Recombinant saccharomyces cerevisiae engineering bacterium for producing dammarendiol and production method of recombinant saccharomyces cerevisiae engineering bacterium
By constructing recombinant Saccharomyces cerevisiae engineering bacteria, the efficient biosynthesis of damanediol was achieved, and the problems of difficulty and cost in the existing technology were solved, providing a foundation for the industrial production of original ginseng diol.
Patent Information
- Application Number
- CN202510695278.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-08-29
AI Technical Summary
The prior art is difficult to efficiently synthesize damatendiol, which leads to the high cost and difficulty of artificial synthesis of original ginseng diol, and is susceptible to pests and diseases during ginseng planting.
By constructing a recombinant Saccharomyces cerevisiae engineered bacteria carrying gene expression cassettes containing hydroxymethylglutaryl CoA reductase and isoprene synthetase encoding genes, the biosynthesis of damerene glycol is achieved, and Saccharomyces cerevisiae is used to efficiently express damerene glycol synthetase.
The efficient biosynthesis of damanediol was achieved, which reduced production costs, laid the foundation for the large-scale industrial production of original ginseng diol, and provided a biosynthesis path for ginseng saponin.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of synthetic biology and genetic engineering, and particularly relates to a recombinant saccharomyces cerevisiae engineered bacterium for producing dammarene diol and a production method. Background Art
[0002] Ginseng is a renowned and precious medicinal herb, both in China and abroad. Its use as a remedy for illnesses is documented as early as the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica). Currently, over 60 triterpenoid saponins have been discovered in ginseng roots. These saponins, derived from ginseng, are known as ginsenosides. With the rapid development of society, economic and cultural advancements have also brought about technological advances. Researchers have delved deeper into the various saponins in ginseng, and numerous experiments have confirmed their diverse therapeutic benefits, including anti-aging, immune system regulation, and central nervous system regulation.
[0003] Protopanaxadiol is the primary active ingredient in the traditional and precious medicinal herbs Panax ginseng and American ginseng. It possesses anti-inflammatory, antioxidant, and broad anti-tumor properties. However, ginseng, the primary source of protopanaxadiol, takes 4-12 years or even longer to grow from cultivation to its current state of efficacy. During this growth process, it is highly susceptible to pests and diseases such as red bark disease and root rot. These factors make it extremely difficult to grow and maintain ginseng at the source.
[0004] Therefore, the artificial synthesis of protopanaxadiol has long been an area of research and interest in biology, chemistry, and medicine. Dammarenediol is a precursor to protopanaxadiol, from which protopanaxadiol can be derived. However, due to the complex chemical structure of these compounds, including multiple chiral structures within their backbones, chemical synthesis is relatively complex. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to address the shortcomings of the above-mentioned prior art and provide a recombinant Saccharomyces cerevisiae engineered strain for producing dammarenediol and a production method. Through synthetic biology and genetic engineering techniques, the present invention first constructs a Saccharomyces cerevisiae strain carrying a gene expression cassette encoding a hydroxymethylglutaryl-CoA reductase gene and an isoprene synthase gene. Based on this, a recombinant Saccharomyces cerevisiae engineered strain carrying a gene expression cassette containing a dammarenediol synthase gene is constructed. This recombinant strain efficiently expresses the dammarenediol synthase enzyme, achieving the biosynthesis of dammarenediol and, in turn, synthesizing dammarenediol, a precursor of the ginsenoside propanaxadiol.
[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a recombinant Saccharomyces cerevisiae engineered bacterium, characterized in that the recombinant Saccharomyces cerevisiae engineered bacterium is a Saccharomyces cerevisiae carrying a gene expression cassette containing a hydroxymethylglutaryl-CoA reductase encoding gene and an isoprene synthase encoding gene.
[0007] The above-mentioned recombinant Saccharomyces cerevisiae engineered strain is characterized in that the gene expression cassette containing the hydroxymethylglutaryl-CoA reductase encoding gene and the isoprene synthase encoding gene has a nucleotide sequence shown in SEQ ID NO: 1.
[0008] The above-mentioned recombinant Saccharomyces cerevisiae engineered bacteria is characterized in that the gene expression cassette is LPP1-TDH3-tHMG1-TDH2-PGK1-ERG20-CYC1-LEU2-LPP2.
[0009] The above-mentioned recombinant Saccharomyces cerevisiae engineered bacteria is characterized in that the Saccharomyces cerevisiae engineered bacteria is Saccharomyces cerevisiae INVSC1-TE.
