Recombinant saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II and construction method of recombinant saccharomyces cerevisiae

Abstract

The invention relates to a recombinant saccharomyces cerevisiae capable of producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II at the same time, and a construction method of the recombinant saccharomyces cerevisiae. The method comprises the following steps: firstly, introducing ERG1, ERG9, ERG20, tHMG1, synPgDDS, synBmCPR1 and a leucine screening tag LEU2 as well as a promoter and a terminator which are derived from yeast into a BY-MVA recombinant bacterium, so as to obtain a recombinant bacterium DM-BmCPR capable of producing dammarendiol; and introducing SynBmCYP068, which is from bacopa monnieri and is subjected to codon optimization, and a Lys screening tag into the DM-BmCPR strain together, so as to obtain the recombinant strain DBmCYP068. The invention also provides a basis for further functional characterization of the downstream modification enzyme gene P450 in the bacopa monnieri biosynthetic pathway.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammaren ediol II and a method for constructing the same. Background Art

[0002] With the intensification of the global aging situation and the current fast-paced lifestyle of people, as well as the high-intensity learning and work pressure, the nervous systems of a considerable number of people are in a sub-healthy state. The dammarane-type triterpenoid saponins in Bacopa monnieri have various pharmacological activities such as sedative-hypnotic, anti-anxiety, anti-depressant, anti-cancer, anti-inflammatory, and anti-Alzheimer's disease (AD). And its potential therapeutic effect on AD has received increasing attention in recent years, making it an important candidate for researching and preventing new drugs and health foods for AD. However, at present in China, Bacopa monnieri is mostly wild resources and the planting scale is not large, which makes it unable to meet the growing market demand. Using the method of biosynthesis to produce the dammarane-type triterpenoid saponins in Bacopa monnieri is an effective solution. However, there is currently no relevant report on the biosynthesis pathway of the dammarane-type triterpenoid saponins in Bacopa monnieri. Therefore, analyzing the biosynthesis pathway of the dammarane-type triterpenoid saponins in Bacopa monnieri is the key to using biosynthesis to produce monomeric compounds. During the process of analyzing the biosynthesis pathway, intermediates are the key to verifying the related enzyme-encoding genes in the biosynthesis pathway. However, the content of these intermediates in plants is extremely low and it is almost impossible to obtain them by extracting plant materials, which seriously affects the development and utilization of intermediates and the analysis of the related biosynthesis pathway.

[0003] Using the principles of synthetic biology to design and transform microorganisms to produce natural products has currently been widely applied to the production of natural products and their biosynthesis pathway intermediates. Among them, Saccharomyces cerevisiae is widely used. For example, the production of ginsenoside CK, Rh2, Rg3, dammarenediol, protopanaxadiol, and protopanaxatriol are all achieved by integrating the genes related to their biosynthesis into the chromosome of Saccharomyces cerevisiae. Among them, the yields of protopanaxadiol, protopanaxatriol, and ginsenoside CK can reach 11 g / L, 5 g / L, and 6 g / L respectively. The production of more and more natural products in Saccharomyces cerevisiae has been realized. These successful examples of using yeast cell factories to produce triterpenoid saponins and their intermediates provide a reference for the future efficient production of more valuable rare saponins by microbial synthesis, and also provide an effective solution for using Saccharomyces cerevisiae to construct chassis cells for these intermediates to produce intermediates or to verify the functions of related enzyme-encoding genes in the natural product biosynthesis pathway through recombinant plasmids.

[0004] At present, there is no relevant report on recombinant Saccharomyces cerevisiae that can produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II simultaneously. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a recombinant Saccharomyces cerevisiae that can produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II simultaneously and a method for constructing the same.

[0006] To achieve the above purpose, the technical scheme adopted by the present invention is as follows:

[0007] A method for constructing a recombinant Saccharomyces cerevisiae that can produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II simultaneously, comprising the following steps:

[0008] Introduce ERG1, ERG9, ERG20, tHMG1, the dammarenediol synthase gene synPgDDS derived from Panax ginseng, the NADPH-cytochrome P450 reductase gene synBmCPR1 derived from Bacopa monnieri, the leucine selection tag LEU2, and yeast-derived promoters and terminators into the BY-MVA recombinant bacterium to obtain a recombinant bacterium DM-BmCPR that can produce dammarenediol;

[0009] Then introduce the codon-optimized SynBmCYP068 gene expression cassette from Bacopa monnieri and the Lys selection tag together into the DM-BmCPR strain to obtain a recombinant bacterium DBmCYP068 that can produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II simultaneously.

[0010] Furthermore, preferably, the expression of the ERG1 gene introduced into BY4742 is controlled by the promoter IDP1 and the terminator TDH2; the expression of the ERG9 gene introduced into BY4742 is controlled by the promoter TEF1 and the terminator CYC1; the expression of the ERG20 gene introduced into BY4742 is controlled by the promoter CPS1 and the terminator ENO2; the expression of the tHMG1 gene introduced into BY4742 is controlled by the promoter PGK1 and the terminator ADH1. A gene expression cassette is constructed and co-transformed with the HIS3 selection tag into the YPRCδ15 locus of Saccharomyces cerevisiae BY4742 by homologous recombination through the lithium acetate transformation method to obtain a recombinant bacterium BY-MVA.

