Recombinant saccharomyces cerevisiae engineering bacterium for producing 7-dehydrocholesterol and application of recombinant saccharomyces cerevisiae engineering bacterium

Abstract

The invention discloses a recombinant saccharomyces cerevisiae engineering bacterium for producing 7-dehydrocholesterol and application, and belongs to the technical field of genetic engineering and biological engineering. On the basis of an original strain, an exogenous gene DHCR24 and key endogenous genes ERG2 and ERG3 are integrated, and a non-path gene ROX1 is knocked out, so that the accumulation of 7-dehydrocholesterol in a saccharomyces cerevisiae body is further improved; the accumulation amount of the 7-dehydrocholesterol is further increased by adding integration of a speed limiting step gene and cell growth promoting genes SET2 and HAP4 and knockout of an RPH1 gene, and the ability of synthesizing the 7-dehydrocholesterol from the beginning in a saccharomyces cerevisiae body is further improved by completing single-copy integration of TPS1, ERG25, ERG26 and ERG27 genes at a saccharomyces cerevisiae GAL80 site. And the yield of 7-dehydrocholesterol reaches 896.2 mg / L after the constructed strain is subjected to shake flask fermentation for 100 hours.

Description

Technical Field

[0001] The invention relates to a recombinant brewer's yeast engineering bacterium for producing 7-dehydrocholesterol and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] 7-Dehydrocholesterol is a sterol present in animals and can be converted into vitamin D3 under ultraviolet irradiation. 7-Dehydrocholesterol is the most important precursor of vitamin D3. Vitamin D3 is widely used in biomedicine, feed breeding and other fields because of its effects on improving the body's immunity and treating bone diseases. In addition, the C25 position of 7-dehydrocholesterol is converted into 25-hydroxy-7-dehydrocholesterol by hydroxylase, and the product generates the active form of vitamin D3, i.e. 25-hydroxyvitamin D3, after ultraviolet irradiation. At present, the productivity of 25-hydroxyvitamin D3 and 25-hydroxy-7-dehydrocholesterol at home and abroad is low, and the price remains high, so efficient biosynthesis of 7-dehydrocholesterol has important practical value. Since vitamin D3 and its derivatives are currently expensive at home and abroad, it is of great significance to achieve high production of 7-dehydrocholesterol, the most important precursor of vitamin D3, in microorganisms.

[0003] 7-dehydrocholesterol is a product of the vitamin D3 metabolic pathway in animals. The function and metabolic pathway of 7-dehydrocholesterol are similar to those of ergosterol in Saccharomyces cerevisiae. The production of 7-dehydrocholesterol in Saccharomyces cerevisiae can be achieved by modifying the ergosterol pathway in Saccharomyces cerevisiae. Therefore, Saccharomyces cerevisiae is an excellent host for the production of drugs such as steroids. This pathway can be achieved by knocking out ERG5 and ERG6 in the ergosterol synthesis pathway and introducing the exogenous gene DHCR24 to produce 7-dehydrocholesterol in Saccharomyces cerevisiae, that is, introducing the 7-dehydrocholesterol production pathway.

[0004] Compared with the traditional chemical method of preparing 7-dehydrocholesterol, which is expensive, has complicated steps and high energy consumption, the production of 7-dehydrocholesterol by microbial metabolism has low pollution, high efficiency and reduces the cost of producing 7-dehydrocholesterol. Since the current microbial production of 7-dehydrocholesterol still has problems such as low yield and high production cost, it is necessary to develop engineered strains with improved production efficiency to achieve the goal of reducing production costs and increasing the accumulation of 7-dehydrocholesterol. Summary of the invention

[0005] The invention provides an engineered yeast strain for efficiently synthesizing 7-dehydrocholesterol. On the basis of the starting strain, the delta (24)-sterol reductase gene DHCR24 and the gene ERG2 or the gene ERG3 from Gallus gallus are integrated and expressed.

[0006] In one embodiment, the engineered yeast Saccharomyces cerevisiae integrates and expresses the δ(24)-sterol reductase gene DHCR24 and the gene ERG2 from Gallus gallus, and integrates and expresses the gene DHCR24 and the gene ERG3.

[0007] In one embodiment, the engineered yeast Saccharomyces cerevisiae also has one or more of the following improvements:

[0008] (1) Reduce the expression of genes ERG5 and ERG6;

[0009] (2) reduce the expression of the gene ROX1;

[0010] (3) Overexpression of one or more of the following endogenous genes of Saccharomyces cerevisiae: ERG2, ERG3, tHMG1, IDI1, ERG1, ERG11, SET2, HAP4, TPS1, ERG25, ERG26, ERG27.

