Yeast engineering bacteria for producing steviol glycosides by fermentation

Abstract

The present application relates to the technical field of biosynthesis, in particular to a kind of whole fermentation production steviol glycoside yeast engineering bacteria and its application.The present application utilizes site-directed mutagenesis technique to carry out mutation to wild type kaurenoic acid 13-hydroxylase, obtains G481L / Q159L mutant, and the mutant is applied to the production of steviol or steviol glycoside, constructs steviol or steviol glycoside synthesis pathway in saccharomyces cerevisiae host, realizes the efficient production of steviol or steviol glycoside, simultaneously by strengthening the level of diterpene precursor synthesis gene (MVA pathway, acetyl coenzyme A, GGPP) of chassis cell, further improves the yield of steviol glycoside, and reduces the cost for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and in particular to an engineered yeast strain for the complete fermentation production of steviol glycosides and its applications. Background Technology

[0002] Steviol glycosides are a general term for stevia extract. They are chemically stable, have only 1 / 300th the calories of sucrose, but possess 300 times the sweetness. Steviol glycosides can replace sucrose as a sweetener in various fields such as food, beverages, medicine, and chemicals. Currently, various sweet components have been isolated from stevia. These are all composed of different types (glucosyl, xylose, and rhamnosyl) of glycosyl groups linked by β-bonds at the C13 or C19 position of steviol, such as rebaudioside A, rebaudioside B, rebaudioside M, rebaudioside D, and rebaudioside E.

[0003] In the existing technology, the production of steviol glycosides mainly adopts three methods: (1) plant extraction method; (2) enzyme conversion method; (3) microbial fermentation synthesis method. Plant extraction involves using extraction processes to obtain steviol glycosides from stevia. However, the natural content of high-end components such as Reb M and Reb D in stevia is extremely low, resulting in low unit yield and significant impact of climate and pests on yield. Chemical synthesis uses chemical catalysis, with steviol as a raw material, to gradually introduce glucose groups at C13 and C19 through chemical glycosylation, yielding steviol glycosides of different sugar chain lengths, such as rebaudioside M and rebaudioside D. However, this method inevitably uses solvents and catalysts during production, and the safety of the product due to residual risks is a widespread problem. Enzymatic conversion utilizes glycosyltransferases to contact the substrate rebaudioside, which is then catalyzed by the enzyme to generate the target rebaudioside. Currently, microbial fermentation synthesis uses genetic engineering to construct genetically engineered strains for de novo synthesis of steviol glycosides. It has advantages such as low cost, unrestricted raw materials, simple extraction process, no seasonality, and low environmental pollution, thus gaining popularity among researchers and becoming the mainstream direction to replace traditional processes.

[0004] Microbial fermentation synthesis can be achieved by constructing a steviol backbone (introducing the enzymes required for steviol production) and a glycosylation module (introducing UDP-glycosyltransferase) in yeast, using glucose as a substrate for fermentation to obtain steviol glycosides. This method realizes the complete synthesis from glucose to the target glycoside without the need for exogenous precursors, and has the advantages of low raw material cost and flexible product adjustment. In this technical route, kaurenoic acid 13-hydroxylase (KAH) is a key enzyme involved in the biosynthesis of diterpenoids. It is responsible for catalyzing the hydroxylation of kaurenoic acid at the 13-carbon atom to form steviol, which is an essential step in steviol biosynthesis and a key node in the formation of the steviol core structure of steviol glycosides. The catalytic efficiency of KAH determines the upper limit of steviol supply, thus affecting the conversion efficiency of the downstream glycosylation module in catalyzing the conversion of steviol to steviol glycosides.

[0005] To further increase the yield of steviol glycosides, it is urgent to improve the catalytic efficiency of key enzymes and, by combining metabolic engineering and gene expression regulation strategies, construct highly efficient steviol glycoside-producing engineered strains to improve the expression level of key enzymes in the synthetic pathway and overcome the rate-limiting bottleneck of the pathway. Summary of the Invention

[0006] In view of this, the present invention provides a kaurene acid 13-hydroxylase mutant and its application in the whole fermentation of steviol glycosides. The mutant has higher activity than the wild type. When applied to the production of steviol or steviol glycosides, a steviol or steviol glycoside synthesis pathway is constructed in the Saccharomyces cerevisiae host to achieve efficient production of steviol or steviol glycosides. At the same time, by enhancing the level of diterpenoid precursor synthesis genes in chassis cells (MVA pathway, acetyl-CoA, GGPP), the yield of steviol glycosides is further improved.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] One of the technical solutions provided by the present invention is a kaurenoic acid 13-hydroxylase (KAH) mutant, which is obtained by the following mutations on the basis of wild-type kaurenoic acid 13-hydroxylase as shown in SEQ ID NO.1: G481L and Q159L.

[0009] Furthermore, the amino acid sequence of the kaurene 13-hydroxylase (KAH) mutant is shown in SEQ ID NO.3.

[0010] The present invention also provides a nucleic acid molecule, wherein the nucleic acid molecule is a nucleotide sequence encoding the kaurene 13-hydroxylase mutant described in one of the above-mentioned technical solutions.

[0011] Furthermore, the nucleic acid molecule includes a DNA molecule with a coding sequence as shown in SEQ ID NO:4; Furthermore, the nucleic acid molecule also includes a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID NO: 4; The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as mRNA or hnRNA.

[0012] The second technical solution provided by the present invention is a recombinant vector or recombinant strain containing the nucleic acid molecules described above.

[0013] In some specific embodiments, the expression plasmids used in the recombinant vector include, but are not limited to: pESC-His, pESC-Leu, and pESC-Ura.

[0014] In some specific embodiments, the host used for the recombinant strain includes, but is not limited to: Saccharomyces cerevisiae, Yersinia lipolytica, Kluyveromyces lactis, and Hansenula polymorpha.

[0015] Preferably, the host used for the recombinant strain is *Saccharomyces cerevisiae*. S. cerevisiae CEN.PK2-1C.

[0016] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution in the kaurene 13-hydroxylase mutant described in the first technical solution.

[0017] The fourth technical solution provided by the present invention is the application of the kaurene acid 13-hydroxylase mutant described in the first technical solution and the recombinant vector or recombinant strain described in the second technical solution in the production of steviol or steviol glycosides.

[0018] In some specific embodiments, the steviol glycosides include, but are not limited to: rebaudioside A, rebaudioside D, rebaudioside M, and rebaudioside I.

[0019] The fifth technical solution provided by this invention is a strain that produces stevia, wherein the strain is obtained by expressing the kaurene 13-hydroxylase mutant described in one of the technical solutions in a host; Furthermore, it also expresses cytochrome P450 reductase CPR in the host; Furthermore, it also expresses kauriene synthase GfKS and kauriene oxidase KO in the host.