[0011] Furthermore, preferably, the expression of the tHMG1 gene introduced into the BY-MVA recombinant bacterium is controlled by the promoter HXT7 and the terminator ADH1, the expression of the synBmCPR1 gene introduced into the BY-MVA recombinant bacterium is controlled by the promoter TEF2 and the terminator TDH2, the expression of the ERG1 gene introduced into the BY-MVA recombinant bacterium is controlled by the promoter IPI1 and the terminator ENO2, the expression of the ERG20 gene introduced into the BY-MVA recombinant bacterium is controlled by the promoter GPM and the terminator CYC1, the expression of the ERG9 gene introduced into the BY-MVA recombinant bacterium is controlled by the promoter PGK1 and the terminator FBA1, and the expression of the synPgDDS gene introduced into the BY-MVA recombinant bacterium is controlled by the promoter TDH3 and the terminator PGT1. A gene expression cassette is constructed and co-transformed with the LEU2 selection tag into the δDNA locus of the BY-MVA recombinant bacterium by homologous recombination through the lithium acetate transformation method to obtain the recombinant bacterium DM-BmCPR capable of producing dammarenediol.

[0012] Furthermore, preferably, a SynBmCYP068 gene expression cassette is constructed using the promoter TEF1, the codon-optimized SynBmCYP068 from Bacopa monnieri, and the terminator PRM5. The SynBmCYP068 gene expression cassette and the Lys selection tag are co-transformed into the NTS2-1 locus of the DM-BmCPR strain by the lithium acetate transformation method to form the recombinant bacterium DBmCYP068 capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

[0013] Furthermore, preferably, the method for constructing the gene expression cassette includes: performing PCR amplification on the promoter, terminator, functional gene, and selection tag respectively using primers with homologous arms, then recovering the target bands to obtain single gene fragments; then using 3 to 6 adjacent basic fragments as templates to perform fusion PCR to obtain fusion fragments and construct a gene expression module.

[0014] Furthermore, preferably, the gene expression module specifically includes:

[0015] Fragment 1: The genes contained therein are UP, ADH1, tHMGR1, HXT7, TEF2;

[0016] Fragment 2: The genes contained therein are synBmCPR1, TDH2, ENO2, ERG1;

[0017] Fragment 3: The genes contained therein are IPI1, GPM, ERG20, CYC1, PGK1, ERG9;

[0018] Fragment 4: The genes it contains are FBA1, TDH3, synPgDDS;

[0019] Fragment 5: The genes it contains are PGT1, LEU2, DN;

[0020] Fragment 6: The genes it contains are NTS2-1-UP, TEF1, SynBmCYP068, PRM5;

[0021] Fragment 7: The gene it contains is Lys;

[0022] Fragment 8: The gene it contains is NTS2-1-DN.

[0023] Furthermore, preferably, the nucleotide sequence of the encoding gene UP is as shown in SEQ ID NO.1;

[0024] The nucleotide sequence of the encoding gene DN is as shown in SEQ ID NO.2;

[0025] The nucleotide sequence of the encoding gene synBmCPR1 is as shown in SEQ ID NO.3;

[0026] The nucleotide sequence of the encoding gene NTS2-1-UP is as shown in SEQ ID NO.4;

[0027] The nucleotide sequence of the encoding gene SynBmCYP068 is as shown in SEQ ID NO.5;

[0028] The nucleotide sequence of the encoding gene NTS2-1-DN is as shown in SEQ ID NO.6.

[0029] The nucleotide sequence of the promoter IDP1 is as shown in SEQ ID NO.7;

[0030] The nucleotide sequence of the promoter CPS1 is as shown in SEQ ID NO.8;

[0031] The nucleotide sequence of the promoter HXT7 is as shown in SEQ ID NO.9;

[0032] The nucleotide sequence of the promoter TEF2 is as shown in SEQ ID NO.10;

[0033] The nucleotide sequence of the promoter IPI1 is as shown in SEQ ID NO.11;

[0034] The nucleotide sequence of the promoter GPM is as shown in SEQ ID NO.12;

[0035] The nucleotide sequence of the terminator TDH2 is shown as SEQ ID NO.13;

[0036] The nucleotide sequence of the terminator ENO2 is shown as SEQ ID NO.14;

[0037] The nucleotide sequence of the terminator ADH1 is shown as SEQ ID NO.15;

[0038] The nucleotide sequence of the terminator FBA1 is shown as SEQ ID NO.16;

[0039] The nucleotide sequence of the terminator PGT1 is shown as SEQ ID NO.17;

[0040] The nucleotide sequence of the terminator PRM5 is shown as SEQ ID NO.18;

[0041] The nucleotide sequence of the screening tag Lys is shown as SEQ ID NO.19.

[0042] The nucleotide sequence of the coding gene tHMGR1 is described in 201210453416X; the nucleotide sequence of the coding gene ERG1 is described in 201210453416X; the nucleotide sequence of the coding gene ERG9 is described in 201210453416X; the nucleotide sequence of the coding gene ERG20 is described in 201210453416X; the nucleotide sequence of the promoter TEF1 is described in 201210453416X; the nucleotide sequence of the promoter PGK1 is described in 201210453416X; the nucleotide sequence of the promoter TDH3 is described in 201210453416X; the nucleotide sequence of the terminator CYC1 is described in 201210453416X; the nucleotide sequence of the screening tag HIS3 is described in 201210453416X; the nucleotide sequence of the screening tag LEU2 is described in 201210453416X; they are the same as those in 201210453416X. The nucleotide sequence of the coding gene synPgDDS is the PgDDS in 201210453416X. It is the same as the PgDDS sequence in 201210453416X.