[0010] Furthermore, the present invention provides a recombinant Saccharomyces cerevisiae engineered bacterium for producing dammarenediol constructed using the above-mentioned recombinant Saccharomyces cerevisiae engineered bacterium, characterized in that the recombinant Saccharomyces cerevisiae engineered bacterium for producing dammarenediol carries a gene expression cassette containing a dammarenediol synthase gene.
[0011] The above-mentioned recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarenediol is characterized in that the gene expression cassette containing the dammarenediol synthase gene has a nucleotide sequence shown in SEQ ID NO: 2.
[0012] The above-mentioned recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarenediol is characterized in that the gene expression cassette is DPP1-TEF1-DS-CYC1-URA3-DPP1.
[0013] The above-mentioned recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarene diol is characterized in that the recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarene diol is the Saccharomyces cerevisiae engineered strain INVSC1-TE-D.
[0014] Furthermore, the present invention provides a method for synthesizing dammarene diol using the above-mentioned recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarene diol, characterized in that it comprises the following steps:
[0015] Step 1: fermenting and culturing a recombinant Saccharomyces cerevisiae engineered strain for producing dammarenediol using YPD fermentation medium;
[0016] Step 2: Collect the fermentation broth to obtain dammarenediol.
[0017] The above method is characterized in that the specific method for obtaining dammarene diol in step 2 includes:
[0018] Step 201: centrifuge the collected fermentation broth, discard the supernatant, and then add sterile water and mix to obtain a suspension;
[0019] Step 202: Transfer the suspension in step 201 to a crushing tube, centrifuge and discard the supernatant, then add zirconium oxide beads and n-hexane solution to the crushing tube and pipette to mix evenly;
[0020] Step 203: Seal the opening of the crushed tube in step 202 with a sealing film, place the tube in a grinder for grinding, evaporate the n-hexane in the crushed tube to dryness, and then add methanol for redissolution to obtain dammarene diol.
[0021] Compared with the prior art, the present invention has the following advantages:
[0022] 1. Dammarenediol is synthesized from 2,3-squalene oxide under the catalysis of dammarenediol synthase. The present invention uses Saccharomyces cerevisiae as a heterologous expression host for dammarenediol synthesis. Saccharomyces cerevisiae is a highly safe model organism and possesses 2,3-squalene oxide synthase, a precursor for dammarenediol synthesis.
[0023] 2. The present invention uses synthetic biology and genetic engineering techniques to first construct a Saccharomyces cerevisiae strain carrying a gene expression cassette containing a gene encoding hydroxymethylglutaryl-CoA reductase and an isoprene synthase. On this basis, a recombinant Saccharomyces cerevisiae strain carrying a gene expression cassette containing a dammarenediol synthase gene is constructed. This strain can efficiently recombinantly express dammarenediol synthase, thereby achieving the biosynthesis of dammarenediol and further synthesizing dammarenediol, a precursor of the ginsenoside propanaxadiol.
[0024] 3. The recombinant Saccharomyces cerevisiae engineered bacteria of the present invention can heterologously produce dammarene diol, reducing production costs, laying the foundation for the large-scale industrial production of protopanaxadiol, and also laying the foundation for the ultimate realization of ginsenoside biosynthesis and large-scale industrial production.
[0025] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a diagram of the LPP1-TDH3-tHMG1-TDH2-PGK1-ERG20-CYC1-LEU2-LPP2 gene expression module.
[0027] Figure 2 This is a diagram of the DPP1-TEF1-DS-CYC1-URA3-DPP1 gene expression module.
[0028] Figure 3 PCR verification of the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE.
[0029] Figure 4 PCR verification of the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE-D.
[0030] Figure 5 This is a liquid chromatogram of dammarene diol produced in Example 4 of the present invention. DETAILED DESCRIPTION
[0031] The methods of the present invention are further described below with reference to examples. However, the examples are for illustrative purposes only and are not intended to be limiting. Experimental methods in the following examples where specific conditions are not specified can generally be performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual compiled by Sambrook et al., or under the conditions recommended by the manufacturer. Those skilled in the art will be able to better understand and grasp the present invention with the help of the examples. However, the scope of protection and claims of the present invention is not limited to the examples provided.
[0032] The Saccharomyces cerevisiae INVSC1 used in the following examples was purchased from Beina Biotechnology.