[0020] The sixth technical solution provided by the present invention is a yeast engineered strain for the production of steviol glycosides by total fermentation. The engineered strain is obtained by further expressing one or more of UDP-glucosyltransferases UGT11, UGT12, UGT13, UGT14, UGT15, UGT16 or UGT17 in the host to construct a production pathway from steviol to rebaudioside. In some specific embodiments, the host strain further includes any one or more of the following gene edits: (1) Overexpression of 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1 and / or isoprene pyrophosphate isomerase IDI1; the expression is single-copy or multiple-copy expression; (2) Knock out the transcriptional regulatory protein GAL80; (3) Downregulate squalene synthase ERG9; (4) Overexpression of fenestration pyrophosphate synthase ERG20 and / or geraniol geraniol pyrophosphate synthase BTS1; (5) Overexpression of gerany-gerany pyrophosphate synthase CrtE; the expression is single-copy or multiple-copy expression; In some specific embodiments, the GenBank number of the cytochrome P450 reductase CPR is: CAA46815.1; In some specific embodiments, the GenBank number of the kauriene synthase GfKS is: BAA84917.1; In some specific embodiments, the GenBank number of the kauriene oxidase KO is: AAC39507.1; In some specific embodiments, the GenBank number of the 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1 is: M22002.1; In some specific embodiments, the NCBI Reference Sequence of the isoprene pyrophosphate isomerase IDI1 is: NP_015208.1; In some specific embodiments, the NCBI Reference Sequence of the transcriptional regulatory protein GAL80 is: NM_001182409.1; In some specific embodiments, the GenBank number of the squalene synthase ERG9 is: X59959.1; In some specific embodiments, the GenBank number of the farnesyl pyrophosphate synthase ERG20 is: J05091.1; In some specific embodiments, the GenBank number of the geraniol geraniol pyrophosphate synthase BTS1 is: U31632.1; In some specific embodiments, the NCBI Reference Sequence of the geraniol geraniol pyrophosphate synthase CrtE is: WP_010888034.1; In some specific embodiments, the GenBank numbers of the UDP-glucosyltransferases UGT11, UGT12, UGT13, and UGT14 are AWU66064.1, AWU66065.1, AAM53963.1, and ACT33422.1, respectively. In some specific embodiments, the NCBI Reference Sequence of the UDP-glucosyltransferase UGT15 is: XP_015629141.1; In some specific embodiments, the nucleotide sequence of the UDP-glucosyltransferase UGT16 is shown in SEQ ID NO. 5; In some specific embodiments, the nucleotide sequence of the UDP-glucosyltransferase UGT17 is shown in SEQ ID NO. 6.

[0021] Furthermore, the host includes, but is not limited to: Saccharomyces cerevisiae, Yersinia lipolytica, Kluyveromyces lactis, or Hansenula polymorpha; preferably Saccharomyces cerevisiae CEN.PK2-1C.

[0022] The seventh technical solution provided by the present invention is the application of the strain described in the fifth technical solution in the production of stevioside.

[0023] The eighth technical solution provided by this invention is the application of the engineered bacteria described in the sixth technical solution in the production of steviol glycosides through total fermentation; In some specific embodiments, steviol glycosides are produced using glucose or sucrose as a substrate and the steviol glycoside-producing strains described above. Furthermore, the steviosides include: rebaudioside A, rebaudioside D, rebaudioside M, and rebaudioside I.

[0024] The present invention has the following beneficial effects: 1. This invention utilizes site-directed mutagenesis to mutate wild-type kaurene acid 13-hydroxylase to obtain the G481L / Q159L mutant, and applies the mutant to the production of steviol or steviol glycosides; the KAH mutant of this invention has higher activity than the wild type, and the yield of steviol or steviol glycosides is significantly improved.

[0025] 2. This invention enhances the level of chassis diterpene precursor synthesis genes: by overexpressing and integrating multiple copies of tHMG1 and IDI1, the flux of the yeast endogenous MVA pathway is enhanced; by knocking out GAL80, the yeast strain is freed from galactose control and responds to glucose concentration; by downregulating ERG9, competitive pathways are reduced; by overexpressing ERG20 and BTS1, the flux of GGPP is enhanced, improving the synthesis efficiency of downstream diterpenoids; by overexpressing CrtE, the supply of upstream terpene precursor GGPP is enhanced, ultimately obtaining an engineered yeast strain that produces steviol glycosides through total fermentation using glucose or sucrose as substrates, further increasing the yield of steviol glycosides and reducing costs for industrial production. Detailed Implementation

[0026] This invention discloses a kaurene acid 13-hydroxylase mutant and its application in the fully fermented steviol glycosides. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0027] In this article, amino acids are represented by single-letter or three-letter codes, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0028] In this document, mutation sites are represented in the form "XaY", where a represents the position of an amino acid in SEQ ID NO.1, X represents the wild-type amino acid at position a in SEQ ID NO.1, and Y represents the amino acid after mutation at position a in SEQ ID NO.1. For example, in this invention, "G481L / Q159L" indicates that glycine G at position 481 of SEQ ID NO.1 is mutated to leucine L, and glutamine Q at position 159 is mutated to leucine L.

[0029] The following identifiers are used in this invention and its embodiments: The “S series” strains, namely the strains numbered S001-S002, are chassis bacteria that produce stevioside. "M series" strains, that is, strains numbered M0XX, are Reb M production strains, such as M001 and M002; "D series" strains, that is, strains numbered D0XX, are Reb D production strains, such as D001 and D002; "Series I" strains, i.e. strains numbered I0XX, are Reb I production strains, such as I001 and I002; "Series A" strains, i.e. strains numbered A0XX, are Reb A production strains, such as A001 and A002.

[0030] The present invention provides a kaurenoic acid 13-hydroxylase mutant, which is obtained by the following mutation on the basis of wild-type kaurenoic acid 13-hydroxylase as shown in SEQ ID NO.1: G481L / Q159L.

[0031] The wild-type kaurenoic acid 13-hydroxylase KAH of this invention is derived from Arabica mustard ( Arabidopsisthaliana The amino acid sequence is shown in SEQ ID NO. 1: ; The nucleic acid sequence of the gene encoding the wild-type kaurenoic acid 13-hydroxylase KAH is shown in SEQ ID NO. 2; The KAH (G481L / Q159L) mutant of the present invention has the amino acid sequence shown in SEQ ID NO. 3; ; The nucleic acid sequence of the coding gene of the KAH(G481L / Q159L) mutant of the present invention is shown in SEQ ID NO. 4.

[0032] Other gene or protein sequence information involved in this application is as follows: UGT16, UDP-glucosyltransferases, has the nucleotide sequence shown in SEQ ID NO. 5; UGT17, UDP-glucosyltransferases, has the nucleotide sequence shown in SEQ ID NO. 6.

[0033] The culture medium used in this application is as follows: YPGal liquid medium: Yeast extract 10 g / L, Peptone 20 g / L, Galactose 20 g / L; YPD liquid medium: Yeast extract 10g / L, Peptone 20g / L, Glucose 20g / L; SC-URA plate medium: glucose 20 g / L, agar powder 15 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, histidine 0.02 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L; LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L; SC-His medium: glucose 20 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, uracil 0.02 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L; SC-His plate medium: glucose 20 g / L, agar powder 15 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, uracil 0.02 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L; SC plate medium containing 5-FOA (5-fluoroorotic acid): glucose 20 g / L, 5-fluoroorotic acid 1 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, histidine 0.02 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L, uracil 0.02 g / L, agar powder 15 g / L.

[0034] The strains and vectors involved in the experiments of this invention are as follows: Chassis cells: Saccharomyces cerevisiae ( S. cerevisiae CEN.PK2-1C. This application uses *Saccharomyces cerevisiae* (CEN.PK2-1C). S. cerevisiae The strain CEN.PK2-1C (from the China Microbial Culture Bank, number Bio-116324) was used as the starting strain for construction and modification.

[0035] pESC-His plasmid was purchased from Hangzhou Baosai Biotechnology.

[0036] pESC URA plasmid was purchased from Shanghai Qincheng Biotechnology Co., Ltd.

[0037] pCAS9-lacZ plasmid was purchased from Beijing Solarbio Technology Co., Ltd.

[0038] The pESC URA27 plasmid was constructed using the pESC URA plasmid as a template. The construction process of the pESC URA27 plasmid is as follows: Using the pESC URA plasmid as a template, and pESG-GAL2-7-F2 and pESG-GAL2-7-R2 as primers, the vector fragment was amplified by PCR; using the pESC URA plasmid as a template, and PESG-GAL2-7-F1 and pESC-GAL2-7-R as primer pairs, and PESG-GAL2-7-F and pESC-GAL2-7-R1 as primer pairs, the multiple cloning site fragment was amplified by PCR; using the S. cerevisiae CEN.PK2-1C genome as a template, and using pGAL2-F and pGAL2-R, and pGAL7-F and pGAL7-R as primer pairs, the GAL2 and GAL7 promoter fragments were amplified by PCR; using S. cerevisiae CEN.PK2-1C genome as a template, the GAL2 and GAL7 promoter fragments were obtained by PCR; Using the cerevisiae CEN.PK2-1C genome as a template, and with TTPS1-F and TTPS1-R, and TPGK1-F and TPGK1-R as primer pairs, PCR amplification was performed to obtain TPS1 and TPGK1 terminator fragments. These fragments were then ligated to a vector using homologous recombinase to obtain the plasmid pESC URA27. The primers used are detailed in Table 2.