[0043] The present invention also protects the recombinant bacterium DM - BmCPR obtained above; the recombinant bacterium DBmCYP068 obtained above.

[0044] The present invention also provides the use of the above - mentioned recombinant bacterium DM - BmCPR or the above - mentioned recombinant bacterium DBmCYP068 in the production of dammarenediol or 23 - OH - Dammarenediol II and 25 - OH - Dammarenediol II.

[0045] The present invention also provides a method for producing dammarenediol or 23-OH-Dammarenediol II and 25-OH-Dammarenediol II, which is characterized in that the recombinant bacterium DM-BmCPR or the recombinant bacterium DBmCYP068 is used to obtain dammarenediol or 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

[0046] The names of the proteins and genes involved in the present invention are specifically as follows:

[0047] The synthesized synBmCPR1 is the encoding gene of NADPH-cytochrome P450 reductase 1 derived from Bacopa monnieri, and the protein encoded by it is NADPH-cytochrome P450 reductase;

[0048] The synthesized synPgDDS is the encoding gene of dammarenediol synthase derived from Panax ginseng, and the protein encoded by it is dammarenediol-Ⅱ synthase;

[0049] tHMG1 is the encoding gene of 3-hydroxy-3-methylglutaryl-CoA reductase 1 derived from a part of Saccharomyces cerevisiae, and the protein encoded by it is 3-hydroxy-3-methylglutaryl-CoA reductase 1 of Saccharomyces cerevisiae;

[0050] ERG1 is the encoding gene of squalene epoxidase of Saccharomyces cerevisiae, and the protein encoded by it is squalene epoxidase;

[0051] ERG9 is the encoding gene of squalene synthase of Saccharomyces cerevisiae, and the protein encoded by it is squalene synthase;

[0052] ERG20 is the encoding gene of farnesyl pyrophosphate synthase of Saccharomyces cerevisiae, and the protein encoded by it is farnesyl pyrophosphate synthase;

[0053] The synthesized SynBmCYP068 is the encoding gene of cytochrome P450 enzyme derived from Bacopa monnieri, and the protein encoded by it is cytochrome P450 enzyme;

[0054] The screening label of the recombinant bacterium BY-MVA is HIS3;

[0055] The screening tags of the recombinant bacterium DM-BmCPR are HIS3 and LEU2;

[0056] The screening tags of the recombinant bacterium DBmCYP068 are HIS3, LEU2 and Lys;

[0057] The recombinant bacterium DM-BmCPR obtained by the above method is also within the protection scope of the present invention;

[0058] The recombinant bacterium DBmCYP068 obtained by the above method is also within the protection scope of the present invention;

[0059] The media used for fermentation of the above recombinant bacteria BY-MVA, DM-BmCPR and DBmCYP068 are liquid media, and the media are all defective media, namely SC-His medium, SC-His-Leu medium and SC-His-Leu-Lys medium respectively. When fermenting, glucose with a final concentration of 2% (mass percentage) needs to be added to each. The formula of each component is shown in Table 1:

[0060] Table 1 Formula of fermentation medium

[0061]

[0062] The above fermentation conditions are 30 °C, 220 rpm / min, and shake culture for 5 days.

[0063] The δ and NTS2-1 sites are 1-10 random positions among multiple δ and NTS2-1 genes on the chromosome of Saccharomyces cerevisiae.

[0064] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0065] First, in the present invention, the upstream genes ERG1, ERG9, ERG20 and tHMG1 of the mevalonate pathway in Saccharomyces cerevisiae and synBmCPR1 and synPgDDS are imported into the recombinant bacterium BY-MVA together to obtain an intermediate recombinant bacterium DM-BmCPR that can produce dammarenediol; then, SynBmCYP068 is imported into DM-BmCPR to form a recombinant bacterium DBmCYP068 that can simultaneously produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II. The recombinant bacteria provided by the present invention lay a foundation for the artificial synthesis of dammarenediol synthase and 23-OH-Dammarenediol II and 25-OH-Dammarenediol II. Description of the drawings

[0066] Figure 1 It is a schematic diagram for constructing the gene expression cassette of the DM-BmCPR engineering bacterium;

[0067] Figure 2 Schematic diagram of the construction of the gene expression cassette of the engineered strain DBmCYP068;

[0068] Figure 3 Results of gel electrophoresis detection of PCR amplification of the fusion fragment of the gene expression cassette of the engineered strain DM-BmCPR;

[0069] Figure 4 Results of gel electrophoresis detection of PCR amplification of the fusion fragment of the gene expression cassette of the engineered strain DBmCYP068;

[0070] Figure 5 TLC thin-layer analysis of the fermentation product of the engineered strain DM-BmCPR of the present invention;

[0071] Figure 6 HPLC detection results of the fermentation product of the engineered strain DM-BmCPR of the present invention;

[0072] Figure 7 LC-MS detection results of the fermentation product of the engineered strain DM-BmCPR of the present invention;

[0073] Figure 8 HPLC quantitative detection results of the fermentation product of the engineered strain DM-BmCPR of the present invention;

[0074] Figure 9 TLC thin-layer analysis results of the fermentation product of the engineered strain DBmCYP068 of the present invention;

[0075] Figure 10 HPLC detection results of the fermentation product of the engineered strain DBmCYP068 of the present invention;

[0076] Figure 11 LC-MS detection results of the fermentation product of the engineered strain DBmCYP068 of the present invention;

[0077] Figure 12 HPLC quantitative detection results of the fermentation product of the engineered strain DBmCYP068 of the present invention. Detailed implementation manners

[0078] The present invention will be further described in detail below in conjunction with the embodiments.

