the above-mentioned kaurene synthase-like mutant.
[0006] The third object of the present invention is to provide a recombinant plasmid containing the above encoding gene.
[0007] The fourth object of the present invention is to provide a genetically engineered bacterium containing the above-mentioned recombinant plasmid.
[0008] The fifth object of the present invention is to provide applications of the above-mentioned genetically engineered bacteria.
[0009] The technical solution of the present invention is summarized as follows:
[0010] The kaurene synthase-like mutant is a kaurene synthase-like amino acid sequence shown in SEQ ID NO. 1, wherein the asparagine at position 504 is mutated to threonine or the tyrosine at position 518 is mutated to cysteine.
[0011] A gene encoding the above-mentioned kaurene synthase-like mutant.
[0012] A recombinant plasmid containing the above encoding gene.
[0013] A genetically engineered bacterium containing the above-mentioned recombinant plasmid.
[0014] The application of the above genetically engineered bacteria in fermentation to prepare tanshinone diene and its downstream products.
[0015] The present invention has the following beneficial effects: The kaurene synthase-like mutants (N504T) and (Y518C) can be used to prepare highly valuable hypotanshinone dienes and their downstream products. This invention offers advantages such as high economic benefits, environmental friendliness, simple processes, and high catalytic efficiency, and has broad prospects for industrialization.
[0016] The present invention realizes the heterologous expression of a mutant of kaurene synthase, breaks through the bottleneck problem of heterologous synthesis of tanshinone diene, constructs a tanshinone diene microbial production chassis cell, and constructs a genetically engineered strain Sc027 / PESC-URA-IrCPS1-IrKSL1 (N504T) ( Figure 3 N504T), Sc027 / PESC-URA-IrCPS1-IrKSL1(Y518C) ( Figure 3 The fermentation yields of tanshinone dienes in 50 ml shake flasks of Y518C reached 13.9 mg / L and 16.2 mg / L, respectively, which were increased by 31.3% and 53.0% compared with the wild-type expression strain. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a site of amino acid mutation similar to that encoded by the kaurene synthase gene IrKSL1.
[0018] Figure 2 Schematic diagram of the expression plasmid (pESC-URA-IrCPS1-IrKSL1) similar to the kaurene synthase gene IrKSL1.
[0019] Figure 3 This is a graph showing the production of tanshinone diene by genetically engineered strains. DETAILED DESCRIPTION
[0020] The present invention discovered a codon-optimized kaurene synthase-like gene, IrKSL1 (SEQ ID NO. 2), from Isodon rubescens (corresponding amino acid sequence shown in SEQ ID NO. 1). By utilizing the characteristic that amino acid sequences of similar kaurene synthases with the same function share similar evolutionary conserved regions, site-directed mutagenesis was performed on the nucleotide sequence of the IrKSL1 gene, and kaurene synthase-like mutant genes, IrKSL1 (N504T) and (Y518C), were screened. Using the expression plasmid pESC-URA-IrCPS1 (synthesized by Jinweizhi Biotechnology Co., Ltd., IrCPS1: SEQ ID NO. 5) as a vector and Saccharomyces cerevisiae Sc0272 as a host, heterologous expression of the kaurene synthase-like mutants was achieved, significantly increasing the yield of tanshinone diene in Saccharomyces cerevisiae.
[0021] The Saccharomyces cerevisiae Sc0272 strain was derived from the commercially available CEN.PK2-1C strain (Source: Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering); the CYB5, ERG19, ERG8, ERG12, ERG10, tHMG1 (SEQ ID NO. 3), IDI1, ERG13, ERG20, ALDH1, CPR1, mERG20 (SEQ ID NO. 4), and PaCrtE genes were inserted into the CEN.PK2-1C genome via homologous recombination (gene accession numbers are shown in Table 1). The specific transformation from CEN.PK2-1C to Sc0272 was as follows:
[0022] leu2-3,112:G418 R _P GAL7 -CYB5_T ERG19 (RC)-ERG19(RC)-P GAL1 (RC)_P GAL10 -ERG8-T ERG8 ;
[0023] his3Δ1:HIS3_P GAL7 -ALDH1-T TDH1 _T ERG12 (RC)-ERG12(RC)-P GAL1 (RC)_P GAL10 -ERG10-TERG10 ;
[0024] ade1Δ:T HMG1 (RC)-tHMG1(RC)-P GAL1 (RC)_P GAL10 -IDI1-T IDI1 _ADE1;
[0025] ura3-52:T HMG1 (RC)-tHMG1(RC)-P GAL1 (RC)_P GAL10 -ERG13-T ERG13 ;
[0026] trp1-289:T HMG1 (RC)-tHMG1(RC)-P GAL1 (RC)_P GAL10 -ERG20-T ERG20 _TRP1;
[0027] gal1 / 10 / 7Δ:natA_P GAL3 -CPR1-T CYC1 ;
[0028] DPP1Δ::PGAL7-mERG20-TADH1;
[0029] LPP1Δ::PGAL7-PaCrtE-TCYC1.
[0030] Table 1 Gene accession number (NCBI)
[0031]
[0032] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
[0033] Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Primers and sequences were synthesized by Genewise Biotechnology Co., Ltd.
[0034] Example 1
[0035] Using the sequence alignment tool MEGA-X and the WEB-LOGO website, the amino acid sequence encoded by the kaurene synthase-like gene IrKSL1 (SEQ ID NO. 2) from Rubescens and optimized for Saccharomyces cerevisiae was compared with the amino acid sequences encoded by kaurene synthase-like genes from other sources, and the mutation sites N504 and Y518 were screened out. Figure 1 .
