Beta-elemene synthase and application thereof in preparation of beta-elemene
By expressing and fusing the β-elemonene synthase gene in Saccharomyces cerevisiae, the problem of low production efficiency of β-elemonene in the prior art is solved, and efficient and economical β-elemonene biosynthesis is achieved.
Patent Information
- Application Number
- CN202510093883.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art is difficult to efficiently produce the anti-tumor drug β-elene through biological factories, and traditional extraction methods are limited and chemical synthesis is complex.
Design a β-elene synthase and transfer it into Saccharomyces cerevisiae to improve the biosynthesis efficiency of β-elene by expressing and fusing specific genes such as ERG20 and β-elene synthase genes.
High yield of β-elene production was achieved, the shake flask output reached 748.1 mg/L, and the titer in the fermenter reached 14.60 g/L, which significantly improved the biosynthesis efficiency of β-elene.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of genetic engineering, and particularly relates to a β-elemene synthase and its application in the preparation of β-elemene. Background Art
[0002] β-elemene is an oxygen-free sesquiterpene olefin, and is an oily monomer isolated from the volatile oil of the medicinal plant Curcuma wenyujin; its chemical name is 1-methyl-1-vinyl-2,4-diisopropyl cyclohexane, and the structural formula is as follows Figures 1-7 shown. As an anti-tumor drug, the main active ingredient of elemene terpenoids is β-elemene; in addition, elemene, together with paclitaxel, vinblastine, harringtonine, etc., is listed as an important plant-derived anti-tumor drug.
[0003] Regarding the good pharmacological activity of β-elemene in anti-tumor, scholars have conducted certain research on its mechanism of action and found that its mechanism of action is mainly to inhibit the growth of tumor cells, induce apoptosis of tumor cells, enhance immunity, relieve cancer pain, and improve microcirculation, etc.; it is mainly used in the clinical treatment of intracranial tumors, lung cancer, liver cancer, gastric cancer, ovarian cancer, rectal cancer, etc. In addition, relevant research has also found that β-elemene has radiosensitization and analgesic effects and can be applied to radiotherapy and chemotherapy as well as cardiovascular diseases. This further illustrates that β-elemene, as a highly effective anti-cancer drug derived from plants, has good application value and market prospects.
[0004] Synthetic biology is an important means to solve the sustainable utilization of the effective components of traditional Chinese medicine. The representative substance of the sesquiterpene compounds, which are the effective components in Artemisia argyi, is β-elemene, which has significant anti-cancer activity and is widely used in the fields of clinical medicine, cosmetics and food. However, due to the limited extraction of such resource-based components from plants and the complexity of chemical synthesis, it is not advisable to obtain sesquiterpenes on a large scale by traditional methods. Using microbial cell factories to produce sesquiterpene compounds is a very promising solution. Summary of the Invention
[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a β-elemene synthase.
[0006] Another purpose of the present invention is to provide the application of the above β-elemene synthase in the preparation of β-elemene.
[0007] Another purpose of the present invention is to provide an engineered bacterium for producing β-elemene.
[0008] The purpose of the present invention is achieved by the following technical solutions:
[0009] A β-elemene synthase is at least one of the following:
[0010] (a) A protein composed of an amino acid sequence shown in at least one of SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5;
[0011] (b) A sequence having a sequence homology of more than 90% with the amino acid sequence shown in at least one of SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5 and having the same protein function.
[0012] The above-mentioned β-elemene synthase can be expressed in an engineered bacterium and produce β-elemene using farnesyl pyrophosphate as a substrate.
[0013] The engineered bacterium is an engineered bacterium with high expression of farnesyl pyrophosphate (FPP); preferably, it is the engineered bacterium EL-Y0.
[0014] The above-mentioned β-elemene synthase, the nucleotide sequence of its encoding gene is obtained according to the codon encoding rule.
[0015] The above-mentioned β-elemene synthase, its encoding gene is a nucleotide sequence shown in at least one of SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6.
[0016] The application of the above-mentioned β-elemene synthase in the production of β-elemene.
[0017] A β-elemene synthase gene, which can produce β-elemene when transferred into Saccharomyces cerevisiae, and its amino acid sequence is at least one of SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6.
[0018] The application of the above-mentioned β-elemene synthase gene in the production of β-elemene.
