Recombinant bacterium for producing cycloastragenol as well as preparation method and application of recombinant bacterium

Abstract

The invention relates to a recombinant bacterium for producing cycloastragenol. The recombinant bacterium is a strain which is obtained by overexpressing the following genes: a cyclase gene OCS3, an epoxidase gene ERG1, a P450 enzyme gene CYP88D25, a P450 enzyme gene CYP71D756, a P450 enzyme gene CYP88D7, a dioxygenase gene OGD1 and a flavin reductase gene AtCPR1. The yield of the obtained recombinant yeast strain of cycloastragenol in a shake flask reaches 23mg / L, and the recombinant yeast strain is suitable for practical popularization and application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering fermentation technology, and particularly relates to a recombinant bacterium for producing cycloastragenol as well as a preparation method and application thereof. BACKGROUND

[0002] Cycloastragenol is a cycloartane type triterpenoid saponin with a chemical formula of C 30 H 50 O5, a CAS number of 78574-94-4, and a relative molecular mass of 490.71. Cycloastragenol and its glycosylated derivatives, as the main effective components of Astragalus membranaceus in China, have a wide range of applications. Cycloastragenol is the only telomerase activator found so far, which can delay telomere shortening by increasing telomerase, and is believed to have anti-aging effects. Its glycosylated derivative, astragaloside IV, has also been developed into a dietary supplement for enhancing immunity and an antiviral drug for treating animals.

[0003] Currently, the production method of cycloastragenol mainly involves extracting astragaloside IV from Astragalus membranaceus, and then hydrolyzing the glycosidic bond to obtain cycloastragenol. This method has a high cost of extraction and purification, and it is also difficult to achieve large-scale industrial production due to the long growth cycle and low content of plants. In 2024, the biosynthetic pathway of cycloastragenol and astragaloside IV was first elucidated, which can be divided into the following parts: MVA pathway, cycloartanol synthesis pathway, P450 post-modification pathway, and glycosylation post-modification pathway. The MVA pathway provides sufficient FPP (farnesyl pyrophosphate) for the entire synthesis process; two molecules of FPP are condensed to form squalene under the action of epoxidase and cyclase OSC3, and then the triterpenoid skeleton cycloartanol is generated; cycloastragenol is converted from cycloartanol under the catalysis of multiple P450 enzymes and dioxygenases; finally, a glycosidic bond is formed between UDP-glucose or UDP-xylose and cycloastragenol under the catalysis of glycosyltransferase, thereby synthesizing astragaloside IV. Researchers have successfully synthesized cycloastragenol and astragaloside IV in tobacco using this biosynthetic pathway, with the yield of astragaloside IV reaching 2.224 mg / g of dry weight.

[0004] Although there have been reports of using tobacco for the heterologous synthesis of cycloastragenol, the yield is still relatively low, and there are also difficulties in large-scale production. Using microbial fermentation to obtain cycloastragenol and astragaloside IV is a more competitive and promising production method. Compared with plant extraction, microbial synthesis has the advantages of low cost, short production cycle, and easy access to raw materials, and is the most promising method. In the article <Research Progress in Yeast Synthesis of Cycloastragenol> by Yuan Yingzi et al., the synthesis of cycloastragenol by recombinant Saccharomyces cerevisiae is reviewed, and it is pointed out that improving the synthesis yield and yield of cycloastragenol in yeast is currently a challenge. SUMMARY

[0005] To solve the above problems, the present application provides a recombinant strain for producing cycloartanol, wherein the recombinant strain is a strain overexpressing the following genes:

[0006] a cyclase gene OCS3, an epoxidase gene ERG1, a P450 enzyme gene CYP88D25, a P450 enzyme gene CYP71D756, a P450 enzyme gene CYP88D7, a dioxygenase gene OGD1, and a flavin reductase gene AtCPR1.

[0007] The cyclase gene OCS3 has a GenBank number MT080939.1 in NCBI.

[0008] The epoxidase gene ERG1 has a GenBank number NM_001181304.1 or XM_503994.2 in NCBI.

[0009] The P450 enzyme gene CYP88D25 has a GenBank number OQ365041 in NCBI.

[0010] The P450 enzyme gene CYP71D756 has a GenBank number OQ365043 in NCBI.

[0011] The P450 enzyme gene CYP88D7 has a GenBank number OQ365042 in NCBI.

[0012] The dioxygenase gene OGD1 has a GenBank number OQ365044 in NCBI.

[0013] The nucleotide sequence of the flavin reductase gene AtCPR1 is the CDS: 134-2200 of GenBank number NM_001203894.1 in NCBI.

[0014] Further, the strain also overexpresses a glycosyltransferase GT15 and a glycosyltransferase GT36.

[0015] The glycosyltransferase GT15 has a GenBank number OQ365045 in NCBI.

[0016] The glycosyltransferase GT36 has a GenBank number OM913794 in NCBI.

[0017] Further, the strain is Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Corynebacterium glutamicum, Escherichia coli or Bacillus subtilis.

[0018] Further, when the strain is Saccharomyces cerevisiae, the strain further overexpresses P450 reductase gene NCP1 and endoplasmic reticulum-related transcription factor INO2, and is blocked to express lanosterol synthesis gene ERG7, transcriptional repressor ROX1, epoxide hydrolase gene LAP2 and epoxide hydrolase gene YNR064C.

[0019] The GenBank number of the P450 reductase gene NCP1 in NCBI is NM_001179172.1.