[0039] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0040] The present invention will be further illustrated below with reference to the embodiments.

[0041] The relevant information regarding the enzymes and encoding genes involved in the examples is shown in Table 1 below: Table 1. Information on the enzymes involved and their encoding genes.

[0042] The primer sequences used to construct the strains in the examples are shown in Table 2: Table 2 Primer Table

[0043] It should be noted that the construction methods provided in the embodiments of the present invention are exemplary and not restrictive. Those skilled in the art can use any technical means to achieve the final gene editing purpose.

[0044] The present invention will be further explained and illustrated below through specific embodiments.

[0045] Example 1: Obtaining the Kaurene 13-hydroxylase KAH mutant Kaurene acid 13-hydroxylase (KAH) catalyzes the conversion of kaurene acid to steviol, while CPR (cytochrome P450 reductase) can convert NADPH to NADP. + CPR is essential for KAH activity; with the support of CPR, KAH continues to function, catalyzing the synthesis of steviol using kaurenoic acid as a substrate. Therefore, we first constructed a recombinant expression plasmid pESC-His-KAH-CPR expressing KAH and CPR.

[0046] 1.1 Construction of recombinant expression plasmid pESC-His-KAH-CPR Using pESC-His as a vector, the recombinant expression plasmid pESC-His-KAH-CPR was constructed. The primers involved are shown in Table 2 above.

[0047] Specifically: Using the wild-type KAH gene fragment as a template and KAH-F and KAH-R as primers, the KAH fragment was amplified by PCR. Simultaneously, using the pESC-His plasmid as a template and TCYC1-circle-F and TCYC1-circle-R as primers, the linearized pESC-His vector fragment was amplified by PCR. The vector and the inserted fragment shared the same homologous arms. Following the kit instructions, the KAH fragment and the linearized pESC-His vector fragment were subjected to homologous recombination. After incubation at 50 °C for 1 h, the mixture was transformed. E.coli DH5α competent cells were plated on Amp-resistant plates and cultured overnight at 37 °C. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB medium containing Amp for sequencing verification. The recombinant expression plasmid pESC-His-KAH with correct sequencing results was extracted for later use.

[0048] Using the same method as described above, the difference is that "using the CPR gene fragment as a template, CPR-F and CPR-R as primers, PCR amplification is performed to obtain the CPR fragment; using the pESC-His-KAH plasmid as a template, TADH1-circle-F and TADH1-circle-R as primers, PCR amplification is performed to obtain the linearized vector fragment, and the vector and the inserted fragment also have the same homologous arms," ​​thus constructing pESC-His-KAH-CPR for later use.

[0049] 1.2 Obtaining mutants Using the KAH nucleotide sequence shown in SEQ ID NO.2 as a template gene fragment, mutation primers KAHmut-F and KAHmut-R were designed based on the G481L / Q159L mutation site. Using the KAH gene fragment as a template, PCR amplification was performed using KAH-F and KAHmut-R, and KAHmut-F and KAH-R as primers, respectively, to obtain the upstream and downstream of the KAH mutant fragment. The coding gene of the KAH (G481L / Q159L) mutant (shown in SEQ ID NO.4) was obtained by overlap PCR. The wild-type KAH gene was replaced with the coding gene of the KAH (G481L / Q159L) mutant to construct the expression plasmid, resulting in pESC-His-KAH(G481L / Q159L)-CPR.

[0050] The PCR system and PCR procedure are shown in Tables 3 and 4 below: Table 3 PCR System

[0051] Table 4 PCR Procedure

[0052] 1.3 Validation of mutant enzyme activity 1.3.1 Construction of recombinant strains Using electroporation, the recombinant plasmid pESC-His-KAH-CPR obtained in 1.1 and the recombinant mutant plasmid pESC-His-KAH(G481L / Q159L)-CPR obtained in 1.2 were transformed into yeast S. cerevisiae CEN.PK2-1C competent cells, respectively. The cells were plated on SC-HIS plates and cultured at 30℃ for 2-3 days. Positive clones were screened to obtain strain P1 containing the recombinant plasmid pESC-His-KAH-CPR and strain P2 containing the recombinant mutant plasmid pESC-His-KAH(G481L / Q159L)-CPR.

[0053] 1.3.2 Enzyme activity verification Single clones of strain P1 or P2 were picked and inoculated into glass tubes containing 5 mL of SC-His medium, and cultured at 30℃ and 220 rpm for 24 hours; the seed OD was measured. 600 The seed culture was transferred to 250 mL shake flasks containing 50 mL SC-His medium at an initial OD of 0.2, with three replicates per group. The culture was carried out at 30°C and 220 rpm for 48 hours, centrifuged at 3000 rpm for 5 min at room temperature, the supernatant was removed, and the cells were washed three times with sterile water and collected. 50 mL of YPGal liquid medium containing 2% galactose was added, and fermentation was induced at 28°C and 220 rpm for 24 hours.

[0054] After induction, the cells were centrifuged at 3,000 rpm for 5 min and the supernatant was discarded. The bacterial cells were collected and weighed. Each 1 g of bacterial cells was resuspended in 3 mL of 50 mM Tris-HCl buffer (pH 7.5), and 1 mM kauronic acid and 5 mM NADPH were added. The mixture was reacted at 30ºC and 250 rpm for 2 h. An equal volume of methanol was added to quench the reaction. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane, and the stevioside conversion rate was determined by HPLC.

[0055] The results are shown in Table 5 below: Table 5 Results of mutant enzyme activity verification

[0056] As shown in Table 5, compared with the control group P1 strain, the molar conversion rate of kaurene acid to steviol catalyzed by the experimental group P2 strain increased from 70.5% to 96.8%, indicating that the KAH (G481L / Q159L) mutant has higher activity than the wild-type KAH. The mutant KAH (G481L / Q159L) was selected for subsequent full fermentation steviol glycoside experiments.

[0057] Example 2 Construction of Stevioside Synthetic Strains (Chassis Strains) Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, integrated expression was performed. GfKS (Kaurene synthase encoding gene) , KO (Kauriene oxidase encoding gene) , KAH (or KAH (G481L / Q159L )) , CPR A chassis strain for synthesizing stevioside was constructed.

[0058] In Saccharomyces cerevisiae cells, the MVA pathway converts cytoplasmic acetyl-CoA into isopentenyl pyrophosphate (IPP) and dimethylallyldiphosphate (DMAPP) through a multi-step enzymatic reaction. Subsequently, IPP and DMAPP condense to form the precursor geraniylgeraniyl pyrophosphate (GGPP). GGPP is then converted into steviol by the action of GfKS, KO, KAH (or KAH (G481L / Q159L)) and CPR. The catalytic reactions involved are shown in Table 6 below.