[0019] A Saccharomyces cerevisiae engineered bacterium with high production of β-elemene has the following characteristics:
[0020] It is obtained by using Saccharomyces cerevisiae EL-Y0 as the starting strain, transferring the tHMGR and UPC2-1 genes, knocking out the NDT80 gene and overexpressing the HIF-1α and ARNT genes, and then transferring the ERG20 gene and the β-elemene synthase gene.
[0021] The transfer of the tHMGR and UPC2-1 genes is to integrate the tHMGR and UPC2-1 genes into the TY4 locus of the Saccharomyces cerevisiae chromosome.
[0022] The transfer of the ERG20 gene and the β-elemene synthase gene is to use (GGGS) 2After fusing ERG20 and β - elemene synthase gene with a linker peptide to obtain a fusion fragment, the fusion fragment was integrated into the TY1 locus of the Saccharomyces cerevisiae chromosome.
[0023] The knockout of the NDT80 gene and overexpression of the HIF - 1α and ARNT genes refer to knocking out the NDT80 gene in the Saccharomyces cerevisiae genome and integrating the HIF - 1α and ARNT genes into the original position of the NDT80 gene.
[0024] The engineered strain EL - Y0 was obtained by using Saccharomyces cerevisiae CEN.PK2 - 1C as the starting strain and introducing TEF1p - ERG1 - PGK1t, ADH1p - ERG13 - ADH1t, ADH1p - ERG12 - ADH1t, TEF2p - ERG19 - CYC1t, PGK1p - IDI1 - PGK1t, TDH3p - AtCPR1 - TDH3t, TEF1p - ERG10 - PGK1t, TDH3p - ERG8 - TDH3t, PGK1p - tHMGR - ADH1t, TDH3p - ERG20+ERG9 - CYC1t at the His3 locus, and introducing TDH3p - ERG20+ERG9 - CYC1t, TEF1t - ERG10 - PGK1t, TDH3p - ERG8 - TDH3t, PGK1p - tHMGR - ADH1t, TEF1p - ERG1 - PGK1t at the Trp locus. The preparation method has been disclosed in Wu S, Zhang F, Xiong W, et al. An Unexpected Oxidosqualene Cyclase Active Site Architecture in the Iris tectorum Multifunctional α - Amyrin Synthase. DOI: 10.1021 / acscatal.0c03231.
[0025] A method for constructing an engineered Saccharomyces cerevisiae strain with high - yield β - elemene, comprising the following steps:
[0026] (1) Using the Saccharomyces cerevisiae EL - Y0 as the starting strain, connecting the GAL1p promoter, ERG20 gene, (GGGS) 2 linker peptide, β - elemene synthase gene and CYC1t terminator in sequence to construct a fusion fragment, integrating it into the TY1 locus of the bacterial genome, and screening to obtain the strain EL - A1;
[0027] (2) The PGK1p promoter, HIF-1α gene, CYC1t terminator, TEF1p promoter, ARNT gene, and ADH1t were ligated in sequence to construct the PGK1p-HIF-1α-CYC1t-TEF1p-ARNT-ADH1t fragment. The NDT80 gene in the genome of the knockout strain EL-A1 was knocked out, and the fragment was integrated into the original position of the NDT80 gene. After screening, the strain EL-A2 was obtained;
[0028] (3) The GAL10p promoter, tHMGR gene, ADH1t terminator, GAL1p promoter, UPC2-1 gene, and CYC1t terminator were ligated in sequence to construct the GAL10p-tHMGR-ADH1t-GAL1p-UPC2-1-CYC1t fragment, which was integrated into the TY4 locus of the strain EL-A2. After screening, the engineered Saccharomyces cerevisiae strain with high-yield β-elemene was obtained.
[0029] The nucleotide sequence of the (GGGS) 2 linker peptide is as shown in SEQ ID NO.8.
[0030] The gene of the β-elemene synthase is a gene capable of expressing and producing β-elemene, preferably the gene corresponding to the nucleotide sequence shown in at least one of SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6.
[0031] The Saccharomyces cerevisiae is the engineered strain EL-Y0.
[0032] The integration is to transform the fragment into the cell using the lithium acetate transformation method. The fragment is transferred together with the targeting fragment containing the homologous fragment and the selection marker, and integrated with the target site through homologous recombination.
[0033] The nucleotide sequence of the GAL80 gene is as shown in the NCBI database sequence accession number 854954.
[0034] The nucleotide sequence of the tHMGR gene is as shown in the NCBI database sequence accession number MW753215.1.