[0020] The GenBank number of the endoplasmic reticulum-related transcription factor INO2 gene in NCBI is NM_001180431.1.

[0021] The GenBank number of the lanosterol synthesis gene ERG7 in NCBI is NM_001179202.2.

[0022] The GenBank number of the transcriptional repressor ROX1 in NCBI is NM_001184162.1.

[0023] The GenBank number of the epoxide hydrolase LAP2 in NCBI is NM_001182884.1.

[0024] The GenBank number of the epoxide hydrolase gene YNR064C in NCBI is NM_001183241.1.

[0025] The GenBank number of the epoxide enzyme gene ERG1 in NCBI is NM_001181304.1.

[0026] Further, the Saccharomyces cerevisiae includes a high-yield squalene chassis strain Saccharomyces cerevisiae Sq55; the strain Saccharomyces cerevisiae Sq55 is a yeast engineering strain Sq61 described in patent CN119752658A.

[0027] Furthermore, when the strain is Yarrowia lipolytica, the expression of the lanosterol synthesis gene ERG7 is also blocked; the GenBank number of the lanosterol synthesis gene ERG7 in NCBI is: XM_504990.3;

[0028] The cyclooxygenase gene ERG1 has a GenBank accession number of XM_503994.2 in NCBI;

[0029] The Yarrowia lipolytica is the squalene-producing Yarrowia lipolytica.

[0030] The present invention also provides a method for producing cycloastragenol, which is fermented using the aforementioned recombinant bacteria.

[0031] Furthermore, the fermentation step is:

[0032] The seed liquid of the recombinant bacteria is inoculated into a fermentation medium for shake flask fermentation or fermentation in a fermenter, and the fermentation liquid is collected, the cell wall is broken, and the liquid is extracted with ethyl acetate.

[0033] Furthermore, the formula of the fermentation medium is:

[0034] Glucose 20g / L, potassium dihydrogen phosphate 2.2g / L, dipotassium hydrogen phosphate 2.9g / L, yeast powder 10g / L, peptone 20g / L, and the rest is water.

[0035] Furthermore, the shake flask fermentation conditions are as follows: the inoculation amount of the seed liquid is 1%; the fermentation temperature is 25-35° C., the rotation speed is 100-300 rpm, and the fermentation time is 60-120 h.

[0036] The recombinant bacteria producing cycloastragenol of the present invention uses a squalene-producing strain as the starting strain, promotes the synthesis of the triterpene skeleton by fusion expression of the ERG1 and OSC3 genes, integrates multiple copies of the P450 enzyme genes CYP88D25, CYP88D7, CYP71D756 and the dioxygenase gene OGD1, overexpresses the cytochrome P450 reductase gene AtCPR1 and the endogenous CPR gene NCP1 of Saccharomyces cerevisiae, and the cytochrome P450 reductase gene as a key coenzyme to transfer electrons for the P450 enzyme, thereby achieving the production of cycloastragenol in Saccharomyces cerevisiae. In addition, the production of cycloastragenol is further improved by metabolic modification strategies such as knocking out competitive pathways, regulating the size of the endoplasmic reticulum, and knocking out negative regulatory transcription factors. Ultimately, the yield of the recombinant yeast strain producing cycloastragenol obtained by the present invention in a shake flask reached 23 mg / L. The present invention also site-specifically integrates two glycosyltransferases, GT15 and GT36, into an engineered strain with high cycloastragenol production. After the fermentation broth is concentrated, a trace amount of astragaloside IV is detected, which is suitable for practical promotion and application.

[0037] Obviously, according to the above content of the present application, other various forms of modification, replacement or change can be made according to the common technical knowledge and usual means in the art without departing from the above basic technical idea of the present application.

[0038] The above content of the present application will be further explained in detail through the following specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the present application is limited to the following examples. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Structural formula of cycloastragenol and its glycosyl derivatives

[0040] Figure 2 Biosynthetic pathway of cycloastragenol and its glycosyl derivatives

[0041] Figure 3 pHHQC02-ERG1-GSlinker-OSC3 fusion expression plasmid map

[0042] Figure 4 Multi-copy site integration into yeast genome map (A, CYP88D25 and CYP71D756 expression cassette; B, CYP88D7 and OGD1 expression cassette)

[0043] Figure 5 pHHQC05-AtCPR1-Ncp1 expression cassette plasmid map

[0044] Figure 6 pHHQC06-INO2 expression cassette plasmid map

[0045] Figure 7 pHHQC06-GT15 and pHHQC06-GT36 expression cassette plasmid map

[0046] Figure 8 HPLC analysis of cycloastragenol synthesized by standard and yeast engineering strains (A, standard map, B, yeast engineering strain map)

[0047] Figure 9 Accumulation of cycloastragenol and squalene by recombinant yeast strains Sq55, Cy04, Cy05, Cy06, Cy07 after 72h cultivation in shake flask

[0048] Figure 10 Yarrowia lipolytica expression cassette plasmid map

[0049] Figure 11 Yield of cycloastragenol and squalene by Yarrowia lipolytica recombinant strains after 72h cultivation in shake flask

[0050] Figure 12LC-MS analysis of astragaloside IV DETAILED DESCRIPTION

[0051] The raw materials and reagents used in the specific embodiments of the application can be obtained by market purchase, wherein the pBBS17 plasmid used is the pBBS17 plasmid described in patent CN116716196A; the high-yield squalene chassis strain Saccharomyces cerevisiae Sq55 is the yeast engineering strain Sq61 in CN119752658A.