[0033] Example 1 Construction of Gene Expression Cassettes Encoding Hydroxymethylglutaryl-CoA Reductase and Isoprene Synthase
[0034] Snapgene software was used to construct a gene expression cassette LPP1-TDH3-tHMG1-TDH2-PGK1-ERG20-CYC1-LEU2-LPP2 containing the tHMG1 gene, ERG20 gene, LPP1 upstream and downstream homology arms, selection marker LEU2 gene, TDH3 promoter, CYC1 terminator, PGK1 promoter, and TDH2 terminator;
[0035] The gene sequence of the expression cassette is SEQ ID NO: 1, which was synthesized by Xi'an Qingke Biotechnology Co., Ltd. and connected to the PRS304 vector.
[0036] The LPP1-TDH3-tHMG1-TDH2-PGK1-ERG20-CYC1-LEU2-LPP2 gene expression cassette map is as follows Figure 1 shown.
[0037] SEQ ID NO: 1:
[0038]
[0039] Example 2 Construction of Recombinant Overexpressing Engineering Bacteria for Hydroxymethylglutaryl Coenzyme A Reductase and Isoprene Synthase
[0040] Use primer F: 5'-TAATGGAATCAACCAACCCTT-3',
[0041] R:5'-TCGGTGGAAAAACGCCACAG-3' was amplified by PCR.
[0042] Amplification conditions were 98°C, 10 min; 98°C, 30 s, 58°C, 30 s, 30 cycles; 72°C, 4 min; 72°C, 10 min.
[0043] The PCR products were purified and recovered using PCR purification reagents and stored at -20°C for future use.
[0044] Prepare Saccharomyces cerevisiae INVSC1 competent cells, add 240 μL of 50% polyethylene glycol 3350 solution, 36 μL of 1 mol / L lithium acetate solution, 25 μL of single-stranded DNA solution, and 50 μL of LPP1-TDH3-tHMG1-TDH2-PGK1-ERG20-CYC1-LEU2-LPP2 gene expression cassette fragment obtained through the purification process in sequence, mix thoroughly, and culture at 30°C for 30 minutes. Heat shock at 42°C for 25 minutes and then cool immediately. Then, add 200 μL of sterile water to suspend the cells, spread on SC-LEU plates to screen positive transformants, select transformants to extract yeast genome, and then perform PCR to verify the target gene tHMG1-ERD20 fragment (see Figure 3 ), and the successfully transformed strain was named the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE.
[0045] Example 3 Construction of Dammarenediol Synthase Recombinant Expression Engineering Bacteria
[0046] The DPP1-TEF1-DS-CYC1-URA3-DPP1 gene expression cassette containing DPP1 upstream and downstream homology arms, the screening marker URA3 gene, the TEF1 promoter, and the CYC1 terminator was constructed using snapgene software. The gene sequence of the expression cassette is SEQ ID NO: 2 and was synthesized by Xi'an Qingke Biotechnology Co., Ltd. and ligated into the PRS304 vector. The gene expression cassette map is shown in Figure 2. Figure 2 shown.
[0047] SEQ ID NO: 2:
[0048]
[0049] Use primer F: 5'-TATATATAGATAGAAACCCAAC-3',
[0050] R:5'-TCTAGGCTGTTTATAGATTGA-3' was amplified by PCR.
[0051] Amplification conditions were 98°C, 10 min; 98°C, 30 s, 58°C, 30 s, 30 cycles; 72°C, 4 min; 72°C, 10 min.
[0052] The PCR products were purified and recovered using PCR purification reagents and stored at -20°C for future use.
[0053] Prepare Saccharomyces cerevisiae INVSC1-TE competent cells, add 240 μL of 50% polyethylene glycol 3350 solution, 36 μL of 1 mol / L lithium acetate solution, 25 μL of single-stranded DNA solution, and 50 μL of DPP1-TEF1-DS-CYC1-URA3-DPP1 gene expression cassette fragment obtained through the purification process in sequence, mix thoroughly, and incubate at 30°C for 30 minutes, heat shock at 42°C for 25 minutes, and then cool immediately. Then, add 200 μL of sterile water to suspend the cells, spread on SC-URA plates to screen positive transformants, select transformants to extract yeast genome, and then perform PCR to verify the target gene DS fragment (see Figure 4 ), and the successfully transformed strain was named the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE-D.
[0054] Example 4 Fermentation and production of dammarene diol
[0055] Step 1: Ferment the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE-D in YPD fermentation medium at 30°C and 200 rpm;
[0056] Step 2: collecting the fermentation broth to obtain dammarene diol; specifically comprising:
[0057] Step 201: 40 mL of the collected fermentation broth was placed in a centrifuge tube, centrifuged at 6000 rpm for 5 min, and the supernatant was discarded. 1 mL of sterile water was then added to the centrifuge tube and mixed to obtain a suspension.