[0035] The nucleotide sequence of the ERG20 gene is as shown in the NCBI database sequence accession number NC 001142.9.
[0036] The nucleotide sequence of the UPC2-1 gene is as shown in the NCBI database sequence accession number NM_001180521.1.
[0037] The nucleotide sequence of the HIF-1α gene is as shown in the NCBI database sequence accession number NM_001243084.2.
[0038] The nucleotide sequence of the ARNT gene is as shown in the NCBI database sequence accession number NM_001668.4.
[0039] The nucleotide sequence of the NDT80 gene is as shown in the NCBI database sequence accession number 856524.
[0040] A method for producing β - elemene, comprising the following steps:
[0041] (1) Transfer the β - elemene synthase gene into an engineered bacterium, inoculate it into a culture medium for cultivation, and add an extraction solution during the cultivation.
[0042] (2) Cultivate and separate to obtain the extraction solution, and further purify it to obtain β - elemene.
[0043] The engineered bacterium described in step (1) is at least one of the strains EL - A1, EL - A2, and the Saccharomyces cerevisiae engineered bacterium with high - yield β - elemene.
[0044] The strain EL - A1 uses Saccharomyces cerevisiae EL - Y0 as the starting strain, and after fusing ERG20 with the β - elemene synthase gene using (GGGS) 2 as a linker peptide to obtain a fusion fragment, it is integrated into the TY1 locus of the Saccharomyces cerevisiae chromosome.
[0045] The engineered bacterium EL - A2 uses the strain EL - A1 as the starting strain, knocks out the NDT80 gene in the genome of the strain, and integrates the HIF - 1α and ARNT genes into the original position of the NDT80 gene.
[0046] The Saccharomyces cerevisiae engineered bacterium with high - yield β - elemene uses the strain EL - A1 as the starting strain, and integrates the tHMGR and UPC2 - 1 genes into the TY4 locus of the Saccharomyces cerevisiae chromosome.
[0047] The extraction solution described in step (1) is dodecane, and the addition time is 12 h after the start of fermentation, and the addition amount is 10% of the total volume of the fermentation broth.
[0048] The cultivation described in step (1) is carried out in a defective SD liquid medium at 28 - 32 °C for 1 - 7 days.
[0049] The cultivation described in step (1) is induction cultivation, and when the initial carbon source is exhausted, glucose and galactose with a mass ratio of 1:1 are added for feeding.
[0050] The present invention has the following advantages and effects compared with the prior art:
[0051] Based on the EL-Y0 chassis bacterium with high-yield precursor FPP, through the flexible linker (GGGS) 2 The FPP synthase ERG20 and the β-elemene synthase AarTPS72 were fused and expressed, and the genes HIF-1, UPC2-1, and tHMGR were overexpressed to increase the metabolic flux of the MVA pathway, thereby improving the biosynthesis of β-elemene; the engineered bacterium EL16 producing β-elemene was successfully obtained; finally, the shake-flask production of β-elemene was 748.1 mg / L. To further evaluate the performance of this engineered bacterium in fermenter fermentation, fed-batch fermentation of the engineered bacterium EL16 was carried out in a 5 L bioreactor, and finally the titer of β-elemene was 14.60 g / L. Description of the Drawings
[0052] Figure 1 It is the GC-MS detection result diagram of the expression products of AarTPS72, AarTPS07, and AarTPS60.
[0053] Figure 2 It is the quantitative analysis result diagram of the expression products of AarTPS72, AarTPS07, and AarTPS60.
[0054] Figure 3 It is the expression vector map and plasmid extraction electrophoresis result diagram of the fusion plasmid.
[0055] Figure 4 It is the schematic diagram of the gene construction method in Example 2.
[0056] Figure 5 It is the detection result diagram of the fermentation products of strain EL12.
[0057] Figure 6 It is the schematic diagram of the gene construction method in Example 3.
[0058] Figure 7 It is the detection result diagram of the fermentation products of strain EL14.
[0059] Figure 8 It is the schematic diagram of the gene construction method in Example 4.
[0060] Figure 9 It is the detection result diagram of the fermentation products of strain EL16.
[0061] Figure 10 It is the experimental result diagram of fed-batch fermentation of β-elemene of the engineered bacterium in Example 5. Detailed Embodiments
[0062] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.