[0052] Example 1 Construction of Saccharomyces cerevisiae engineering strain for producing cycloastragenol and astragaloside IV according to the application

[0053] (1) Fusion expression of ERG1 gene (GenBank No. NM_001181304.1) and OSC3 gene (GenBank No. MT080939.1)

[0054] The OSC3 gene encoding cyclase from Astragalus membranaceus was amplified by PCR using the cDNA of Astragalus membranaceus as a template and OSC3-GSlinker-F / OSC3-R as primers, and the PCR product was detected by 1.0% agarose gel electrophoresis and purified by a clean-up kit;

[0055] The PCR product of the yeast endogenous gene ERG1 was obtained by PCR using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template and ERG1-F / ERG1-GSlinker-R as primers, and the PCR product was detected by 1.0% agarose gel electrophoresis and purified by a clean-up kit;

[0056] The empty plasmid pBBS17 was double-digested with BamH1 and HindIII, and the digested product was detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered by gel cutting and purification; then the purified gene fragment was ligated with the linearized plasmid pBBS17 using the multi-fragment one-step cloning kit of Abudantek (37℃, 60min); the ligation product was transformed into E. coli DH5α to obtain the transformation product; the transformation product was spread on LB solid medium (added with a final concentration of 100mg / L ampicillin) to obtain single colonies, which were then cultured in a shaker flask at 37℃, 220rpm for 8-12h, and the plasmid was extracted for sequencing verification, and the correct one was the fusion expression plasmid pHHQC02-ERG1-GSlinker-OSC3, see Figure 3 ;

[0057] PCR products of homologous arms LAP2-UP and LAP2-Dn were obtained using primers LAP2-UF / LAP2-UR, LAP2-DF / LAP2-DR with Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as template, and the PCR products were detected by 1.0% agarose gel electrophoresis and purified by clean-up kit;

[0058] PCR product of ERG1-GSlinker-OSC3 expression cassette was obtained using primers LAP2-F / LAP2-R with plasmid pHHQC02-ERG1-GSlinker-OSC3 as template, and the PCR products were detected by 1.0% agarose gel electrophoresis and purified by clean-up kit;

[0059] The purified products of homologous arms LAP2-UP and LAP2-Dn and the purified product of ERG1-GSlinker-OSC3 expression cassette were used to integrate the ERG1 fusion OSC3 expression cassette into the LAP2 locus of the high-squalene-producing chassis strain Saccharomyces cerevisiae Sq55 by CRISPR / Cas9 gene editing technology, to complete the overexpression of ERG1 and OSC3 while knocking out the epoxide hydrolase gene LAP2 (GenBank No. NM_001182884.1), and obtain the yeast engineering strain Cy01.

[0060] (2) Multi-copy integration of cytochrome P450 enzyme gene

[0061] The P450 genes CYP88D25 (GenBank No. OQ365041), CYP88D7 (GenBank No. OQ365042), CYP71D756 (GenBank No. OQ365043) and dioxygenase gene OGD1 (GenBank No. OQ365044) derived from Astragalus membranaceus were PCR amplified using primers CYP88D25-F / CYP88D25-R, CYP88D7-F / CYP88D7-R, CYP71D756-F / CYP71D756-R and OGD1-F / OGD1-R, respectively, with the cDNA of Astragalus membranaceus as template, and the PCR products were detected by 1.0% agarose gel electrophoresis and purified by clean-up kit;

[0062] The target plasmid pBBS17 was double digested with BamHI and HindIII, and the digested product was detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered and purified by cutting the gel; then the purified CYP88D25 gene fragment was connected with the linearized plasmid pBBS17 using the Novagen one-step cloning kit (37°C, 30min); the connection product was transformed into E. coli DH5α to obtain the transformed product; the transformed product was spread on LB solid medium (containing a final concentration of 100mg / L ampicillin) to obtain a single colony, which was then cultured in a shaking flask at 37°C, 220rpm for 8-12h, and the plasmid was extracted for sequencing verification. If the verification is correct, the P450 enzyme gene CYP88D25 expression plasmid pHHQC03-CYP88D25 is obtained.

[0063] The target plasmid pHHQC03-CYP88D25 was double digested with NotI and BcuI, and the digested product was detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered and purified by cutting the gel; then the purified CYP71D756 gene fragment was connected with the linearized plasmid pHHQC03-CYP88D25 using the Novagen one-step cloning kit (37°C, 30min); the connection product was transformed into E. coli DH5α to obtain the transformed product; the transformed product was spread on LB solid medium (containing a final concentration of 100mg / L ampicillin) to obtain a single colony, which was then cultured in a shaking flask at 37°C, 220rpm for 8-12h, and the plasmid was extracted for sequencing verification. If the verification is correct, the P450 enzyme gene CYP88D25 and CYP71D756 expression plasmid pHHQC03-CYP88D25-CYP71D756 is obtained.

[0064] According to the same method for constructing the plasmid pHHQC03-CYP88D25-CYP71D756, the purified gene fragments CYP88D7 and OGD1 were used to obtain the P450 enzyme gene CYP88D7 and dioxygenase gene OGD1 expression plasmid pHHQC04-CYP88D7-OGD1.