[0059] Table 6

[0060] 2.1 Construction of S000 strain (integration expression at site 1114a) GfKS, CPR ) The target gene was integrated into the genome of *Saccharomyces cerevisiae* using the CRISPR / Cas9 gene editing system. A plasmid carrying a gene expressing the Cas9 protein and an sgRNA sequence targeting the corresponding site was constructed for targeted gene integration. The specific construction steps are as follows: (1) Construction of the knockout plasmid pCas9-1114a First, the gRNA sequence of the 1114a integration site was predicted using the online website Zhang Lab's CRISPOR (http: / / crispor.tefor.net / ), and a highly efficient gRNA sequence without off-target effects was selected. Using pESC URA as a template and primers 1114a-gRNA-F and gRNA-R (universal primers) as primer pairs, an expression cassette containing gRNA (including the URA3 expression cassette, which serves as a screening tag for Saccharomyces cerevisiae) was cloned. Using pCAS9-lacZ as a template and primers cas9-gRNA-F and cas9-gRNA-R (universal primers) as primer pairs, the vector backbone of the gRNA plasmid was cloned. The expression cassette fragment and the vector backbone were then combined using recombinase to construct the complete plasmid pCas9-1114a.

[0061] (2) Construction of Donor DNA Donor DNA construction method containing specific genes: Using a correctly sequenced gene expression plasmid as a template, amplification of the complete expression cassette containing the specific gene was performed using primer pairs (50 bp homologous arms). After PCR, verification was performed using a small-well gel; if the band size was correct, ethanol precipitation was performed to obtain the donor fragment. Specifically: Using the GFKS gene as a template and GFKS-F and GFKS-R as primers, the GFKS gene fragment was amplified. Using the pESC-His plasmid as a template and TCYC1-circle-F and TCYC1-circle-R as primers (universal primers), the linearized pESC-His plasmid fragment was amplified. The GFKS gene fragment and the linearized pESC-His plasmid fragment obtained above were used to construct the PESC-GFKS plasmid using the same method as in 1.1. Using the CPR gene as a template, and CPR-F and CPR-R as templates, the CPR gene fragment was amplified by PCR. Using the PESC-GFKS plasmid as a template, and TADH1-circle-F and TADH1-circle-R as primers (universal primers), the linearized PESC-GFKS plasmid fragment was amplified by PCR. The CPR gene fragment and the linearized PESC-GFKS plasmid fragment obtained above were used to construct the PESC-GFKS-CPR expression plasmid using the same method as in 1.1.

[0062] Using PESC-GFKS-CPR plasmid as a template and 1114a-DONOR-F and 1114a-DONOR-R as primers, the donor fragment was amplified by PCR. After PCR, the fragment was verified using a small-well gel. If the band size was correct, ethanol precipitation was performed to obtain the donor fragment: up-T. CYC1 -GfKS-P GAL1 -P GAL10 -CPR-T ADH1 -down.

[0063] (3) Strain construction 1) Preparation and transformation of competent cells The plasmid pCas9-1114a obtained in step (1) and the Donor DNA:up-T obtained in step (2) were combined. CYC1 -GfKS-P GAL1 -P GAL10 -CPR-T ADH1 -down was co-transfected into S. cerevisiae CEN.PK2-1C competent cells, specifically: ① Pick a single colony of yeast S. cerevisiae CEN.PK2-1C from the plate and inoculate it into 5 mL of YPD medium. Incubate at 30℃ for about 12 h. Then transfer 2 mL to 50 mL of YPD medium and incubate at 30℃ until the OD is about 2.0 (about 4 h, OD can be between 1.3 and 2.0). ② Transfer the bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 3000 rpm for 5 min, and discard the supernatant; ③ Add 25 mL of sterile water, centrifuge at 3000 rpm for 5 min, and discard the supernatant; ④ Repeat step ③; ⑤ Resuspend the bacterial cells in 1 mL of sterile water, take 100 μL of bacterial solution and dispense it into a 1.5 mL sterile Eppendorf tube, centrifuge at 12000 rpm for 1 min, and discard the supernatant; ⑥ Add the following to a 1.5 mL tube in the following order: 240 μL PEG3350, 36 μL LiAC, 50 μL SSDNA, 5 ng Cas9-gRNA plasmid (pCas9-1114a), and 15 ng Donor fragment (up-T). CYC1 -GfKS-P GAL1 -P GAL10 -CPR-T ADH1 -down segment); ⑦ Mix the above reagents with the bacterial cells, heat shock them in a metal bath at 42℃ for 45 min, centrifuge at 12000 rpm for 1 min to remove the supernatant, add 1 mL of YPD liquid, and incubate at 30℃ for 2 h. ⑧ Centrifuge at 12000 rpm for 1 min, remove the supernatant, and wash twice with 1 mL of sterile water; ⑨ Resuspend the bacterial cells in 1 mL of sterile water, take an appropriate amount of bacterial solution and spread it on an SD-auxotrophic plate (using the above plasmid system, it is a URA-auxotrophic plate SD-URA), and incubate at 30℃ for 3-4 days until single colonies appear.

[0064] 2) Positive clones were screened by colony PCR and sequenced for verification. The PCR-verified clones were re-streaked onto SC plates containing 5-FOA (5-fluoroorotic acid) and incubated at 30°C to eliminate the Cas9 plasmid. The clones that grew on SC (containing 5-FOA) plates were streaked onto YPD and SC-URA plates and incubated at 30°C. The bacteria that grew on YPD plates but not on SC-URA plates were identified as gene-edited bacteria. These clones were selected and incubated on YPD liquid medium at 30°C for 24 h, and the bacterial strain (20% glycerol) was then preserved to obtain the chassis strain S000.

[0065] 2.2 Construction of strains S001-S002 (integrated expression at the 106a site) KO, KAH (or KAH (G481L / Q159L )) Based on the chassis strain S000 obtained in section 2.1, KO (kaurene oxidase) and KAH (or KAH) were further integrated and expressed. (G481L / Q159L) ), and obtained strains S001 and S002 that produce stevioside.

[0066] The same method as steps (1)-(3) in section 2.1 was used to construct the knockout plasmid and donor DNA, and only the corresponding primers or templates were replaced. The plasmid was then integrated and expressed at the 106a site of the genome. KO and KAH (G481L / Q159L) The primers involved are listed in Table 2. Step (3) involves mixing the knockout plasmid pCas9-106a with the donor DNA:up-T. CYC1 -KO-P GAL1 -P GAL10 -KAH(G481L / Q159L)-T ADH1 -down was used to transform the S000 strain obtained in 2.1 to obtain strain S001, which was then stored for later use (20% glycerol).

[0067] The same method as steps (1)-(3) in section 2.1 was used to construct the knockout plasmid and donor DNA, and only the corresponding primers or templates were replaced. The plasmid was then integrated and expressed at the 106a site of the genome. KO and KAH, The primers involved are listed in Table 2. Step (3) involves mixing the knockout plasmid pCas9-106a with the donor DNA:up-T. CYC1 -KO-P GAL1 -P GAL10 -KAH-T ADH1 -down was used to transform the strain S000 to obtain strain S002, which was then stored for later use (20% glycerol).

[0068] The strain information constructed in this embodiment is shown in Table 7 below: Table 7. Strain Construction Information

[0069] Example 3: Total Synthesis of Reb M I. Strain Construction 3.1 Construction of a fully synthetic Reb M strain (integration of UGT11, UGT12, UGT13, and UGT15 at site 1414a; integration of UGT17 at site 208a) Using S001 or S002 obtained in Example 2 above as the chassis strain, UGT11, UGT12, UGT13, UGT15, and UGT17 were further integrated and expressed to construct a Reb M-producing strain. The specific construction process of the strain is as follows: (1) The same method as steps (1)-(3) in section 2.1 is used, and the primers involved are listed in Table 2. The difference is that step (1) is a two-site integration (site 1414a and site 208a), specifically: Construction method of dual-site gRNA-cas9 plasmid: First, construct pCas9-1414a plasmid (the difference is that 1414a-gRNA-F and gRNA-R are used as primer pairs) and pCas9-208a plasmid (the difference is that 208a-gRNA-F and gRNA-R are used as primer pairs) according to the method of step (1) in part 2.1, and complete the sequencing.