[0063] In the following embodiments, if the specific test conditions are not specified, they are generally in accordance with conventional test conditions or the test conditions recommended by the reagent company. Materials, reagents, etc. used, unless otherwise specified, are all reagents and materials obtained through commercial channels.
[0064] Example 1 Screening, Vector Construction and Expression of Artemisia argyi TPSs
[0065] Based on the previous research in our laboratory, several potential terpene synthase (TPS) candidate genes were isolated from the root, stem and leaf tissues of Artemisia argyi, a genus of Compositae, which may have the ability to synthesize β - elemene. They are AarTPS72, AarTPS07 and AarTPS60 respectively. To verify their functions, these genes were integrated into plasmids and transferred into Saccharomyces cerevisiae for expression in this experiment. The specific steps are as follows:
[0066] 1.1 Vector Construction
[0067] In this experiment, using Artemisia argyi cDNA as a template, the candidate genes of Artemisia argyi AarTPSs (AarTPS72, AarTPS07 and AarTPS60) were amplified. The reaction system, primer sequences and procedures are shown in Tables 1 - 3 below. After the amplified target fragments were purified and recovered, the yeast expression vector pESC - URA was double - digested with the restriction endonucleases BamHI and NheI from Thermo Fisher Scientific Company, so that the linearized vector and the purified PCR product have the same sticky ends. Specifically, refer to Tables 4 - 5.
[0068] Table 1 Gene Amplification System
[0069]
[0070] Table 2 Gene Amplification Reaction Procedure
[0071]
[0072] Table 3 Primers Involved in Vector Construction
[0073]
[0074] Table 4 pESC - URA Double - Digestion System
[0075]
[0076] Put the double digestion system into a PCR instrument, incubate at 37°C for 30 min, then at 80°C for 5 min to inactivate the enzymes and terminate the reaction. Subsequently, use the ClonExpress II One Step Cloning Kit from Nanjing Novoprotein to perform homologous recombination on the linearized vector and the purified PCR product. The reaction system is shown in Table 5, and the reaction condition is 37°C for 30 min.
[0077] Table 5 Homologous recombination reaction system
[0078]
[0079]
[0080] Insert them into the yeast expression vector pESC-URA between the BamHI and NheI restriction sites under the control of the GAL1 promoter. Then transform the recombinant product into Escherichia coli DH5α, screen the positive transformants by colony PCR for the grown single colonies, extract the plasmids and perform sequencing identification. The identification results prove that 4 yeast expression vectors have been successfully constructed, namely pESC-URA-GAL1p-AarTPS72-CYC1t, pESC-URA-GAL1p-AarTPS07-CYC1t, pESC-URA-GAL1p-AarTPS60-CYC1t.
[0081] 1.2 Transformation and verification of the vector
[0082] Using EL-Y0 as the starting strain, competent yeast cells were prepared; the engineered strain EL-Y0 was obtained by transferring TEF1p-ERG1-PGK1t, ADH1p-ERG13-ADH1t, ADH1p-ERG12-ADH1t, TEF2p-ERG19-CYC1t, PGK1p-IDI1-PGK1t, TDH3p-AtCPR1-TDH3t, TEF1p-ERG10-PGK1t, TDH3p-ERG8-TDH3t, PGK1p-tHMGR-ADH1t, TDH3p-ERG20+ERG9-CYC1t into the His3 locus and TDH3p-ERG20+ERG9-CYC1t, TEF1t-ERG10-PGK1t, TDH3p-ERG8-TDH3t, PGK1p-tHMGR-ADH1t, TEF1p-ERG1-PGK1t into the Trp locus of the starting strain Saccharomyces cerevisiae CEN.PK2-1C. The preparation method of the strain has been disclosed in Wu S, Zhang F, Xiong W, et al. An Unexpected Oxidosqualene Cyclase Active Site Architecture in the Iristectorum Multifunctional α-Amyrin Synthase. DOI: 10.1021 / acscatal.0c03231.
[0083] The plasmids prepared in 1.1 were respectively transformed into the competent cells by the lithium acetate chemical transformation method and spread on the solid medium of SD-Ura. After waiting for 2-3 days, when monoclonal colonies grew out, positive monoclonal colonies were picked, and PCR sequencing was used to verify whether the target gene was successfully transformed. After verification, three engineered strains EL01 (AarTPS72), EL02 (AarTPS07), and EL03 (AarTPS60) were obtained.