[0065] Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template, the homologous arms rDNA-UP and rDNA-Dn PCR products were obtained by PCR using primers rDNA-UF / rDNA-UR, rDNA-DF / rDNA-DR, and the gene fragments were purified by 1.0% agarose gel electrophoresis and clean-up kit.

[0066] The CYP88D25 and CYP71D756 expression cassette PCR products were obtained by PCR using the plasmid pHHQC03-CYP88D25-CYP71D756 as a template and primers rDNA-F / rDNA-R, and the PCR products were detected by 1.0% agarose gel electrophoresis and purified by a clean-up kit;

[0067] The LEU2 gene expression cassette PCR product was obtained by PCR using the Saccharomyces cerevisiae S288C as a template and primers LEU2-F / LEU2-R, and the PCR product was detected by 1.0% agarose gel electrophoresis and purified by a clean-up kit;

[0068] The purified products of the homologous arms rDNA-UP and rDNA-Dn, the CYP88D25 and CYP71D756 expression cassette, and the LEU2 expression cassette were transformed into the yeast engineering strain Cy01 competent cells to obtain a transformation product; the transformation product was coated on an SD-ΔLEU solid medium and incubated in an inverted incubator at 30°C for about 48 h to obtain a transformant. The transformant is the yeast engineering strain Cy02 in which the CYP88D25 gene and the CYP71D756 gene are integrated into the multicopy site of the yeast engineering strain Cy01 by using the endogenous homologous recombination of the yeast. Figure 4 A).

[0069] In the same manner as in the construction of the yeast engineering strain Cy02, the CYP88D7 and OGD1 expression cassette PCR products were obtained by PCR using the above P450 enzyme and dioxygenase expression plasmid pHHQC04-CYP88D7-OGD1 as a template, and the P450 enzyme gene CYP88D7 and the dioxygenase gene OGD1 were integrated into the multicopy site of the yeast engineering strain Cy02 by using the endogenous homologous recombination of the Saccharomyces cerevisiae to obtain the yeast engineering strain Cy03 Figure 4 B).

[0070] (3) Overexpression of the AtCPR1 gene (GenBank No. NM_001203894.1 (CDS: 134-2200)) and the endogenous NCP1 gene (GenBank No. NM_001179172.1)

[0071] The gene AtCPR1 of cytochrome P450 reductase from Arabidopsis thaliana is amplified by PCR using the cDNA of Arabidopsis thaliana as template and the primer pair AtCPR1-F / AtCPR1-R, and the PCR product is detected by 1.0% agarose gel electrophoresis and purified by clean-up kit;

[0072] The target plasmid pBBS17 is double digested by BamHI and HindIII, and the digested product is detected by 1.0% agarose gel electrophoresis and the linearized vector fragment is purified by gel cutting and recovering. Then the purified AtCPR1 gene fragment is ligated with the linearized plasmid pBBS17 by using the One Step Cloning Kit of Novagen (37℃, 30min), and the ligation product is transformed into E. coli DH5α to obtain the transformed product. The transformed product is spread on LB solid medium (with final concentration of 100mg / L ampicillin) to obtain single colonies, which are cultured in a shaker flask at 37℃, 220rpm for 8-12h, and then the plasmid is extracted and sequenced to verify. If the verification is correct, the expression plasmid pHHQC05-AtCPR1 of P450 reductase gene AtCPR1 is obtained.

[0073] The Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome is used as template to obtain the PCR product of the yeast endogenous P450 reductase gene NCP1 by PCR using the primer pair NCP1-F / NCP1-R, and the PCR product is detected by 1.0% agarose gel electrophoresis and purified by clean-up kit.

[0074] The target plasmid pHHQC05-AtCPR1 is double digested by NotI and SacI, and the digested product is detected by 1.0% agarose gel electrophoresis and the linearized vector fragment is purified by gel cutting and recovering. Then the purified NCP1 gene fragment is ligated with the linearized plasmid pHHQC05-AtCPR1 by using the One Step Cloning Kit of Novagen (37℃, 30min), and the ligation product is transformed into E. coli DH5α to obtain the transformed product. The transformed product is spread on LB solid medium (with final concentration of 100mg / L ampicillin) to obtain single colonies, which are cultured in a shaker flask at 37℃, 220rpm for 8-12h, and then the plasmid is extracted and sequenced to verify. If the verification is correct, the expression plasmid pHHQC05-AtCPR1-NCP1 of P450 reductase genes AtCPR1 and NCP1 is obtained (see Figure 5 );

[0075] PCR products of homologous arms YNR064C-UP and YNR064C-Dn were obtained using primers YNR064C-UF / YNR064C-UR, YNR064C-DF / YNR064C-DR with Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as template, and the PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified by clean-up kit;

[0076] PCR products of AtCPR1 and NCP1 expression cassette were obtained using primers YNR064C-F / YNR064C-R with plasmid pHHQC05-AtCPR1-NCP1 as template, and the PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified by clean-up kit;

[0077] The purified products of homologous arms YNR064C-UP and YNR064C-Dn, the purified products of AtCPR1 and NCP1 expression cassette were integrated into YNR064C site of engineering strain Cy03 by CRISPR / Cas9 gene editing technology, and AtCPR1 and NCP1 expression cassette was integrated into YNR064C site of engineering strain Cy03, and the knockout of epoxy hydrolase gene YNR064C (GenBank No. NM_001183241.1) was completed, and the yeast engineering strain Cy04 overexpressing AtCPR1 and NCP1 was obtained.