[0070] The specific construction process of the pCAS-1414a-208a plasmid is as follows: Using the pCas9-1414a plasmid as a template, and 1414a-D-F1 and D-R1 (universal primers) as primer pairs, the fragment 1414a-d-1 containing 1414-gRNA and a gRNA terminator is cloned; using the pCas9-208a plasmid as a template, and D-F2 (universal primers) and DR-mut (universal primers) as primer pairs, the fragment P-SNR52 is cloned by PCR. Simultaneously, using the pCas9-208a plasmid as a template, and D-F2-mut (universal primers) and D-R2 (universal primers) as primer pairs, PCR is performed... Fragment 208a-URA3 was cloned. After verifying the size of fragments P-SNR52 and 208a-URA3, they were recovered by gel extraction and used as templates for fusion PCR. Using primers D-F2 and D-R2 as primer pairs, fragment 208a-d-2 containing gRNA expression cassette and URA3 expression cassette was cloned. Based on the above fragments, plasmid pCAS-1414a-208a was constructed using GoldenGate. The plasmid backbone used was pCAS9-lacZ plasmid; fragment 1: 1414a-d-1; fragment 2: 208a-d-2. The GoldenGate program is shown in Table 8 below.

[0071] Table 8 Golden-gate Program and Architecture

[0072] (2) Using the UGT11 gene fragment as a template and UGT11-F and UGT11-R as primer pairs, the UGT11 fragment was amplified. Simultaneously, using the pESC-His plasmid as a template and TCYC1-circle-F and TCYC1-circle-R as primers, the linearized pESC-His vector fragment was amplified by PCR. The vector and the inserted fragment had the same homologous arms. Following the kit instructions, the UGT11 fragment and the linearized pESC-His vector fragment were subjected to homologous recombination. After reacting at 50 °C for 1 h, the fragment was transformed. E.coli DH5α competent cells were plated on Amp-resistant plates and cultured overnight at 37 °C. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB medium containing Amp for sequencing verification. The recombinant expression plasmid pESC-UGT11 with correct sequencing results was extracted for later use.

[0073] (3) Using the same method as in step (2) above, with UGT12 as a template and UGT12-F and UGT12-R as primer pairs, the UGT12 fragment was amplified by PCR. At the same time, with pESC-UGT11 plasmid as a template and TADH1-circle-F and TADH1-circle-R as primer pairs, the linearized vector fragment was amplified by PCR. The vector and the inserted fragment also have the same homologous arms. The PESC-UGT11-UGT12 plasmid was constructed.

[0074] (4) Using the same method as in steps (2) and (3) above, replace UGT11 and UGT12 with UGT13 and UGT15 gene fragments, replace pESC-His plasmid with pESC-URA27 plasmid as template, and replace the corresponding primers to construct PESC-UGT13-UGT15 plasmid. The difference is that the primers for amplifying UGT13 are UGT13-F and UGT13-R, the primers for amplifying UGT15 are UGT15-F and UGT15-R, and the primers for amplifying the linearized vector fragment are TTPS1-circle-F and TTPS1-circle-R, TPGK1-circle-F and TPGK1-circle-R.

[0075] (5) Using the same method as in step (2) above, the UGT117 gene fragment and pESC-His plasmid were used as templates, and the primers were replaced to construct the PESC-UGT17 plasmid. The difference is that the primers for amplifying UGT17 are UGT17-F and UGT17-R, and the primers for linearizing pESC-His are TADH1-circle-F and TADH1-circle-R.

[0076] (6) The above plasmids PESC-UGT11-UGT12, PESC-UGT13-UGT15 and PESC-UGT17 are used for the amplification of the target genes UGT11, UGT12, UGT13, UGT15 or UGT17 in DonorDNA construction. Design the donor DNA using the same method as in step (2) of section 2.1, only replacing the corresponding primers or template (primer sequences are shown in Table 2), as follows: Using PESC-UGT11-UGT12 plasmid as template, and 1414a-DONOR-F and UGT11-12-R as primer pairs, amplify to obtain T CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 Fragment; using PESC-UGT13-UGT15 plasmid as template and UGT13-15-F and 1414a-DONOR-R as primer pairs, T was amplified to obtainTPS1 -UGT13-P GAL7 -P GAL2 -UGT15-T PGK1 Fragment; UGT11-12-R and UGT13-15-F primers contain the same 20bp homologous arm fragment, after amplification T CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 Fragments and T TPS1 -UGT13-P GAL7 -P GAL2 -UGT15-T PGK1 The fragment contained homologous arms. Using PESC-UGT17 plasmid as a template and 208a-DONOR-F / R primers, the Donor DNA:up-T was amplified. CYC1 -UGT17-P GAL1 -P GAL10 -T ADH1 -down.

[0077] (7) Using 1414a-DONOR-F and 1414a-DONOR-R as primer pairs, and fragment T as template. CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 and T TPS1 -UGT13-P GAL7 -P GAL2 -UGT15-T PGK1 Donor DNA containing UGT11, UGT12, UGT13, and UGT15 is formed by overlapping: up-T CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 -T TPS1 -UGT13-P GAL7 -P GAL2 -UGT15-T PGK1 -down.

[0078] The dual-site knockout plasmid pCAS-1414a-208a and Donor DNA:up-T were used. CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 -T TPS1 -UGT13-P GAL7 -P GAL2 -UGT15-TPGK1 -down and up-T CYC1 -UGT17-P GAL1 -P GAL10 -T ADH1 -down was transformed into either S001 or S002 chassis strains to obtain Reb M production strains M001 and M002, respectively, and the strains were preserved for later use (20% glycerol).

[0079] 3.2 Overexpression and multi-copy integration of tHMG1 and IDI1 Using strains M001 and M002 obtained in section 3.1 as chassis strains, tHMG1 and IDI1 were further overexpressed and multiplied to enhance the throughput of the yeast endogenous MVA pathway. The specific construction process is as follows: 3.2.1 Overexpression of tHMG1 and IDI1 (overexpression site 1622b) The same method as steps (1)-(3) in section 2.1 was used. The primers (see Table 2) and templates (the template for amplifying the tHMG1 and IDI1 gene fragments was the Saccharomyces cerevisiae CEN.PK2-1C genome) were replaced according to the expression site and the target gene to construct the knockout plasmid pCas9-1622b and donor DNA:up-T CYC1 -tHMG1-P GAL1 -P GAL10 -IDI-T ADH1 -down. Step (3) is to separately add Donor DNA:up-T CYC1 -tHMG1-P GAL1 -P GAL10 -IDI-T ADH1 The -down and pCas9-1622b plasmids were co-transformed into strains M001 and M002 by electroporation, resulting in strains M003 and M003-1, respectively. The strains were then preserved for later use (20% glycerol).

[0080] 3.2.2 Multi-copy integration of tHMG1 and IDI1 Using the same method as in 3.2.1, multiple copies of tHMG1 and IDI1 were integrated into the YPRCΔ15c site at M003 or M003-1. Step (3) involves sequentially inserting the knockout plasmid pCas9-YPRCΔ15c and Donor DNA:up-T CYC1 -tHMG1-P GAL1 -P GAL10 -IDI-T ADH1 -down was transformed into strains M003 and M003-1, resulting in strains M004 and M004-1, which were then preserved for future use (20% glycerol).

[0081] Using the same method as in 3.2.1, multiple copies of tHMG1 and IDI1 were integrated at site 308a in M004 or M004-1. Step (3) involves inserting the knockout plasmid pCas9-308a and Donor DNA:up-T CYC1 -tHMG1-P GAL1 -P GAL10 -IDI-T ADH1 -down was transformed into strains M004 and M004-1, resulting in strains M005 and M005-1, which were then preserved for later use (20% glycerol).

[0082] 3.3 Knockout of GAL80 Using the M005 and M005-1 strains obtained in 3.2 above as chassis strains, GAL80 was further knocked out, allowing the yeast strains to be free from galactose control and respond to glucose concentration.