[0084] 1.3 Shake flask fermentation
[0085] Three positive transformants were picked from the expansion plate of the engineered strain obtained in 1.2 into shake flasks containing 3 mL of SD medium with the corresponding selection markers and cultured overnight at 30 °C and 200 rpm to form a seed solution. The seed solution was inoculated into a shake flask containing 50 mL of SD-Ura medium at an inoculation amount of 1% (v / v). During this period, galactose was added for induction expression, and 5 mL of dodecane extractant was added (to avoid the volatilization of metabolites during the induction expression process), and cultured on a yeast shaker for 5 d.
[0086] After the shake flask fermentation was completed, the fermentation broth and the extractant were placed in a 50 mL centrifuge tube and centrifuged at 5000 rpm for 5 min. The upper n-dodecane organic phase was aspirated and appropriately diluted with n-hexane and filtered. The metabolites in the dilution were then detected using GC-MS.
[0087] Preparation of standard curve: using n-dodecane as solvent, a β-elemene standard stock solution with a solubility of 10 mg / mL was prepared, and then β-elemene standard solutions with solubility of 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL were prepared according to different dilution ratios, and they were placed in injection vials for GC-MS detection; the standard curve of β-elemene was obtained by processing the β-elemene concentration and peak area data, which was used for subsequent quantitative analysis of β-elemene samples.
[0088] 1.4 Results and Discussion
[0089] The experimental results are as follows Figure 1 As shown in the figure, compared with the control group, AarTPS72, AarTPS07 and AarTPS60 added three new product peaks, among which there was a main product at about 16.22min, which was identified as β-elemene by comparing the retention time and mass spectrum with the β-elemene standard (germaene A is prone to Cope rearrangement to synthesize β-elemene under high temperature conditions); there were also three minor product compounds P2, P3 and P4. Using the NIST17 mass spectrum database, it was found that they had similarities of more than 90% with compounds a-selinene, 8-Isopropenyl-1,5-dimethyl-cyclodeca-1,5-diene and neointermedeol, respectively. Therefore, it was preliminarily speculated that the structures of compounds P2, P3 and P4 were the three search compounds respectively; the specific structures need to be further purified and identified.
[0090] The prepared samples were 1 H-NMR, 13 C-NMR, DEPT135 and other nuclear magnetic resonance detections, the spectrum is shown in Appendix 4, and the carbon and hydrogen spectrum data are shown in Table 3-12. It was found that the nuclear magnetic resonance data of P4 were consistent with the neointermedeol compounds reported in the literature. Therefore, the main product of AarTPS72, AarTPS07 and AarTPS60 is β-elemene, which was identified as a multifunctional germacrene A synthase; this is consistent with the result that AarTPS72, AarTPS07 and AarTPS60 are close to germacrene A synthase in phylogeny.
[0091] The results of GC-MS quantitative analysis are as followsFigure 4 As shown in Figure 4 , by comparing the yields of three synthases mainly producing β-elemene, it was found that AarTPS72 had the highest catalytic efficiency. The shake-flask yield of β-elemene in strain EL01 was 82.7 mg / L, and gene AarTPS72 was selected for the construction of the engineered yeast strain for subsequent production of β-elemene.
[0092] Example 2 Construction of an Engineered Strain with Fusion Expression of ERG20
[0093] 2.1 Construction of the Fusion Plasmid
[0094] In this experiment, (GGGS) 2 was selected as the linker peptide to construct the fusion plasmid of ERG20 (farnesyl pyrophosphate synthase) and AarTPSs for producing β-elemene.
[0095] First, using the ERG20-(GGGS) 2 plasmid as a template, the primer ERG20-F and ERG20-(GGGS)2-AarTPS72-R in Table 6 were used to amplify the target fragment, the first half fragment ERG20-(GGGS) 2 -AarTPS72, i.e., ERG20-(GGGS) 2 (SEQ ID NO.7).
[0096] Second, using the pESC-URA-GAL1p-AarTPS72-CYC1t obtained in Example 1 as a template, the primers ERG20-(GGGS)2-AarTPS72-F and pESC-URA-AarTPS72-R were used to amplify the second half fragment (GGGS) 2 -AarTPS72.
[0097] After that, the front and rear fragments were subjected to overlap PCR experiment through homologous arms. The specific steps of overlap PCR: Referring to the reaction system in Table 1, 1 μL of ERG20-(GGGS) 2 and (GGGS) 2 -AarTPS72 were respectively added equally to the gene amplification system, and the reaction was carried out in a PCR instrument according to the amplification program in Table 2 to obtain the overlapping fragment ERG20-(GGGS) 2 -AarTPS72.