[0078] (4) Metabolic modification to block downstream competitive pathways

[0079] A key precursor in the biosynthesis of cycloartane is 2,3-epoxy-squalene, and the endogenous yeast catalyzes the formation of lanosterol by lanosterol synthase gene ERG7, and further forms the component of yeast cell wall ergosterol, which leads to the consumption of precursors. In order to minimize the consumption of substrates, it is necessary to completely block ERG7.

[0080] PCR products of homologous arms del-ERG7-UP and del-ERG7-Dn were obtained using primers ERG7-UF / del-ERG7-UR, del-ERG7-DF / ERG7-DR with Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as template, and the PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified by clean-up kit;

[0081] The purified products of homologous arms del-ERG7-UP and del-ERG7-Dn were transformed into the yeast engineering strain Cy04 competent cells, and the lanosterol synthase ERG7 gene (GenBank No.: NM_001179202.2) of Cy04 was knocked out by using the CRISPR / Cas9 gene editing technology, to obtain the yeast engineering strain Cy05.

[0082] (5) Adjusting the size of endoplasmic reticulum and knocking out negative transcription factor

[0083] The endoplasmic reticulum-related transcription factor INO2 gene PCR product was obtained by PCR using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template and primers INO2-F / INO2-R, and the PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by a clean-up kit;

[0084] The target plasmid pBBS17 was double-digested with BamHI and HindIII, and the digested product was detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was purified by gel cutting and recovery. Then the purified INO2 gene fragment was ligated with the linearized plasmid pBBS17 using a Novagen one-step cloning kit (37°C, 30min); the ligation product was transformed into E. coli DH5α to obtain the transformation product; the transformation product was plated on LB solid medium (containing a final concentration of 100mg / L ampicillin) to obtain a single colony, which was then cultured in a shaker flask at 37°C, 220rpm for 8-12h, and the plasmid was extracted for sequencing verification. The correct verification obtained the endoplasmic reticulum-related transcription factor INO2 expression plasmid pHHQC06-INO2( Figure 6 );

[0085] The homologous arms ROX1-UP and ROX1-Dn PCR products were obtained by PCR using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template and primers ROX1-UF / ROX1-UR, ROX1-DF / ROX1-DR, and the PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified by a clean-up kit;

[0086] The endoplasmic reticulum-related transcription factor INO2 expression cassette PCR product was obtained by PCR using plasmid pHHQC06-INO2 as a template and primers ROX1-F / ROX1-R, and the PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by a clean-up kit;

[0087] According to the same method of constructing the yeast engineering strain Cy01, the endoplasmic reticulum-associated transcription factor IN02 (GenBank No. NM_001180431.1) was integrated into the ROX1 site of Cy05, and the overexpression of IN02 and the knockout of the transcriptional repressor ROX1 (GenBank No. NM_001184162.1) were completed, thereby obtaining the yeast engineering strain Cy06.

[0088] (6) Integrating glycosyltransferase to produce astragaloside

[0089] The genes encoding glycosyltransferases GT15 (GenBank No. OQ365045) and GT36 (GenBank No. OM913794) from Astragalus membranaceus were amplified using the cDNA of Astragalus membranaceus as a template and the primer pairs GT15-F / GT15-R and GT36-F / GT36-R, respectively, to obtain the gene fragments of GT15 and GT36. The PCR products were detected by 1.0% agarose gel electrophoresis, and the gene fragments were purified by a clean-up kit.

[0090] According to the same method of constructing pHHQC05-AtCPR1-NCP1, the purified GT15 and GT36 gene fragments and the empty plasmid pBBS17 were subjected to enzyme digestion and one-step cloning ligation to obtain the expression plasmid pHHQC07-GT15-GT36 of the glycosyltransferase genes GT15 and GT36. Figure 7 ).

[0091] The homologous arms GAL1-7-UP and GAL1-7-Dn were obtained by PCR using the Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template and the primer pairs GAL1-7-UF / GAL1-7-UR and GAL1-7-DF / GAL1-7-DR. The PCR products were detected by 1.0% agarose gel electrophoresis, and the gene fragments were purified by a clean-up kit.

[0092] The glycosyltransferase gene expression cassettes of GT15 and GT36 were obtained by PCR using the plasmid pHHQC07-GT15-GT36 as a template and the primer pairs, respectively. The PCR products were detected by 1.0% agarose gel electrophoresis, and the gene fragments were purified by a clean-up kit.

[0093] According to the same method of constructing the yeast engineering strain Cy01, the expression cassettes of glycosyltransferases GT15 and GT36 were sequentially integrated into the GAL1-7 site of the yeast engineering strain Cy06, and the expression of glycosyltransferases was completed, and the yeast engineering strains Cy07 were obtained.

[0094] Example 2 The present application constructs a yeast engineering strain for producing cycloastragenol and astragaloside A using Yarrowia lipolytica as a chassis

[0095] The gene editing method of CRISPR-Cas9 was used to integrate the target gene into the genome of Yarrowia lipolytica. This method requires the construction of a pCRISPRyl plasmid carrying a gene expressing Cas9 protein and an sgRNA sequence containing an integration site PAM sequence, and a pHR plasmid containing an integration site homologous arm and a donor. The pCRISPRyl plasmid and pHR plasmid used are constructed according to Wheeldon et al., ACS Synth. Biol. 2017, 6, 402-409; since the sites used for gene integration are constructed in advance, only the expression frame for overexpression needs to be connected between the left and right homologous arms containing the corresponding site.