[0079] Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For those materials or equipment whose manufacturers are not specified, they are all conventional products that can be obtained by purchase.

[0080] Example 1 Cloning of Gene Elements and Construction of Gene Expression Fragment Modules

[0081] The cloning of gene elements and the construction of gene expression fragment modules are divided into the following three steps:

[0082] (1) Yeast genomic DNA extraction

[0083] Pick a single colony of Saccharomyces cerevisiae BY4742 on YPD solid medium and place it in 10 mL of YPD liquid medium. Incubate at 30 °C and 220 rpm / min for 24 h. Then, centrifuge at 10000 g for 5 min to collect the cells in a 2 mL centrifuge tube and wash twice with ddH 2 O water. Extract the genomic DNA of the above-mentioned yeast BY4742 using a yeast genomic DNA extraction kit (DP307, Tiangen Biochemical Technology (Beijing) Co., Ltd.). The specific steps are carried out according to the instructions. Finally, obtain the yeast genomic DNA and store it at -20 °C for later use.

[0084] (2) Codon optimization of the sequences of PgDDS, BmCPR1, and BmCYP068

[0085] The genes encoding dammarenediol synthase synPgDDS, cytochrome P450 reductase synBmCPR1, and the encoding gene SynBmCYP068 that can produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in the present invention are all synthesized by Tsingke Biotechnology Co., Ltd. (Tianjin, China) after codon optimization for in vivo expression in yeast.

[0086] (3) Gene sequence amplification and construction of gene expression cassettes

[0087] The schematic diagram of the construction of the gene expression cassette of the DM-BmCPR engineering bacteria in the present invention is as shown in Figure 1 shown, and the schematic diagram of the construction of the gene expression cassette of the DBmCYP068 engineering bacteria is as shown in Figure 2 shown. The primers used for cloning gene elements and constructing gene expression modules are listed in Tables 2 and 3. The general procedure for constructing each yeast strain is described as follows:

[0088] 1) PCR amplify each promoter, codon-optimized gene, terminator, and selection tag using the primers with homologous arms in Table 2, and use High-Fidelity DNA Polymerase (NEB: M0491) for gene element cloning. The PCR reaction system is as follows: High-Fidelity DNA Polymerase 12.5 μL, cDNA 1 μL, primers F and R (10 pmol / L) 0.8 μL each, ddH 2 O 9.5 μL; The PCR reaction program is 95°C for 5 min; 95°C for 30 s, 60°C for 15 s, 72°C for 2 min, for 35 cycles; 72°C for 5 min.

[0089] Table 2 Primers for the fragments used to construct the yeast chassis of DM-BmCPR and DBmCYP068

[0090]

[0091]

[0092]

[0093] 2) After the PCR amplification is completed, agarose gel electrophoresis is performed for detection. After confirming successful amplification, the target band is recovered. The gene is recovered from the gel using the Universal DNA Purification and Recovery Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The recovered gene fragment is measured for its recovery concentration using a NanoReady ultra-micro ultraviolet-visible spectrophotometer and stored at -20°C in the refrigerator for later use.

[0094] 3) The single gene fragments obtained in the above steps, adjacent gene fragments all have a homologous sequence of about 60 bp. Using 2 - 4 adjacent single gene fragments as templates, fusion PCR is carried out (the genes and amplification primers contained in the fusion fragments are shown in Table 3), and each fusion fragment is obtained (as shown in Figure 3 and Figure 4 ). The fusion PCR reaction system is: High-Fidelity DNA Polymerase 12.5 μL, mixed template 1 μL, primers F and R (10 pmol / L) 0.8 μL each, ddH 2 O 9.5 μL; The PCR reaction program is 95°C for 5 min; 95°C for 30 s, 60°C for 15 s, 72°C for 5 min, for 35 cycles; 72°C for 5 min.

[0095] The fusion fragment is recovered from the gel using the Universal DNA Purification and Recovery Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The recovered fusion fragment is measured for its recovery concentration using a NanoReady ultra-micro ultraviolet-visible spectrophotometer and stored at -20°C in the refrigerator for later use.

[0096] Table 3 Genes and primers contained in the fusion fragments of the DM-BmCPR chassis and the DBmCYP068 chassis

[0097]

[0098]

[0099] Example 2 Construction of Genetic Engineering Bacterium DM-BmCPR of Saccharomyces cerevisiae

[0100] After the construction of the upstream gene fusion fragments (Fragment1 - Fragment5) of the mevalonate pathway, they were co-transformed into the BY-MVA recombinant bacterium (this bacterium already exists) by the lithium acetate transformation method (LiAc / ssDNA). Since each adjacent fragment shares approximately 60 bp of homologous sequence, they will be ligated together by yeast homologous recombination and integrated into the chromosomal δDNA locus. The specific steps are as follows:

[0101] (1) The starting bacterium, the BY-MVA recombinant bacterium, was cultured overnight in SC-HIS liquid medium until the OD 600 was 0.6 - 1.0. 1 mL was taken and dispensed into 2 1.5 mL EP tubes respectively. Centrifuge at 10000 rpm / min for 1 min, discard the supernatant, wash the cells with sterile water, centrifuge at 10000 rpm / min for 1 min, and discard the supernatant; repeat the washing of the cells with sterile water once.