[0098] Referring to the method in Example 1, ERG20-(GGGS) 2-AarTPS72 was inserted into the yeast expression vector pESC-URA between the BamHI and NheI restriction sites under the control of the GAL1 promoter. After correct sequencing, the positive transformants obtained the plasmid pESC-URA-ERG20-(GGGS) through plasmid extraction experiments. 2 -AarTPS72, and the plasmid map is as Figure 3 shown in A of Figure 3 . The electrophoresis result after plasmid extraction is as
[0099] shown in B of
[0100] Using the plasmid pCfb2988 as a template, the fragment TY1-TRP-UP end was amplified by the primers pTY1-TRP-ERG20-UP-F and pTY1-ERG20-(GGGS) 2 -R in Table 6, and the fragment TY1-TRP-DOWN end was amplified using the primers pTY1-TRP-ERG20-DOWN-F and pTY1-TRP-ERG20-DOWN-R.
[0101] Using the fusion plasmid pESC-URA-ERG20-(GGGS) 2 -AarTPS72 obtained in 2.1 as a template, the fragment TY1-TRP-AarTPS72 (TRP-GAL1p-ERG20-(GGGS) 2 -AarTPS72-CYC1t was amplified using primers. The specific construction can refer to Figure 4 ).
[0102] Table 6 Primers involved in the fusion expression of ERG20
[0103]
[0104] 2.3 Product expression and detection
[0105] The target fragment obtained in 2.1 was purified and recovered. Referring to the method in Example 1, using the yeast strain EL-Y0 as the starting strain, the engineering strain EL12 was obtained through transformation and screening, and then the product was induced to express.
[0106] The product after induced expression was detected and quantitatively analyzed by GC-MS, and the results are shown in Figure 5 . As can be seen from Figure 5 , the flask yield of the β-elemene-producing strain EL12 obtained after fusion expression was 246.5 mg / L, which was 2.98 times higher than that of the strain EL01.
[0107] Example 3 Construction of an engineered strain overexpressing HIF-1
[0108] 3.1 Construction of recombinant fragments
[0109] In order to further improve the yield of β - elemene in strain EL12, the gene of hypoxia - inducible factor - 1 (HIF - 1) complex in the strain was overexpressed in this experiment. The specific steps are as follows:
[0110] Using the yeast genome of strain CEN.PK2 - 1C as a template, PCR amplification was carried out with NDT80 - UP - F and NDT80 - UP - R, NDT80 - DOWN - F and NDT80 - DOWN - R paired in Table 7 respectively to obtain NDT80 - UP and NDT80 - DOWN fragments.
[0111] Using plasmid pTP - URA - HIF - 1α - ARNT as a template (this plasmid and its preparation method have been disclosed in the literature Lin, X., An, T., Fu, D. et al. Optimization of central carbon metabolism by Warburg effect of human cancer cell improves triterpenes biosynthesis in yeast. Adv. Biotechnol. 1, 4 (2023).), the NDT80 - PGK1p - HIF - 1α - CYC1t - TEF1p - ARNT - ADH1t fragment was amplified using primers NDT80 - ADH1t - F and NDT80 - CYC1t - R.
[0112] Table 7 Primers involved in overexpressing HIF - 1
[0113]
[0114] 3.2 Product expression and detection
[0115] The three fragments obtained in 3.1 were purified and recovered. Referring to the method of Example 1, they were transferred into the yeast strain EL12 obtained in Example 2 together with the Cas9 plasmid pCRCT-GTR-URA3-△NDT80 (the construction method of this plasmid refers to the literature Zhang, Y., Wang, J., Wang, Z. et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10, 1053 (2019). https: / / doi.org / 10.1038 / s41467-019-09005-3, the plasmid backbone pCRCT was purchased from New Bio, and the gRNA sequence is shown in SEQ ID NO. 9) (the specific construction method can refer to Figure 6 ). After transformation and screening, the engineered strain EL14 was obtained, and then the product was induced to express.
[0116] The experimental results are as Figure 7 shown. The β-elemene yield in the shake flask of the strain EL14 obtained by heterologous expression of the HIF-1 gene reached 518.9 mg / L, which was 210% of that of the strain EL12.