[0096] (1) Co-overexpression of ERG1 (GenBank No. XM_503994.2) and OSC3 (GenBank No. MT080939.1) in Yarrowia lipolytica

[0097] ①OSC3 and ERG1 genes were amplified from Astragalus membranaceus cDNA and Yarrowia lipolytica genome, respectively, using primer pairs OSC3-F / OSC3-R and ERG1-F / ERG1-R. Subsequently, the corresponding promoters and terminators were amplified from the Yarrowia lipolytica genome using primer pairs OSC3-TEF-F / OSC3-TEF-R, XPR2-F(OSC3) / XPR2-R(OSC3), ERG1-TEF-F / ERG1-TEF-R, and XPR2-F(ERG1) / XPR2-R(ERG1), respectively. The open reading frames, corresponding promoters and terminators were connected by overlap. Subsequently, the two fragments successfully connected were connected to the left and right homologous arms of the pHR-C3 plasmid linearized by Spe I, and the plasmid pHR_C3_OSC3+ERG1 was obtained, i.e., OSC3 and ERG1 were overexpressed at the C3 site.

[0098] ② The previously constructed plasmid pCRISPRyl-C3 for simultaneous overexpression of ERG1 and OSC3 at the C3 locus and the plasmid pHR_C3_OSC3+ERG1 were co-transformed into competent cells of the laboratory-constructed squalene-producing recombinant strain SQ4. Subsequently, the cells were plated on YNB plates and cultured in a 30°C constant temperature incubator for 3-4 days. Positive single clones were screened by colony PCR. The correct single clones verified by PCR were inoculated into YPD liquid medium and cultured for 16 hours. After that, they were streaked onto YPD plates containing 5-FOA (5-fluoroorotic acid) and cultured at 30°C for 24 hours to eliminate the LEU and URA tags. Subsequently, the single colonies on the 5-FOA plate were inoculated into YPD, YNB+LEU, and YNB+URA liquid culture media to verify whether the screening marker was successfully removed. The bacteria with successful removal were preserved, and the remaining liquid was used for genome extraction. Finally, the genome was used as a template for verification with primers, and the amplified bands were sequenced. The strain with correct sequencing was YLC-1.

[0099] (2) Overexpression of cytochrome P450 enzyme genes in the cycloastragenol biosynthesis pathway (CYP88D25, GenBank No. OQ365041; CYP71D756, GenBank No. OQ365043; CYP88D7, GenBank No. OQ365042), the reduction chaperone AtCPR (GenBank No. NM_001203894.1 (CDS: 134-2200)), and the cycloastragenol synthase gene (OGD1, GenBank No. OQ365044)

[0100] The same method as in “(1) Co-overexpression of ERG1 and OSC3 in Yarrowia lipolytica” was used to construct plasmids pHR-AXP-CYP88D25, pHR-XPR2-CYP88D7, pHR-A3-CYP71D756, pHR-MFE1-AtCPR, and pHR-D17-CYPOGD1 ( Figure 10 ), and these pHR plasmids and the corresponding pCRISPRyl plasmid combinations were sequentially co-transformed into recombinant strain YLC-1 to obtain strain YLC-2; into recombinant strain YLC-2 to obtain strain YLC-3; into recombinant strain YLC-3 to obtain strain YLC-4; into recombinant strain YLC-4 to obtain strain YLC-5; and into recombinant strain YLC-5 to obtain strain YLC-6. The primers used in the construction process are shown in Table 1.

[0101] (3) Metabolic modification blocks downstream competitive pathways

[0102] The upper and lower homologous arms of ERG7 were amplified from the genome of Yarrowia lipolytica by using primer pair ERG7-LH-F / ERG7-LH-R and ERG7-LH-F / ERG7-RH-R, respectively, and the purified two fragments were ligated with the backbone part of pHR plasmid to obtain the Donor plasmid pHR-ΔERG7 for knocking out ERG7. The pCRISPRyl was linearized by using primer pair ERG7-sg-F / ERG-sg-R, and then transformed into DH5α. The single colony on the plate was picked and sequenced. The correct sequencing result was the Cas9 plasmid pCRISPRyl-ΔERG7 for knocking out ERG7 (GenBank No.: XM_504990.3).

[0103] The plasmid pHR-ΔERG7 and the plasmid pCRISPRyl-ΔERG7 were co-transformed into YLC-6 to obtain the recombinant strain YLC-7. The ERG7 in the strain was completely destroyed, resulting in the blockage of the biosynthesis pathway of lanosterol.

[0104] (4) Integrating glycosyltransferases (GT15, GenBank No. OQ365045; GT36, GenBank No. OM913794) to produce astragaloside B

[0105] The same method as that in the step of "(1) Co-overexpressing ERG1 and OSC3 in Yarrowia lipolytica" was used to construct the plasmids pHR-C5-GT15, pHR-B1-GT36( Figure 10 ), respectively. These pHR plasmids and the corresponding pCRISPRyl plasmids were combined and co-transformed into the recombinant strain YLC-7 in sequence to obtain the strain YLC-8, and then transformed into the recombinant strain YLC-8 to obtain the strain YLC-9. The primers used in the construction process are shown in Table 1.