[0102] (2) In the laminar flow hood, prepare the following transformation system: 240 μL of 50% PEG4000 (filtered and sterilized by W / V), 36 μL of 1.0 M LiAc (filtered and sterilized), 10 μL of SSDNA (2.0 mg / mL), 2 ng of each of the fusion fragments (Fragment1 - Fragment 5), and make up to 360 μL with sterile water. Resuspend the cells obtained in step (1) with the mixed transformation system, incubate at 30 °C for 40 min; heat shock at 42 °C for 40 min. Take 200 μL of the transformed bacterial solution and spread it on 2 SC-His-Leu solid screening media respectively; the screening culture conditions are: place it upside down in a 30 °C incubator for 2 - 3 days. Wait for the transformants to grow, select 8 monoclonal colonies from each plate, name them strains DM-BmCPR 1-1 - 1-8, DM-BmCPR 2-1 - 2-8, and culture them in 500 μL of SC-His-Leu liquid medium at 30 °C and 220 rpm / min for 1 day.

[0103] (3) Transfer the above DM-BmCPR 1-1 - 1-8 and DM-BmCPR 2-1 - 2-8 bacterial solutions into 50 mL of SC-His-Leu liquid medium respectively, and culture them at 30 °C and 220 rpm / min for 5 days.

[0104] (4) Collect the bacterial cells at 5000 rpm / min for 10 min, and discard the supernatant. Ultrasonically extract with 4 mL of methanol solution for 30 min, and use TLC thin-layer analysis to detect the production of the product. The developing agent is petroleum ether: ethyl acetate = 5:1 (v / v). The detection results are as Figure 5 shown.

[0105] (5) Detect the product of DM-BmCPR by HPLC. Filter the supernatant of the extracted product through a 0.22 μm organic microporous filter membrane for HPLC. The mobile phase is water (A) - acetonitrile (B), and gradient elution is used: 0 - 8 min, 85% - 90% B; 8 - 15 min, 90% - 92% B; 15 - 20 min, 92% - 94% B; 20 - 25 min, 94% - 97% B; 25 - 35 min, 94% - 100% B; 35 - 45 min, 100% B; the total amount of the A + B mobile phase is 100%; linear gradient elution is adopted; the injection volume: 20 μL; the flow rate: 0.8 mL / min; the detection wavelength is 194 nm, and the detector is a diode array detector. The HPLC detection results are as Figure 6 shown, indicating that the chassis bacteria of DM-BmCPR produced the target product, dihydroartemisinic alcohol (DM).

[0106] The method for obtaining the recombinant strain BY-MVA is specifically as follows:

[0107] Use the promoter IDP1 and terminator TDH2 to control the expression of the ERG1 gene introduced into BY4742; use the promoter TEF1 and terminator CYC1 to control the expression of the ERG9 gene introduced into BY4742; use the promoter CPS1 and terminator ENO2 to control the expression of the ERG20 gene introduced into BY4742; use the promoter PGK1 and terminator ADH1 to control the expression of the tHMG1 gene introduced into BY4742, construct a gene expression cassette, and co-transform the gene expression cassette and the HIS3 selection tag into the YPRCδ15 locus of Saccharomyces cerevisiae BY4742 by homologous recombination through the lithium acetate transformation method to obtain the recombinant strain BY-MVA.

[0108] Example 3 Production of Dihydroartemisinic Alcohol by Engineered Bacteria

[0109] 1. Shake-flask culture of engineered bacteria and product extraction

[0110] Activate the chassis engineering bacterium DM-BmCPR with high-yield dammarenediol (DM) on an SC-His-Leu solid screening culture plate, pick 8 single colonies and ferment the seed liquid in an SC-His-Leu liquid medium (30 °C, 200 rpm, 24 h); transfer it to a 250 mL Erlenmeyer flask containing 50 mL of SC-His-Leu liquid medium, and shake and culture at 30 °C, 200 rpm / min for 5 days to obtain the fermentation product.

[0111] 2. The method for extracting the product and the HPLC detection conditions are the same as those in Example 2.

[0112] 3. Quantitative analysis

[0113] Establish a standard curve using the DM standard product for measuring the yield of the DM-BmCPR chassis cells. Weigh 1.5 mg of the DM standard product and dissolve it in 1.5 mL of methanol to obtain a stock solution of 1 mg / mL. Gradually dilute the DM stock solution with methanol into standard solutions of 0.5 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.01 mg / mL, 0.005 mg / mL, and 0.001 mg / mL. Take 10 μL of each concentration of the standard solution for HPLC analysis, and perform three parallel detections for each concentration. Calculate the peak area of the standard solution under the detection of the ultraviolet wavelength of 194 nm. According to the linear relationship between the peak area and the sample content, obtain the standard curve equation of the DM concentration and the peak area. Use the standard curve for quantitative analysis to measure the content of dammarenediol. The standard curve regression equation: y = 850.924779*X + 1.500552, r 2 = 0.9998, and screen out the DM-BmCPR chassis bacterium with the highest yield.