[0083] The same method as steps (1)-(3) in section 2.1 was used to construct the knockout plasmid pCas9-GAL80 and donor DNA. The primers involved are listed in Table 2. The difference is that the primer pair for amplifying the gRNA of GAL80 is GAL80-gRNA-F and gRNA-R, and step (2) is to construct the knockout donor.

[0084] The specific method for removing the donor is as follows: Knockout fragment: Based on the sequence of the gene to be knocked out, GAL80-donor-F and GAL80-donor-R primer pairs were designed (both F and R were designed to be 59 bp, with 18 bp of sequence overlap, making the full length of the knockout fragment 100 bp). The system was prepared using KODone enzyme as shown in Table 9 below: Table 9 System Composition

[0085] The procedure is as follows: [98℃ 10 s - 55℃ 5 s - 68℃ 5 s] × 35 cycles. After PCR, the results are verified with a small-well gel. If the band size is correct, ethanol precipitation is performed to obtain the knockout fragment up-GAL80-knockout-down.

[0086] Step (3) involves co-transforming the knockout plasmid pCas9-GAL80 and Donor DNA:up-GAL80-knockout-down into strains M005 and M005-1 to obtain strains M008 and M008-1, which are then stored for later use (20% glycerol).

[0087] 3.4 Downregulate ERG9 expression (reducing the expression of the original promoter P) ERG9Replace with P HXT1 ) Using the strain obtained in 3.3 above as the chassis strain, the original promoter P of ERG9 was... ERG9 Replace with P HXT1 This approach further lowers ERG9, reducing competitive pathways.

[0088] The same method as steps (1)-(3) in section 2.1 was used to construct the knockout plasmid pCas9-P. ERG9 For donor DNA, simply replace the corresponding primers or templates; the primers involved are listed in Table 2. The difference lies in the amplification of P... ERG9 The primer pair for the gRNA is pERG9-gRNA-F and gRNA-R. Step (2) uses the Saccharomyces cerevisiae genome as a template to design primers containing the upstream and downstream homologous arms of the Erg9 promoter and P. HXT1 The sequence was amplified using primers pHXT1-ERG9-DONOR-F and pHXT1-ERG9-DONOR-R (see Table 2), and the product was precipitated with alcohol to obtain DONOR DNA: up-P HXT1 -down. Step (3) involves inserting the knockout plasmid pCas9-PERG9 and Donor DNA:up-P HXT1 -down was transformed into strains M008 and M008-1, resulting in strains M009 and M009-1, which were then preserved for later use (20% glycerol).

[0089] 3.5 Overexpression of ERG20 and BTS1 (overexpression site 1021b) Using the M009 and M009-1 strains obtained in section 3.4 above as chassis strains, ERG20 and BTS1 were further overexpressed to enhance GGPP throughput and improve the synthesis efficiency of downstream diterpenoid compounds.

[0090] The same method as steps (1)-(3) in section 2.1 was used to construct the knockout plasmid and donor DNA, only replacing the corresponding primers or templates. The primers involved are listed in Table 2. The amplification template for the ERG20 and BTS1 gene fragments is the Saccharomyces cerevisiae CEN.PK2-1C genome. Step (3) involves converting the knockout plasmid pCas9-1021b and the donor DNA:up-T CYC1 -ERG20-P GAL1 -P GAL10 -BTS1-T ADH1 -down was transformed into strains M009 and M009-1, resulting in strains M010 and M010-1, which were then preserved for later use (20% glycerol).

[0091] 3.6 Multiple copies integrated into CrtE (integration site 911b, or dual site 607C+911b) Using the M010 and M010-1 strains obtained in 3.5 above as chassis strains, further multi-copy integration and overexpression of CrtE were carried out to enhance the supply of upstream terpene precursor GGPP.

[0092] The same method as steps (1)-(3) in section 2.1 is used to construct the knockout plasmid pCas9-911b and donor DNA, only replacing the corresponding primers or templates. The primer pair for amplifying the donor DNA is 911b-DONOR-F and 911b-DONOR-R. Step (3) involves converting the knockout plasmid pCas9-911b and the donor DNA into a 2-T matrix. CYC1 -CrtE-P GAL1 -P GAL10 -T ADH1 -down was used to transform strain M010 to obtain strain M011, which was then stored for later use (20% glycerol).

[0093] Construct the knockout plasmid pCas9-607C-911b using the same method as step (1) in section 3.1, only replacing the corresponding primers. Construct the donor DNA using the same method as in section 2.1, only replacing the corresponding primers or templates. The primers involved are listed in Table 2. Step (3) involves combining the two-site plasmid pCas9-607C-911b and the Donor DNA:up-T CYC1 -CrtE-P GAL1 -P GAL10 -T ADH1 -down and up-T TPS1 -CrtE-P GAL7 -P GAL2 -T PGK1 -down was transformed into strains M010 and M010-1 to obtain strains M012 and M012-1, which were then preserved for later use (20% glycerol).

[0094] The strain information constructed in this embodiment is shown in Table 10 below: Table 10 Information on strain construction in Example 3

[0095] II. Reb M Production Validation Experiment The recombinant strain obtained in the first part was used as the fermentation strain to produce Reb M.

[0096] Experimental methods: Glycerol bacteria were taken from a -80℃ freezer, streaked on YPD plates, and cultured at 30℃ for 2-3 days. Single colonies were picked from the plates and cultured in 10 mL of YPD medium overnight at 30℃ and 250 rpm. The seed culture was transferred at a 5% inoculum to a 250 mL shake flask containing 25 mL of YPD liquid medium and fermented at 30 ºC and 250 rpm, with three replicates per group. After 120 h of fermentation, 5 mL of fermentation broth was added to glass beads and shaken for 30 min. Reb M in the fermentation broth was extracted with anhydrous methanol, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by high performance liquid chromatography.

[0097] High-performance liquid chromatography (HPLC) detection conditions: C18 column (250 mm × 4.6 mm, 5 µm), mobile phase 32% acetonitrile-68% phosphoric acid water (pH 3.0), flow rate 1.0 mL / min, detection wavelength 210 nm, column temperature 40℃, time 30 min, injection volume 5 µL, and external standard method for quantification.

[0098] The production results are shown in Table 11 below: Table 11 Reb M production in Example 3

[0099] As shown in Table 11, the difference between strains M001 and M002 is that M001 expresses the KAH(G481L / Q159L) mutant, while M002 expresses wild-type KAH. The yield of M001 is higher than that of M002. It can be seen that the G481L / Q159L mutant constructed for KAH in this invention has a very significant impact on the production of Reb M, with the yield significantly increasing from 25.812 mg / L to 52.247 mg / L. This also shows that the single technical feature of KAH(G481L / Q159L) mutant can be directly transferred into yeast without any prior modification, which can also increase the yield of Reb M. That is, the synergistic effect of KAH(G481L / Q159L) mutant has the characteristic of not depending on a specific prior modification chassis. Strains M003-M005 were developed by overexpressing tHMG1 and IDI1 on the basis of strain M001, which enhanced the flux of the endogenous MVA pathway in yeast strains. The yield of strain M005 gradually increased compared to M003 and M004, indicating that the yield of Reb M was also improved with the increase of copy number. Among them, M003 was developed by expressing tHMG1 and IDI1 by single copy on the basis of M001, and the yield of M003 was increased by 79.30% compared to M001. The yield of M005 was 3.27 times that of M001, indicating that the yield of tHMG1 and IDI1 was significantly improved when the copy number was 3. The M008 production strain, based on the M005 strain with GAL80 further knocked out, showed a 52.33% increase in yield compared to M005. The M009 strain, based on the M008 strain with further downregulation of ERG9, showed a 52.45% increase in yield compared to M008, indicating that downregulation of ERG9 effectively increases Reb M production. The M010 strain, based on the M009 strain with further overexpression of ERG20 and BTS1, enhanced GGPP flux, resulting in an 8.22% increase in yield compared to M009. These findings demonstrate that global regulatory optimization and competitive pathway inhibition can significantly improve pathway flux and increase Reb M production. The M011-M012 production strains further overexpressed CrtE based on M010. M011 (single-copy integrated CrtE) yielded 591.172 mg / L, while M012 (multiple-copy integrated CrtE) yielded 850.1 mg / L, representing a 97.64% increase compared to M010. This indicates that multiple-copy integrated CrtE significantly improved Reb M yield. Compared to strain M002, M012, through KAH mutation and a series of modifications, increased Reb M yield from the initial 25.81 mg / L to 850.10 mg / L, significantly improving Reb M production. The difference between strains M012-1 and M012 is that M012-1 did not undergo KAH mutation; therefore, strain M012 had a higher Reb M yield than strain M012-1.