[0106] The primer information involved in Examples 1-2 is as follows:

[0107] Table 1 Nucleotide sequences of primers

[0108]

[0109]

[0110]

[0111] The beneficial effects of the present application are further illustrated by the following test examples:

[0112] Test Example 1 Application of recombinant yeast in synthesis of cycloastragenol

[0113] (1) Preparation of culture medium

[0114] YPD medium composition: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L, solvent is deionized water, pH value is natural. YPD plate is added with 2 g / L agar in YPD liquid medium.

[0115] Seed medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L;

[0116] Fermentation medium: glucose 20 g / L, potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 2.9 g / L, yeast extract 10 g / L, peptone 20 g / L.

[0117] (2) Production of cycloartanol at a shake flask level

[0118] The recombinant yeast engineering strains Cy04 / Cy05 / Cy06, YPC-6 / YPC-7 constructed in Examples 1 and 2 were picked and subjected to fermentation test in a shake flask. The specific shake flask fermentation test steps are as follows:

[0119] The single colonies cultured on YPD solid medium for 48 h were inoculated into seed medium, and then subjected to 24 h culture at 30°C with 200 rpm shaking bed oscillation. The cultured seed was inoculated into fermentation medium at 1% (v / v), and then subjected to 72 h culture at 30°C with 200 rpm shaking bed oscillation. After the fermentation was completed, the fermentation liquor was broken, and cycloartanol was obtained by extraction with ethyl acetate.

[0120] (3) Determination of cycloartanol content:

[0121] Preparation of standard sample: 1 g / L cycloartanol standard sample was prepared, and standard samples with concentrations of 10, 30, 50, 70 and 100 mg / L were diluted, respectively. 1 mL of sample was filtered with a filter membrane and then loaded into a sample bottle for determination.

[0122] Preparation of sample: 0.6 mL of fermentation liquor was added with 0.6 mL of ethyl acetate and 0.3 g of zirconium oxide beads, and the cells were broken by a frozen grinder under the following working conditions: 55 HZ, 30 min. After 200 μL of ethyl acetate was taken and dried, 200 μL of ethanol was added for membrane sampling.

[0123] LC-MS detection method:

[0124] Solvent A: [H2O + 0.1% formic acid], solvent B: [60% acetonitrile (CH3CN)]; sample volume: 5 μL; gradient: 60% [B] from 0 to 18 min; flow rate: 1 mL·min -1

[0125] ​The LC-MS detection results of cycloastragenol in the standard sample and the fermentation broth obtained by fermentation of the recombinant yeast engineering strain are shown in Table 1. Figure 8 The recombinant yeast engineering strains Cy06 and YPC-7 were inoculated into the fermentation medium with glucose as the carbon source and fermented for 72 h, and the yield of cycloastragenol in the constructed engineering strains reached 23.2 mg / L and 8 mg / L, respectively. Figure 9 , Figure 11

[0126] Test Example 2 Application of Recombinant Yeast in Synthesis of Astragaloside A

[0127] (1) Preparation of culture medium

[0128] The composition of YPD culture medium is as follows: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, solvent is deionized water, and the pH value is natural. The YPD plate is prepared by adding agar with a final concentration of 2 g / L to the YPD liquid medium.

[0129] Seed culture medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L;

[0130] Fermentation medium: glucose 20 g / L, potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 2.9 g / L, yeast powder 10 g / L, peptone 20 g / L.

[0131] (2) Production of astragaloside A in shake flask

[0132] The recombinant engineering strains Cy07 and YLC-9 constructed in Examples 1 and 2 were picked for fermentation test in shake flask, and the specific shake flask fermentation test steps are as follows:

[0133] The single colonies cultured on YPD solid medium for 48 h were inoculated into the seed culture medium, and incubated at 30°C with 200 rpm shaking for 24 h. The cultured seed was inoculated into the fermentation medium at 1% (v / v), and incubated at 30°C with 200 rpm shaking for 72 h. After fermentation, the fermentation broth was broken and extracted with ethyl acetate to obtain astragaloside A. The astragaloside A in the fermentation broth of the recombinant strains Cy07 and YPC-9 was detected.

[0134] (3) Determination of the content of astragaloside A:

[0135] Preparation of standard sample: 1 g / L astragaloside A standard sample was prepared, and the standard sample was diluted to concentrations of 10, 30, 50, 70 and 100 mg / L, respectively. 1 mL of the sample was filtered with a filter membrane and placed in a sample bottle for testing.

[0136] ​Sample preparation: 0.6 mL of fermentation broth was taken, 0.6 mL of ethyl acetate and 0.3 g of zirconium oxide beads were added, and the cells were broken by a frozen grinder under the following working conditions: 55 HZ, 30 min of breaking, 200 μL of ethyl acetate was taken, and after being dried, 200 μL of ethanol was added to pass through the membrane for sampling.