[0058] Step 202: Transfer the suspension in step 201 to a 2 mL crushed tube, centrifuge and discard the supernatant, then add 1 g of 0.5 mm zirconium oxide beads to the crushed tube, then add 1 mL of n-hexane solution and pipette to mix evenly;
[0059] Step 203: Seal the opening of the crushed tube in step 202 with a sealing film, place the tube in a grinder and grind for 90 minutes, then evaporate the n-hexane in the crushed tube to dryness, and then add 1 mL of methanol to redissolve it to obtain dammarene diol.
[0060] The prepared dammarene diol was subjected to liquid chromatography detection. The liquid chromatography detection method used a Shimadzu C18 column, the mobile phase was acetonitrile: water, the ratio was 95:5, the column temperature was 30℃, the flow rate was set to 1mL / min, and the injection volume was selected to be 20μL. The results are as follows Figure 5 As shown in the figure, the HPLC chromatogram corresponds to that of a dammarenediol standard sample, indicating that the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE-D constructed by the present invention can synthesize the natural product dammarenediol in vivo. The final results show that the recombinant Saccharomyces cerevisiae engineered strain INVSC1-TE-D constructed by the present invention can efficiently synthesize dammarenediol in vivo by co-expressing hydroxymethylglutaryl-CoA reductase, isoprene synthase, and dammarenediol synthase.
[0061] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the invention shall still fall within the scope of protection of the technical solution of the present invention.
Claims
1. A recombinant Saccharomyces cerevisiae engineered bacterium, characterized in that: The recombinant Saccharomyces cerevisiae engineered bacteria is a Saccharomyces cerevisiae carrying a gene expression cassette containing a hydroxymethylglutaryl-CoA reductase encoding gene and an isoprene synthase encoding gene.
2. The recombinant Saccharomyces cerevisiae engineered bacterium according to claim 1, characterized in that: The gene expression cassette containing the hydroxymethylglutaryl-CoA reductase encoding gene and the isoprene synthase encoding gene has a nucleotide sequence shown in SEQ ID NO:
1.
3. The recombinant Saccharomyces cerevisiae engineered bacterium according to claim 1, characterized in that: The gene expression cassette is LPP1-TDH3-tHMG1-TDH2-PGK1-ERG20-CYC1-LEU2-LPP2.
4. The recombinant Saccharomyces cerevisiae engineered bacterium according to claim 1, characterized in that: The engineered yeast Saccharomyces cerevisiae is Saccharomyces cerevisiae INVSC1-TE.
5. A recombinant Saccharomyces cerevisiae engineered bacterium for producing dammarenediol constructed using the recombinant Saccharomyces cerevisiae engineered bacterium according to claim 1, 2, 3 or 4, characterized in that: The recombinant saccharomyces cerevisiae engineered bacteria used for producing dammarenediol carries a gene expression cassette containing a dammarenediol synthase gene.
6. The recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarenediol according to claim 5, characterized in that: The gene expression cassette containing the dammarenediol synthase gene has a nucleotide sequence shown in SEQ ID NO:
2.
7. The recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarenediol according to claim 5, characterized in that: The gene expression cassette is DPP1-TEF1-DS-CYC1-URA3-DPP1.
8. The recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarenediol according to claim 5, characterized in that: The recombinant Saccharomyces cerevisiae engineered bacteria used for producing dammarenediol is the Saccharomyces cerevisiae engineered strain INVSC1-TE-D.
9. A method for synthesizing dammarene diol using the recombinant Saccharomyces cerevisiae engineered bacteria for producing dammarene diol according to claim 5, characterized in that: The following steps are involved: Step 1: fermenting and culturing a recombinant Saccharomyces cerevisiae engineered strain for producing dammarenediol using YPD fermentation medium; Step 2: Collect the fermentation broth to obtain dammarenediol.
10. The method according to claim 9, characterized in that The specific method for obtaining dammarene diol in step 2 includes: Step 201: centrifuge the collected fermentation broth, discard the supernatant, and then add sterile water and mix to obtain a suspension; Step 202: Transfer the suspension in step 201 to a crushing tube, centrifuge and discard the supernatant, then add zirconium oxide beads and n-hexane solution to the crushing tube and pipette to mix evenly; Step 203: Seal the opening of the crushed tube in step 202 with a sealing film, place the tube in a grinder for grinding, evaporate the n-hexane in the crushed tube to dryness, and then add methanol for redissolution to obtain dammarene diol.