[0100] Example 4: Total Synthesis of Reb D 4.1 Construction of a de novo synthetic strain for Reb D synthesis from glucose Using S001 and S002 obtained in Example 2 as chassis strains, UGT11, UGT12, UGT13, UGT14, and UGT15 were integrated and expressed to construct Reb D-producing engineered strains D001 and D002.

[0101] Construction of D001 and D002: Using the same method as in section 3.1, UGT11, UGT12, UGT13, UGT14, and UGT15 were integrated and expressed at the dual-site 1414a and 208a sites of the S001 or S002 chassis strain (UGT11, UGT12, UGT13, and UGT14 were integrated and expressed at site 1414a; UGT15 was integrated and expressed at site 208a). The primers involved are listed in Table 2. The difference lies in constructing the Donor DNA containing the four genes UGT11, UGT12, UGT13, and UGT14, and the Donor DNA containing UGT15. The dual-site plasmid pCAS-1414a-208a and the Donor DNA:up-T were used. CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 -T TPS1 -UGT13-P GAL7 -P GAL2 -UGT14-T PGK1 -down and up-T CYC1 -UGT15-P GAL1 -P GAL10 -T ADH1 -down was transformed into strains S001 and S002, resulting in Reb D producing strains D001 and D002, which were then preserved for future use (20% glycerol).

[0102] 4.2 The strain obtained in 4.1 was further modified (the modification method is the same as in Example 3). Based on Reb D producing strains D001 and D002, the modifications in 3.2-3.6 of Example 3 were repeated. The strain construction was carried out in accordance with the construction operation of the total synthesis Reb M strain in 3.2-3.6 of Example 3. The relevant construction information is shown in Table 12 below.

[0103] Table 12 Information on strain construction in Example 4

[0104] 4.3 Reb D Production Validation Experiment The recombinant strains obtained above were used as fermentation strains to produce Reb D (the experimental methods and detection methods were the same as those in the Reb M production verification experiment in Example 3).

[0105] Experimental methods: Glycerol bacteria were taken from a -80℃ freezer, streaked on YPD plates, and cultured at 30℃ for 2-3 days. Single colonies were picked from the plates and cultured in 10 mL of YPD medium overnight at 30℃ and 250 rpm. A 5% inoculum was transferred to a 250 mL shake flask containing 25 mL of YPD liquid medium and fermented at 30℃ and 250 rpm, with three replicates per group. After 120 h of fermentation, 5 mL of the fermentation broth was added to glass beads and shaken for 30 min. Reb D in the fermentation broth was extracted with anhydrous methanol, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by high-performance liquid chromatography (HPLC). The yield results are shown in Table 13 below. Table 13 Reb D Yield Results for Example 4

[0106] As can be seen from Table 13, the difference between D001 and D002 Reb D producing strains is that D001 expresses the KAH (G481L / Q159L) mutant, while D002 expresses wild-type KAH. Compared with D002, the Reb D yield of D001 increased by 96.07%, which shows that the G481L / Q159L mutant constructed for KAH in this invention also has a significant impact on Reb D production. D003-D005 are based on strain D001, which further overexpresses tHMG1 and IDI1. With the increase of tHMG1 and IDI1 copy number, the yield of D005 (166.256 mg / L) is significantly higher than that of D001 (45.628 mg / L). D008-D012 are based on strain D005, which has undergone a series of modifications including knocking out GAL80, downregulating ERG9, overexpressing ERG20 and BTS1, and integrating multiple copies of CrtE. Compared with strain D002, strain D012 has a yield of 811.218 mg / L after undergoing the KAH mutation and a series of modifications of this invention.

[0107] Example 5: Total Synthesis of Reb I 5.1 Construction of strains for de novo synthesis of Reb I from glucose Using S001 and S002 obtained in Example 2 as chassis strains, UGT11, UGT12, UGT13 and UGT16 were integrated and expressed to construct Reb I-producing engineered strains I001 and I002.

[0108] Construction of strains I001 and I002: Two-site integration was performed using the same method as in section 3.1. UGT11 and UGT12 were integrated into the 1414a site, and UGT13 and UGT16 were integrated into the 208a site. The primers used are listed in Table 2. The resulting two-site Cas9 plasmid pCAS-1414a-208a was constructed. Donor DNA: up-T CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 -down、up-T TPS1 -UGT13-P GAL7 -P GAL2 -UGT16-T PGK1 -down. The dual-site Cas9 plasmid (pCAS-1414a-208a) and two donor DNAs were co-transformed into strains S001 and S002 to obtain Reb I producing strains I001 and I002, respectively. The strains were then stored for later use (20% glycerol).

[0109] 5.2 The strain obtained in 5.1 was further modified (the modification method is the same as in Example 3). Based on Reb I production strains I001 and I002, the modifications in 3.2-3.6 of Example 3 were repeated. The strain construction was carried out in accordance with the construction operation of the total synthesis Reb M strain in 3.2-3.6 of Example 3. The relevant construction information is shown in Table 14 below.

[0110] Table 14. Construction information of strain 5 in Example 5

[0111] 5.3 Reb I Production Validation Experiment Using the recombinant strain obtained in 5.2 as the fermentation strain, Reb I production experiment was carried out (the experimental method, detection method and Reb M production verification experiment in Example 3 are the same).

[0112] Experimental methods: Glycerol bacteria were taken from a -80℃ freezer, streaked on YPD plates, and cultured at 30℃ for 2-3 days. Single colonies were picked from the plates and cultured in 10 mL of YPD medium overnight at 30℃ and 250 rpm. Seed culture was transferred at a 5% inoculum to 250 mL shake flasks containing 25 mL of YPD liquid medium and fermented at 30 ºC and 250 rpm, with three replicates per group. After 120 h of fermentation, 5 mL of fermentation broth was added to glass beads and shaken for 30 min. Reb I in the fermentation broth was extracted with anhydrous methanol, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by high-performance liquid chromatography. The yield results are shown in Table 15 below. Table 15 Reb I Yield Results for Example 5

[0113] As can be seen from Table 15, the difference between I001 and I002 in producing Reb I is that I001 expresses the KAH (G481L / Q159L) mutant, while I002 expresses wild-type KAH. Compared with I002, the Reb I yield of I001 increased by 68.98%, which shows that the G481L / Q159L mutant constructed for KAH in this invention also has a significant impact on the production of Reb I. I003-I005 are based on strain I001, which further overexpresses tHMG1 and IDI1. With the increase of tHMG1 and IDI1 copy number, the yield of I005 (126.732 mg / L) is significantly higher than that of I001 (25.524 mg / L). I008-I0012 are based on strain I005, which has undergone a series of modifications, including knocking out GAL80, downregulating ERG9, overexpressing ERG20 and BTS1, and integrating multiple copies of CrtE. Compared with strain I002, strain I0012 has a yield of 692.815 mg / L after undergoing the KAH mutation and a series of modifications of this invention.