[0036] Example 2
[0037] The construction of a similar kaurene synthase mutant is as follows:
[0038] First, we commissioned Jinweizhi to synthesize a codon-optimized kaurene synthase-like gene, IrKSL1 (shown in SEQ ID No. 2). Using PCR, we used the nucleotide sequence shown in SEQ ID No. 2 as a template and the primer pairs shown in Table 2 as upstream and downstream primers (synthesized by Jinweizhi). We amplified the wild-type IrKSL1 gene and its mutants (N504T) and (Y518C), respectively. The genes were verified by agarose gel electrophoresis and then recovered and purified.
[0039] Table 2 Primer sequence list
[0040]
[0041] Example 3
[0042] The recombinant plasmid containing the gene encoding the kaurene synthase mutant was constructed as follows:
[0043] The pESC-URA-IrCPS1 plasmid was selected, and the plasmid was double-digested with restriction endonucleases BamHI and HindIII, verified by agarose gel electrophoresis, and then recovered and purified to obtain the linearized pESC-URA-IrCPS1 plasmid.
[0044] The wild-type and mutant IrKSL1 genes amplified in Example 2 were then ligated with the linearized pESC-URA-IrCPS1 plasmid using Vazyme's homologous recombinase CloneExpress II. The combined ligation mixtures were incubated at 37°C for 30 minutes to generate the wild-type and mutant IrKSL1 gene plasmids, which were then transformed into competent E. coli cells using the DH5α transfection method.
[0045] PCR verification was performed using IrKSL1-F / IrKSL1-R primers, and the strains corresponding to the positive bands were sent for DNA sequencing. Positive recombinant plasmids containing genes encoding wild-type and mutant (N504T) and (Y518C) kaurene synthase were obtained. The plasmid structures are shown in Figure 2 .
[0046] Example 4
[0047] The genetically engineered bacteria containing the above recombinant plasmid were constructed as follows:
[0048] The wild-type and mutant recombinant plasmids constructed in Example 3 were respectively introduced into the Saccharomyces cerevisiae Sc0272 strain using the lithium acetate transformation method. The Saccharomyces cerevisiae cells carrying the recombinant plasmids were plated onto SC-URA solid culture plates and cultured in a 30°C incubator for 2-3 days to obtain the engineered Saccharomyces cerevisiae strains Sc0272 / PESC-URA-IrCPS1-IrKSL1(N504T), Sc0272 / PESC-URA-IrCPS1-IrKSL1(Y518C), and the wild-type kaurene synthase-like expression strain Sc0272 / PESC-URA-IrCPS1-IrKSL1(WT).
[0049] SC-URA medium formula: 20 g / L glucose, 1.7 g / L YNB (amino-free yeast nitrogen source), 5 g / L ammonium sulfate, 2 g / L default amino acid mixture, solid medium needs to be supplemented with 2% agar powder.
[0050] The default amino acid mixture composition includes:
[0051] Arginine 2g, lysine 2g, alanine 2g, aspartic acid 2g, asparagine 2g, glutamine 2g, phenylalanine 2g, proline 2g, cysteine 2g, serine 2g, glutamic acid 2g, isoleucine 2g, threonine 2g, tryptophan 2g, tyrosine 2g, valine 2g, inositol 2g, glycine 2g, p-toluic acid 0.2g.
[0052] Example 5
[0053] The production of tanshinone diene by shake flask fermentation of an engineered strain of Saccharomyces cerevisiae is as follows:
[0054] The cerevisiae engineered strain Sc027 / PESC-URA-IrCPS1-IrKSL1 (N504T) constructed in Example 4 was selected ( Figure 3 N504T), Sc027 / PESC-URA-IrCPS1-IrKSL1(Y518C) ( Figure 3 Y518C), and Sc0272 / PESC-URA-IrCPS1-IrKSL1(WT) ( Figure 3Shake flask fermentation was performed using a single colony in 3 mL of SC-URA medium and incubated at 220 rpm at 30°C for 20 h. At an initial OD600 of 0.05, the colony was transferred to 50 mL of SC-URA medium and incubated for 96 h in a shake flask. At 24 h, galactose was added to a final concentration of 10 g / L to induce protein expression, and n-dodecane was added to a final volume concentration of 10% for biphasic fermentation. Ethanol was added to a final concentration of 10 g / L at 30 and 54 h to supplement the carbon source required for microbial growth. The fermentation end time was 96 h.
[0055] like Figure 3 As shown, the production of tanshinone diene by mutants N504T and Y518C reached 13.9 mg / L and 16.2 mg / L, respectively, which were increased by 31.3% and 53.0% compared with the wild-type expression strain.
[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention is conventional technology.
Claims
1. A mutant similar to kaurene synthase, characterized by SEQ In the amino acid sequence of the kaurene synthase-like enzyme shown in ID NO. 1, asparagine at position 504 is mutated to threonine or tyrosine at position 518 is mutated to cysteine.
2. A gene encoding the kaurene-like synthase mutant according to claim 1.
3. A recombinant plasmid containing the coding gene according to claim 2.
4. A genetically engineered bacterium containing the recombinant plasmid according to claim 3.
5. Use of the genetically engineered bacteria according to claim 4 in fermenting and preparing tanshinone diene and its downstream products.