[0114] 4. Results

[0115] (1) The HPLC detection results are as Figure 6 shown, and dammarenediol was produced in the DM-BmCPR chassis bacterium;

[0116] (2) The DM-BmCPR chassis bacterium is a recombinant bacterium obtained by introducing the dammarenediol synthase gene PgDDS of Panax ginseng and the gene encoding NADPH-cytochrome P450 reductase 1 into the BY-MVA recombinant bacterium, and overexpressing the related genes ERG20, ERG9, and tHMG1 of the mevalonate pathway; the LC-MS detection results of the extract of the DM-BmCPR chassis bacterium are as Figure 7As shown in the figure, where (a) is the extraction product of the BV-MVA recombinant strain; (b) is the extraction product of the DM-BmCPR chassis bacterium; (c) is dammarenediol; (d) is the mass spectrum of dammarenediol; (e) is the mass spectrum of dammarenediol in the extraction product of the DM-BmCPR chassis bacterium; it can be seen from the figure that the retention times of dammarenediol in the DM-BmCPR chassis bacterium sample and the dammarenediol standard product are 9.89 min and 9.84 min respectively, and they have the same mass spectrum, indicating that there is dammarenediol in the DM-BmCPR chassis extraction product;

[0117] (3) The content of dammarenediol was quantitatively analyzed by using a standard curve, and the highest content of dammarenediol in 8 DM-BmCPR chassis strains reached 109.33 mg / L (Table 4 and Figure 8 ).

[0118] Table 4 Yields of dammarenediol produced by different strains in shake flasks

[0119] Strain Yield (mg / L) DM-BmCPR-1 80.82±0.17 DM-BmCPR-2 90.53±0.34 DM-BmCPR-3 67.94±0.56 DM-BmCPR-4 105.67±0.57 DM-BmCPR-5 107.28±0.36 DM-BmCPR-6 103.91±0.47 DM-BmCPR-7 103.18±0.65 DM-BmCPR-8 109.33±0.36

[0120] Example 4 Construction of the Saccharomyces cerevisiae genetic engineering bacterium DBmCYP068

[0121] After the construction of the dammarenediol skeleton modification gene fusion fragment (Fragment 6 - Fragment 8) was completed, it was co-transformed into the DM-BmCPR yeast chassis engineering bacterium by the lithium acetate transformation method (Li Ac / ssDNA). Similarly, each adjacent fragment shared about 60 bp of homologous sequence, and they would be ligated together by yeast homologous recombination and integrated into the chromosomal NTS2-1 locus. The specific steps are as follows:

[0122] (1) The starting bacterium, the DM-BmCPR chassis bacterium, was cultured overnight in SC-His-Leu liquid medium until OD 600 0.6 - 1.0. 1 mL was taken and dispensed into 2 1.5 mL EP tubes respectively. Centrifuge at 10000 rpm / min for 1 min, discard the supernatant, wash the cells with sterile water, centrifuge at 10000 rpm / min for 1 min, and discard the supernatant; repeat the cell washing once.

[0123] (2) In the laminar flow hood, perform the following transformation system ratio: 240 μL of 50% PEG4000 (filtered and sterilized by filtration, W / V), 36 μL of 1.0 M LiAc (filtered and sterilized by filtration), 10 μL of SSDNA (2.0 mg / mL), 2 ng each of the fusion fragments (Fragment 6 - Fragment 8), and make up to 360 μL with sterile water. Resuspend the cells with the mixed transformation system, incubate at 30 °C for 40 min; heat shock at 42 °C for 40 min, and spread 200 μL of the transformed bacterial solution onto 2 SC-His-Leu-Lys solid screening media (the formula is shown in Table 1); the screening culture conditions are: place it upside down in a 30 °C incubator for 2 - 3 days. Wait for the transformants to grow, select 8 monoclonal colonies from each plate, name them strains DBmCYP068-1 to DBmCYP068-8, and culture them in 500 μL of SC-His-Leu-Lys liquid medium at 30 °C and 220 rpm / min for 1 day.

[0124] (3) Transfer the above DBmCYP068-1 to DBmCYP068-8 bacterial solutions into 50 mL of SC-His-Leu-Lys liquid medium respectively, and culture at 30 °C and 220 rpm / min for 5 days.

[0125] (4) Collect the cells at 5000 rpm / min for 10 min, discard the supernatant. Ultrasonically extract with 4 mL of methanol solution for 30 min, and detect the product formation by TLC thin layer analysis. The developing conditions are: petroleum ether: ethyl acetate = 1.5:1 (v / v), and the detection results are as Figure 9 shown.

[0126] (5) Detect the product of DBmCYP068 by HPLC. Filter the supernatant of the extracted product through a 0.22 μm organic microporous filter membrane for HPLC. The HPLC detection conditions are as follows: The HPLC detection instrument is an Agilent high performance liquid chromatograph, the chromatographic column is an Agilent EC-C18 chromatographic column (4.6×100 mm, 2.7 μm), column temperature: 25 °C; the mobile phase for determination is: water (A) - acetonitrile (B), gradient elution: 0 - 10 min, 70% - 75% B; 10 - 20 min, 75% - 85% B; 20 - 22 min, 85% - 100% B; 22 - 25 min, 100% B; the total of the mobile phase A + B used during elution is 100%; linear gradient elution is used; elution time: 25 min; injection volume: 10 μL; flow rate: 0.8 mL / min; detection wavelength 203 nm, and the detector is a diode array detector. The HPLC detection results are as Figure 10 shown.