[0114] Example 6: Total Synthesis of Reb A 6.1 Construction of strains for de novo synthesis of Reb A from glucose Using S001 and S002 obtained in Example 2 as chassis strains, UGT11, UGT12, UGT13 and UGT14 were integrated and expressed to construct Reb A-producing engineered strains A001 and A002.

[0115] Construction of strains A001 and A002: Two-site integration was performed using the same method as in section 3.1. UGT11 and UGT12 were integrated into the 1414a site, and UGT13 and UGT14 were integrated into the 208a site. The primers used are listed in Table 2. The two-site Cas9 plasmid pCAS-1414a-208a was constructed. Donor DNA: up-T CYC1 -UGT11-P GAL1 -P GAL10 -UGT12-T ADH1 -down、up-T TPS1 -UGT13-P GAL7 -P GAL2 -UGT14-T PGK1 -down. The dual-site Cas9 plasmid pCAS-1414a-208a and two donor DNAs were co-transformed into strains S001 and S002 to obtain Reb A producing strains A001 and A002, respectively. The strains were then stored for later use (20% glycerol).

[0116] 6.2 The strain obtained in 6.1 was further modified (the modification method is the same as in Example 3). Based on Reb A production strains A001 and A002, the modifications in 3.2-3.6 of Example 3 were repeated. The strain construction was carried out in accordance with the construction operation of the total synthesis Reb M strain in 3.2-3.6 of Example 3. The relevant construction information is shown in Table 16 below.

[0117] Table 16 Information on strain construction in Example 6

[0118] 6.3 Reb A Production Validation Experiment The recombinant strain obtained in 6.2 above was used as the fermentation strain to carry out the Reb A production experiment (the experimental method, detection method and Reb M production verification experiment in Example 3 are the same).

[0119] Experimental methods: Glycerol bacteria were taken from a -80℃ freezer, streaked on YPD plates, and incubated at 30℃ for 2-3 days. Single colonies were picked from the plates and incubated overnight at 30℃ and 250 rpm. Seed culture was transferred at a 5% inoculum to 250 mL shake flasks containing 25 mL of YPD liquid culture medium and fermented at 30℃ and 250 rpm, with three replicates per group. After 120 h of fermentation, 5 mL of fermentation broth was added to glass beads and shaken for 30 min. Reb A in the fermentation broth was extracted with anhydrous methanol, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by high-performance liquid chromatography (HPLC). The yield results are shown in Table 17 below. Table 17 Reb A Yield Results for Example 6

[0120] As can be seen from Table 17, the difference between the Reb A producing strains A001 and A002 is that A001 expresses the KAH (G481L / Q159L) mutant, while A002 expresses wild-type KAH. Compared with A002, the Reb A yield of A001 increased by 93.43%, which shows that the G481L / Q159L mutant constructed for KAH in this invention also has a significant impact on the production of Reb A. A003-A005 are based on strain A001, which further overexpresses tHMG1 and IDI1. With the increase of tHMG1 and IDI1 copy number, the yield of A005 (157.035 mg / L) is significantly higher than that of A001 (43.405 mg / L). A008-A0012 are based on strain A005, which has undergone a series of modifications including knocking out GAL80, downregulating ERG9, overexpressing ERG20 and BTS1, and integrating multiple copies of CrtE. Compared with strain A002, strain A0012 has a yield of 795.777 mg / L after undergoing the KAH mutation and a series of modifications of this invention.

[0121] In summary, the KAH mutation (G481L / Q159L) of this invention not only significantly improves Reb M production but also has a similar effect on Reb D, Reb I, and Reb A, with a more significant impact on Reb M production. The M001 strain (expressing the KAH mutant (G481L / Q159L)) produced 102.41% more Reb M than the M002 strain (expressing wild-type KAH). This demonstrates that the KAH (G481L / Q159L) mutant has a universal applicability in improving the production of a series of rebaudioside compounds, including Reb D, Reb I, and Reb A. Furthermore, the modification methods of this invention (multiple copy integration of tHMG1 and IDI1, GAL80 knockout, ERG9 downregulation, ERG20 and BTS1 overexpression, and multiple copy integration of CrtE) can significantly increase the synthesis and production of a series of rebaudioside compounds, including Reb D, Reb I, and Reb A.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A stevioside-producing strain, characterized in that, The strain was obtained by expressing a kaurene acid 13-hydroxylase mutant and cytochrome P450 reductase CPR in a host. The amino acid sequence of the kaurene 13-hydroxylase mutant is shown in SEQ ID NO.3; The host is: brewer's yeast ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ) or Hansenula polymorpha ( Hansenula polymorpha ).

2. The steviol-producing strain as described in claim 1, characterized in that, It also expresses kaurene synthase GfKS and kaurene oxidase KO in the host.

3. The use of the strain described in claim 1 or 2 in the production of steviol.

4. A yeast strain for the complete fermentation production of steviol glycosides, characterized in that, The engineered bacteria were obtained by further expressing one or more of the following in the host to construct a production pathway from steviol to rebaudioside: a kaurene 13-hydroxylase mutant, cytochrome P450 reductase CPR, kaurene synthase GfKS, and kaurene oxidase KO. The amino acid sequence of the kaurene 13-hydroxylase mutant is shown in SEQ ID NO.

3.

5. The engineered yeast strain for the complete fermentation production of steviol glycosides as described in claim 4, characterized in that, The host was also subjected to one or more of the following gene edits: (1) Overexpression of 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1 and / or isoprene pyrophosphate isomerase IDI1; the expression is single-copy or multiple-copy expression; (2) Knock out the transcriptional regulatory protein GAL80; (3) Downregulates the expression of squalene synthase ERG9; (4) Overexpression of fenestration pyrophosphate synthase ERG20 and / or geraniol geraniol pyrophosphate synthase BTS1; (5) Overexpression of geraniol geraniol pyrophosphate synthase CrtE; the expression is single-copy or multiple-copy expression.

6. The engineered yeast strain for the complete fermentation production of steviol glycosides as described in claim 5, characterized in that, The GenBank accession number for the cytochrome P450 reductase CPR is CAA46815.1; The GenBank accession number for the kauriene synthase GfKS is BAA84917.1; The GenBank accession number for the kauriene oxidase KO is AAC39507.1; The GenBank accession number for the 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1 is: M22002.1; The NCBI Reference Sequence for the isoprene pyrophosphate isomerase IDI1 is: NP_015208.1; The NCBI Reference Sequence for the transcriptional regulatory protein GAL80 is: NM_001182409.1; The GenBank accession number for the squalene synthase ERG9 is X59959.1; The GenBank accession number for the farnesyl pyrophosphate synthase ERG20 is J05091.1; The GenBank accession number for the geraniol geraniol pyrophosphate synthase BTS1 is: U31632.1; The NCBI Reference Sequence for the geraniol geraniol pyrophosphate synthase CrtE is: WP_010888034.1; The GenBank numbers for the UDP-glucosyltransferases UGT11, UGT12, UGT13, and UGT14 are AWU66064.1, AWU66065.1, AAM53963.1, and ACT33422.1, respectively. The NCBI Reference Sequence for the UDP-glucosyltransferase UGT15 is: XP_015629141.1; The nucleotide sequence of the gene encoding the UDP-glucosyltransferase UGT16 is shown in SEQ ID NO.5; The nucleotide sequence of the gene encoding the UDP-glucosyltransferase UGT17 is shown in SEQ ID NO.

6.

7. The engineered yeast strain for the complete fermentation production of steviol glycosides as described in claim 4, characterized in that, The host is: Saccharomyces cerevisiae, Yersinia lipolytica, Kluyveromyces lactis, or Hansenula polymorpha.

8. The use of the engineered bacteria according to any one of claims 4-7 in the total fermentation production of steviol glycosides.

9. The application as described in claim 8, characterized in that, The steviosides include: rebaudioside A, rebaudioside D, rebaudioside M, and rebaudioside I.

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