[0117] Example 4 Optimization of the MVA pathway to improve the biosynthesis of β-elemene
[0118] 4.1 Construction of recombinant fragments
[0119] In order to further increase the β-elemene yield in the strain EL14, in this experiment, the MVA pathway was further optimized to improve the biosynthesis of β-elemene. The expression cassettes containing the tHMGR and UPC2-1 genes were respectively integrated into the TY4 multi-copy locus of the genome of the strain EL14 by the method of multi-fragment homologous recombination. The specific steps are as follows:
[0120] Using the plasmid pCfb2798 as a template, PCR amplification was carried out with the primers pTY4-LEU2-UP-F and pTY4-LEU2-UP-R, pTY4-LEU2-DOWN-F and pTY4-LEU2-DOWN-R in Table 8 respectively, to obtain the TY4-LEU2-UP end and TY4-LEU2-DOWN end fragments.
[0121] Using plasmid pESC-URA-tHMGR-UPC2-1 as a template, the TY4-LEU2-tHMGR-UPC2-1 (LEU2-GAL10p-tHMGR-ADH1t-GAL1p-UPC2-1-CYC1t) fragment was amplified using primers pTY4-LEU2-ADH1t-F and pTY4-LEU2-CYC1t-R.
[0122] Table 8 Primers involved in optimizing the MVA pathway
[0123]
[0124] 4.2 Product expression and detection
[0125] The three fragments obtained in 4.1 were purified and recovered. Referring to the method of Example 1, using strain EL14 as the starting strain, the engineered strain EL16 was obtained through transformation and screening, and then the product was induced to express.
[0126] The experimental results are as Figure 9 shown. The β-elemene yield of strain EL16 was 748.1 mg / L, which was 44% higher than that of strain EL14.
[0127] Example 5 Fed-batch fermentation of the engineered strain
[0128] To evaluate the potential of the engineered strain EL16 as a β-elemene cell factory, fed-batch fermentation was carried out in a 5 L bioreactor, and the specific steps are as follows:
[0129] (1) Pick a verified functional single colony from the expansion plate into 3 mL of the corresponding SD medium with a screening marker and culture it at 30 °C with shaking at 200 rpm for 24 h;
[0130] (2) Transfer 500 μL of the bacterial solution to a shake flask containing 50 mL of SD-deficient medium and culture it at 30 °C with shaking at 200 rpm for 24 h;
[0131] (3) Finally, transfer it to 200 mL of SD-deficient medium at an inoculation amount of 1% (v / v) and culture it at 30 °C with shaking at 200 rpm for 24 h. The bacterial solution obtained in this way was used as the seed solution and added to the fermenter. The relevant parameters of the fermenter were: fermentation temperature 30 °C, aeration rate 2 L / min. During the fermentation process, the pH was maintained at 5.5 by automatically adding ammonia water. In addition, the DO (dissolved oxygen) was maintained at about 40% by adjusting the stirring speed. When the initial carbon source was exhausted, feeding (glucose:galactose = 1:1) was started to maintain cell growth and induce expression;
[0132] (4) At 12 h of fermentation, 10% (v / v) of dodecane solvent was added to the fermenter, and the total fermentation time was 120 h. After fermentation, the fermented cells and the upper organic phase were collected by centrifugation for the separation and purification of the subsequent products. The formula of the fermenter and the feeding medium is shown in the following table:
[0133] Table 9 Formula of the Fermenter Medium
[0134]
[0135] Table 10 Composition of the Trace Element Solution
[0136]
[0137] Table 11 Composition of the Vitamin Solution
[0138]
[0139]
[0140] The difference in this experiment is that when the initial carbon source was completely consumed, ethanol was used as the carbon source for feeding, and bacterial samples were taken every 12 h. Then, the cell density (OD 600 ) was measured and GC-MS analysis was performed on each sampling sample. The experimental results are as Figure 10 shown. The results show that due to the addition of dodecane, the cell growth of the engineered strain EL17 was slightly inhibited in the first 24 h, but the growth was rapid in the later stage; the cell density (OD 600 ) of the engineered strain EL16 reached the maximum value of 196 at 120 h of fermentation, and the titer of β-elemene finally reached 14.0 g / L at this time.
[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.
Claims
1. A β-elemene synthase, characterized in that At least one of the following: (a) a protein consisting of an amino acid sequence as shown in at least one of SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5; (b) A sequence having a sequence homology of more than 90% with at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5 and having the same protein function.