[0127] Example 5

[0128] Production of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II by engineered bacteria:

[0129] 1. Shake flask culture of engineered bacteria and product extraction

[0130] Activate the chassis bacterium DBmCYP068 with high production of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II on the SC-His-Leu-Lys solid culture plate, pick 8 single colonies into the SC-His-Leu-Lys liquid medium, and ferment the seed liquid at 30 °C and 200 rpm / min for 24 h; transfer it to a 250 mL Erlenmeyer flask containing 50 mL of SC-His-Leu-Lys liquid medium, and shake culture at 30 °C and 200 rpm / min for 5 days to obtain the fermentation product.

[0131] 2. The method for extracting the product and the HPLC detection conditions are the same as in Example 4.

[0132] 3. Quantitative analysis

[0133] Weigh 1.5 mg of 23-OH-Dammarenediol II and 200-2 standard products and dissolve them in 1.5 mL of methanol to obtain a stock solution of 1 mg / mL. Gradually dilute the stock solutions of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II with methanol to standard solutions of 0.5 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.01 mg / mL. Take 10 μL of each concentration of the standard solution for HPLC analysis, and perform three parallel detections for each concentration. Calculate the peak area of the standard solution under the detection of the ultraviolet wavelength of 203 nm. According to the linear relationship between the peak area and the sample content, obtain the standard curve equations of the concentrations and peak areas of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II. Use the standard curve for quantitative analysis to measure the contents of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II. The standard curve regression equations are: Y 1 = 842.963253*X + 1.368344, r 2 = 0.9998; Y 2 = 256.904583*X + 0.37003351, r 2 = 0.9999, and screen out the chassis bacterium DBmCYP068 with the highest yield (X refers to the concentration of the standard solution, and Y refers to the content of the compound in the measured sample).

[0134] 4. Results

[0135] (1) The TLC thin-layer chromatography analysis and HPLC detection results are as Figure 9 and 10 shown. There is no 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in the DM-BmCPR chassis bacteria, while 23-OH-Dammarenediol II and 25-OH-Dammarenediol II are produced in the DBmCYP068 chassis bacteria;

[0136] (2) The DBmCYP068 chassis bacteria are recombinant bacteria obtained by introducing the gene encoding the dammarenediol side-chain modifying enzyme cytochrome P450 reductase gene BmCYP068 into the DM-BmCPR chassis bacteria; The LC-MS detection results of the extracts of the BmCYP068 chassis bacteria are as Figure 11 shown. Among them, (a) is the ultraviolet absorption diagram of the DM-BmCPR chassis bacteria, DBmCYP068 chassis bacteria, 23-OH-Dammarenediol and 25-OH-Dammarenediol II; (b) is the total ion chromatogram of the DM-BmCPR chassis bacteria, DBmCYP068 chassis bacteria, 23-OH-Dammarenediol II and 25-OH-Dammarenediol II; (c) is the mass spectrum diagram of the 23-OH-Dammarenediol standard; (d) is the mass spectrum diagram of the 25-OH-Dammarenediol II standard; (e) is the mass spectrum diagram of 23-OH-Dammarenediol in the extract of the DBmCYP068 chassis bacteria; (f) is the mass spectrum diagram of 25-OH-Dammarenediol II in the extract of the DBmCYP068 chassis bacteria; It can be seen from the figure that the retention times of 23-OH-Dammarenediol II in the extract of the DBmCYP068 chassis bacteria and the 23-OH-Dammarenediol II standard are 7.81 min and 7.79 min respectively, and the retention times of 25-OH-Dammarenediol II in the extract of the DBmCYP068 chassis bacteria and the 25-OH-Dammarenediol II standard are 3.98 min and 3.97 min respectively, indicating that there are 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in the extract of the BmCYP068 chassis;

[0137] (3) The contents of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II were quantitatively analyzed using a standard curve, and the highest contents of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II in 8 DBmCYP068 chassis strains reached 34.45 mg / L and 248.59 mg / L, respectively (Table 5 and Figure 12 ).

[0138] Table 5 Yields of 23-OH-Dammarenediol II and 25-OH-Dammarenediol II produced by different strains in shake flasks

[0139]

[0140] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II, characterized in that: The steps include: Introducing ERG1, ERG9, ERG20 and tHMG1, the dammarenediol synthase gene synPgDDS from ginseng, the NADPH-cytochrome P450 reductase gene synBmCPR1 from Bacopa monnieri, the leucine selection tag LEU2, and a yeast-derived promoter and terminator into the BY-MVA recombinant bacteria, obtaining the recombinant bacteria DM-BmCPR that can produce dammarenediol; The codon-optimized SynBmCYP068 gene expression cassette and Lys selection tag from Bacopa monnieri were then introduced into the DM-BmCPR strain to obtain the recombinant bacterium DBmCYP068 that can simultaneously produce 23-OH-DammarenediolII and 25-OH-DammarenediolII.

2. The method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II according to claim 1, characterized in that: The expression of the ERG1 gene introduced into BY4742 was controlled by promoter IDP1 and terminator TDH2; the expression of the ERG9 gene introduced into BY4742 was controlled by promoter TEF1 and terminator CYC1; the expression of the ERG20 gene introduced into BY4742 was controlled by promoter CPS1 and terminator ENO2; the expression of the tHMG 1 gene introduced into BY4742 was controlled by promoter PGK1 and terminator ADH 1, and a gene expression cassette was constructed. The gene expression cassette and the HIS3 screening tag were co-transformed into the YPRCδ15 site of Saccharomyces cerevisiae BY4742 through homologous recombination and lithium acetate transformation method to obtain the recombinant bacterium BY-MVA.