[0137] LC-MS detection method:

[0138] Solvent A: [H2O + 0.1% formic acid], solvent B: [60% acetonitrile (CH3CN)]; injection volume: 5 μL; gradient: from 0 to 18 min 60% [B]; using 1 mL·min -1 of flow rate; chromatographic column: Welchrom column C18 (4.6 x 250 mm x 5 μm)

[0139] After the fermentation was completed, no astragaloside A was detected in the fermentation broth of the recombinant bacteria Cy07 and YPC-9, and then, after the fermentation broth was concentrated by 20 times, a small amount of astragaloside A was detected. Figure 12

[0140] In summary, the present application provides a recombinant bacteria for producing cycloastragenol, which is a squalene-producing strain as a starting strain, and the genes in the synthesis pathway are stably integrated into the yeast genome by using CRISPR / Cas9 technology, including fusion expression of ERG1 and OSC3 genes, multi-copy integration of P450 enzyme genes CYP88D25, CYP88D7, CYP71D756, OGD1, overexpression of cytochrome P450 reductase gene AtCPR1 and endogenous CPR gene NCP1 of Saccharomyces cerevisiae, realizing de novo synthesis of cycloastragenol. And through metabolic modification strategies such as knocking out competitive pathways, adjusting the size of endoplasmic reticulum, knocking out negative regulatory transcription factors, the yield of cycloastragenol is further improved. Subsequently, on the basis of the recombinant strain with further improved cycloastragenol yield, glycosyltransferase is integrated, realizing de novo synthesis of astragaloside A Figures 1-2 ), laying a foundation for subsequent biosynthesis of triterpenoids.​

Claims

1. A recombinant bacterium producing cycloastragenol, characterized in that: The recombinant bacteria are strains that overexpress the following genes: Cyclase gene OCS3, cyclooxygenase gene ERG1, P450 enzyme gene CYP88D25, P450 enzyme gene CYP71D756, P450 enzyme gene CYP88D7, dioxygenase gene OGD1, flavin reductase gene AtCPR1; The cyclase gene OCS3 has a GenBank accession number of MT080939.1 in NCBI; The cyclooxygenase gene ERG1 has a GenBank accession number of NM_001181304.1 or XM_503994.2 in NCBI; The P450 enzyme gene CYP88D25 has a GenBank accession number of OQ365041 in NCBI; The P450 enzyme gene CYP71D756 has a GenBank accession number of OQ365043 in NCBI; The P450 enzyme gene CYP88D7 has a GenBank accession number of OQ365042 in NCBI; The dioxygenase gene OGD1 has a GenBank accession number of OQ365044 in NCBI; The nucleotide sequence of the flavin reductase gene AtCPR1 is CDS: 134-2200 of GenBank No. NM_001203894.1 in NCBI.

2. The recombinant bacterium according to claim 1, characterized in that: The strain also overexpressed glycosyltransferase GT15 and glycosyltransferase GT36; The glycosyltransferase GT15 has a GenBank accession number of OQ365045 in NCBI; The glycosyltransferase GT36 has a GenBank accession number of OM913794 in NCBI.

3. The recombinant bacterium according to claim 1 or 2, characterized in that: The strain is Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Corynebacterium glutamicum, Escherichia coli or Bacillus subtilis.

4. The recombinant bacterium according to claim 3, characterized in that: When the strain is Saccharomyces cerevisiae, it also overexpresses the P450 reductase gene NCP1 and the endoplasmic reticulum-related transcription factor INO2, and blocks the expression of the lanosterol synthesis gene ERG7, the transcription repressor ROX1, the epoxide hydrolase gene LAP2, and the epoxide hydrolase gene YNR064C; The P450 reductase gene NCP1 has a GenBank accession number of NM_001179172.1 in NCBI; The GenBank number of the endoplasmic reticulum-related transcription factor INO2 gene in NCBI is NM_001180431.1; The GenBank number of the lanosterol synthesis gene ERG7 in NCBI is: NM_001179202.2; The GenBank number of the transcriptional repressor ROX1 in NCBI is NM_001184162.1; The GenBank number of the epoxy hydrolase LAP2 in NCBI is NM_001182884.1; The epoxide hydrolase gene YNR064C has a GenBank accession number of NM_001183241.1 in NCBI; The cyclooxygenase gene ERG1 has a GenBank accession number of NM_001181304.1 in NCBI.

5. The recombinant bacterium according to claim 4, characterized in that: The brewer's yeast includes a chassis strain Saccharomyces cerevisiae Sq55 with high squalene production; the strain Saccharomyces cerevisiae Sq55 is the yeast engineering strain Sq61 described in patent CN119752658A.

6. The recombinant bacterium according to claim 3, characterized in that: When the strain is Yarrowia lipolytica, the expression of the lanosterol synthesis gene ERG7 is also blocked; the GenBank number of the lanosterol synthesis gene ERG7 in NCBI is: XM_504990.3; The cyclooxygenase gene ERG1 has a GenBank accession number of XM_503994.2 in NCBI; The Yarrowia lipolytica is the squalene-producing Yarrowia lipolytica.

7. A method for producing cycloastragenol, characterized in that: The method is fermented by using the recombinant bacteria described in any one of claims 1 to 6.

8. The method according to claim 7, wherein: The fermentation steps are: The seed liquid of the recombinant bacteria is inoculated into a fermentation medium for shake flask fermentation or fermentation in a fermenter, and the fermentation liquid is collected, the cell wall is broken, and the liquid is extracted with ethyl acetate.

9. The method according to claim 8, characterized in that: The formula of the fermentation medium is: Glucose 20g / L, potassium dihydrogen phosphate 2.2g / L, dipotassium hydrogen phosphate 2.9g / L, yeast powder 10g / L, peptone 20g / L, and the rest is water.

10. The method according to claim 8, characterized in that: The shaking flask fermentation conditions are as follows: the inoculation amount of the seed liquid is 1%; the fermentation temperature is 25-35° C., the rotation speed is 100-300 rpm, and the fermentation time is 60-120 hours.

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