2. The β-elemene synthase according to claim 1, characterized in that: The β-elemene synthase encoding gene is a nucleotide sequence shown by at least one of SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.
6.
3. An engineered strain of Saccharomyces cerevisiae that produces high yield of β-elemene, characterized in that: Using Saccharomyces cerevisiae as the starting strain, the tHMGR and UPC2-1 genes were transferred, the NDT80 gene was knocked out, the HIF-1α and ARNT genes were overexpressed, and the ERG20 gene and β-elemene synthase gene were transferred to obtain it.
4. The engineered yeast of claim 3, characterized in that: The said transfer of tHMGR and UPC2-1 genes is to integrate tHMGR and UPC2-1 genes into the TY4 site of the Saccharomyces cerevisiae chromosome; The ERG20 gene and the β-elemene synthase gene are transferred by using (GGGS)2 as a connecting peptide to fuse the ERG20 and β-elemene synthase genes to obtain a fusion fragment, and then integrating the fusion fragment into the TY1 site of the Saccharomyces cerevisiae chromosome; The knockout of NDT80 gene and overexpression of HIF-1α and ARNT genes is to knock out the NDT80 gene in the Saccharomyces cerevisiae genome and integrate the HIF-1α and ARNT genes into the original position of the NDT80 gene.
5. A β-elemene synthase gene, which is capable of producing β-elemene when introduced into Saccharomyces cerevisiae, characterized in that: Its amino acid sequence is at least one of SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.
6.
6. Use of the β-elemene synthase according to any one of claims 1 to 2, the engineered bacteria according to any one of claims 3 to 4, and the β-elemene synthase gene according to claim 5 in producing β-elemene.
7. A method for constructing an engineered strain of Saccharomyces cerevisiae that produces high β-elemene yield, characterized in that The steps include: (1) Using Saccharomyces cerevisiae as the starting strain, the GAL80 gene was knocked out to obtain the strain EL-B0; (2) The GAL1p promoter, ERG20 gene, (GGGS)2 connecting peptide, β-elemene synthase gene and CYC1t terminator were sequentially connected to obtain a fusion fragment, which was integrated into the TY1 site of the strain EL-A0 genome, and the strain EL-A1 was obtained after screening; (3) The PGK1p promoter, HIF-1α gene, CYC1t terminator, TEF1p promoter, ARNT gene and ADH1t were sequentially connected to obtain the PGK1p-HIF-1α-CYC1t-TEF1p-ARNT-ADH1t fragment, the NDT80 gene in the genome of strain EL-A1 was knocked out, and the fragment was integrated into the position of the original NDT80 gene. After screening, strain EL-A2 was obtained; (4) The GAL10p promoter, tHMGR gene, ADH1t terminator, GAL1p promoter, UPC2-1 gene and CYC1t terminator were sequentially connected to construct the GAL10p-tHMGR-ADH1t-GAL1p-UPC2-1-CYC1t fragment, which was integrated into the TY4 site of strain EL-A2, and an engineered Saccharomyces cerevisiae strain with high β-elemene production was obtained after screening.
8. The construction method according to claim 7, characterized in that: The nucleotide sequence of the (GGGS)2 connecting peptide is shown in SEQ ID NO.8; The integration is to transform the fragment into the cell using the lithium acetate transformation method, and the fragment is transferred together with the positioning fragment containing the homologous fragment and the screening marker, and integrated with the target site through homologous recombination.
9. The β-elemene synthase according to any one of claims 1 to 2, the engineered bacteria according to any one of claims 3 to 4, the β-elemene synthase gene according to claim 5, and the construction method according to any one of claims 7 to 8, characterized in that: The brewer's yeast is an engineered bacterium EL-Y. The nucleotide sequence of the GAL80 gene is shown in NCBI database sequence accession number 854954; The nucleotide sequence of the HIF-1α gene is shown in the NCBI database sequence accession number NM_001243084.2; The nucleotide sequence of the ARNT gene is shown in the NCBI database sequence accession number NM_001668.4; The nucleotide sequence of the NDT80 gene is shown in NCBI database sequence accession number 856524.
10. A method for producing β-elemene, characterized in that The steps include: (1) transferring the β-elemene synthase gene into an engineered bacterium, inoculating it into a culture medium for cultivation, and adding an extract during the cultivation; (2) Cultivate and separate to obtain an extract, which is then further purified to obtain β-elemene.