3. The method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II according to claim 1, characterized in that: The expression of the tHMG1 gene introduced into the BY-MVA recombinant bacteria was controlled by promoter HXT7 and terminator ADH1, the expression of the synBmCPR1 gene introduced into the BY-MVA recombinant bacteria was controlled by promoter TEF2 and terminator TDH2, the expression of the ERG1 gene introduced into the BY-MVA recombinant bacteria was controlled by promoter IPI 1 and terminator ENO2, the expression of the ERG20 gene introduced into the BY-MVA recombinant bacteria was controlled by promoter GPM and terminator CYC1, the expression of the ERG9 gene introduced into the BY-MVA recombinant bacteria was controlled by promoter PGK1 and terminator FBA1, and the expression of the synPgDDS gene introduced into the BY-MVA recombinant bacteria was controlled by promoter TDH3 and terminator PGT1 to construct a gene expression cassette, and the gene expression cassette and the LEU2 screening tag were co-transformed into the δDNA site of the BY-MVA recombinant bacteria through homologous recombination and lithium acetate transformation method to obtain the recombinant bacteria DM-BmCPR that can produce dammarenediol.

4. The method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II according to claim 1, characterized in that: The SynBmCYP068 gene expression cassette was constructed using the promoter TEF1, codon-optimized SynBmCYP068 from Bacopa monnieri and terminator PRM5. The SynBmCYP068 gene expression cassette and the Lys selection tag were co-transformed into the NTS2-1 site of the DM-BmCPR strain using the lithium acetate transformation method to form the recombinant bacterium DBmCYP068 that can simultaneously produce 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

5. The method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II according to any one of claims 1 to 4, characterized in that: The method for constructing the gene expression cassette comprises: performing PCR amplification on the promoter, terminator, functional gene and screening tag respectively using primers with homology arms, and then recovering the target band to obtain a single gene fragment; and then using adjacent 3 to 6 basic fragments as templates to perform fusion PCR to obtain a fusion fragment to construct a gene expression module.

6. The method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II according to claim 5, characterized in that: The gene expression module specifically includes: Fragment 1: contains genes UP, ADH1, tHMGR1, HXT7, and TEF2; Fragment 2: contains genes synBmCPR1, TDH2, ENO2, and ERG1; Fragment 3: contains genes IPI1, GPM, ERG20, CYC1, PGK1, and ERG9; Fragment 4: contains genes FBA1, TDH3, and synPgDDS; Fragment 5: contains genes PGT1, LEU2, and DN; Fragment 6: Contains genes NTS2-1-UP, TEF1, SynBmCYP068, and PRM5; Fragment 7: contains the gene Lys; Fragment 8: Contains the gene NTS2-1-DN.

7. The method for constructing a recombinant Saccharomyces cerevisiae capable of simultaneously producing 23-OH-Dammarenediol II and 25-OH-Dammarenediol II according to claim 6, characterized in that: The nucleotide sequence encoding the gene UP is shown in SEQ ID NO.1; The nucleotide sequence encoding gene DN is shown in SEQ ID NO.2; The nucleotide sequence encoding the gene synBmCPR1 is shown in SEQ ID NO.3; The nucleotide sequence encoding the gene NTS2-1-UP is shown in SEQ ID NO.4; The nucleotide sequence encoding the gene SynBmCYP068 is shown in SEQ ID NO.5; The nucleotide sequence encoding gene NTS2-1-DN is shown in SEQ ID NO.

6. The nucleotide sequence of promoter IDP1 is shown in SEQ ID NO.7; The nucleotide sequence of promoter CPS1 is shown in SEQ ID NO.8; The nucleotide sequence of promoter HXT7 is shown in SEQ ID NO.9; The nucleotide sequence of promoter TEF2 is shown in SEQ ID NO.10; The nucleotide sequence of promoter IPI1 is shown in SEQ ID NO.11; The nucleotide sequence of the promoter GPM is shown in SEQ ID NO.12; The nucleotide sequence of terminator TDH2 is shown in SEQ ID NO.13; The nucleotide sequence of terminator ENO2 is shown in SEQ ID NO.14; The nucleotide sequence of terminator ADH1 is shown in SEQ ID NO.15; The nucleotide sequence of terminator FBA 1 is shown in SEQ ID NO.16; The nucleotide sequence of terminator PGT1 is shown in SEQ ID NO.17; The nucleotide sequence of terminator PRM5 is shown in SEQ ID NO.18; The nucleotide sequence of the screening tag Lys is shown in SEQ ID NO.

19.

8. The recombinant bacterium DM-BmCPR is obtained by the method of claim 3; or the recombinant bacterium DBmCYP068 is obtained by the method of claim 3.

9. Use of the recombinant bacterium DM-BmCPR or the recombinant bacterium DBmCYP068 according to claim 8 in the production of dammarene diol or 23-OH-Dammarenediol II and 25-OH-Dammarenediol II.

10. A method for producing dammarene diol or 23-OH-Dammarenediol II and 25-OH-Dammarenediol II, characterized in that: The recombinant bacterium DM-BmCPR or the recombinant bacterium DBmCYP068 according to claim 8 is used to obtain dammarene diol or 23-OH-DammarenediolII and 25-OH-DammarenediolII.

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