[0011] In one embodiment, the engineered Saccharomyces cerevisiae bacteria integrate multiple copies of the gene DHCR24.

[0012] In one embodiment, the engineered Saccharomyces cerevisiae bacteria integrate 4 copies of the gene DHCR24.

[0013] In one embodiment, the genes ERG1, ERG11 and DHCR24 are integrated at the Ty1 site.

[0014] In one embodiment, the genes tHMG1 and IDI1 are integrated at the Ty3 site.

[0015] In one embodiment, the genes SET2 and HAP4 are integrated at the RPH1 locus.

[0016] In one embodiment, the genes TPS1, ERG25, ERG26 and ERG27 are integrated at the GAL80 locus.

[0017] In one embodiment, the starting strain includes but is not limited to Saccharomyces cerevisiae C800.

[0018] In one embodiment, the promoter P TDH1 Promote the expression of DHCR24 gene.

[0019] In one embodiment, the nucleotide sequence of the gene DHCR24 is shown as SEQ ID NO.1.

[0020] In one embodiment, the gene DHCR24 and the gene ERG2 are integrated at the ERG5 locus.

[0021] In one embodiment, the gene DHCR24 and the gene ERG3 are integrated at the ERG6 locus.

[0022] In one embodiment, the endogenous gene ROX1 of Saccharomyces cerevisiae is knocked out; the nucleotide sequence of the gene ROX1 is shown in SEQ ID NO.6.

[0023] In one embodiment, the promoter P TDH1 Initiate DHCR24 gene expression.

[0024] In one embodiment, the promoter P TEF1 Initiate the expression of ERG2 gene; the nucleotide sequence of the gene ERG2 is shown in SEQ ID NO.4.

[0025] In one embodiment, the promoter P TDH3 Initiate the expression of ERG3 gene; the nucleotide sequence of the gene ERG3 is shown in SEQ ID NO.5.

[0026] In one embodiment, the promoter P GAL1 Initiate the expression of tHMG1 gene; the nucleotide sequence of the gene tHMG1 is shown in SEQ ID NO.7.

[0027] In one embodiment, the promoter P GAL10 Initiate the expression of IDI1 gene; the nucleotide sequence of the gene IDI1 is shown in SEQ ID NO.8.

[0028] In one embodiment, the promoter P TEF1 Initiate the expression of ERG1 gene; the nucleotide sequence of the gene ERG1 is shown in SEQ ID NO.12.

[0029] In one embodiment, the promoter P PGK1 Initiate the expression of ERG11 gene; the nucleotide sequence of the gene ERG11 is shown in SEQ ID NO.13.

[0030] In one embodiment, the promoter P TDH3 The expression of the SET2 gene is initiated; the nucleotide sequence of the gene SET2 is shown in SEQ ID NO.9.

[0031] In one embodiment, the promoter P TDH1 Initiate the expression of HAP4 gene; the nucleotide sequence of the gene HAP4 is shown in SEQ ID NO.10.

[0032] In one embodiment, the promoter P GAL7Initiate the expression of TPS1 gene; the nucleotide sequence of the gene TPS1 is shown in SEQ ID NO.19.

[0033] In one embodiment, the promoter P TEF1 Initiate the expression of ERG25 gene; the nucleotide sequence of the gene ERG25 is shown in SEQ ID NO.20.

[0034] In one embodiment, the promoter P PGK1 Initiate the expression of ERG26 gene; the nucleotide sequence of the gene ERG26 is shown in SEQ ID NO.21.

[0035] In one embodiment, the promoter P TDH3 Initiate the expression of ERG27 gene; the nucleotide sequence of the gene ERG27 is shown in SEQ ID NO.22.

[0036] In one embodiment, the promoter P TEF1 The nucleotide sequence of the promoter is shown in SEQ ID NO.14; PGK1 The nucleotide sequence of the promoter is shown in SEQ ID NO.15; GAL1 or promoter P GAL10 The nucleotide sequence of the promoter is shown in SEQ ID NO.16; TDH1 The nucleotide sequence of the promoter is shown in SEQ ID NO.17; TDH3 The nucleotide sequence of the promoter is shown in SEQ ID NO.18; GAL7 The nucleotide sequence is shown in SEQ ID NO.24.

[0037] The invention also provides a microbial preparation containing the engineered yeast of saccharomyces cerevisiae.

[0038] The invention also provides a method for preparing 7-dehydrocholesterol, which comprises culturing the engineering yeast Saccharomyces cerevisiae in a culture medium.

[0039] In one embodiment, the engineered yeast Saccharomyces cerevisiae is inoculated into a fermentation medium and fermented at 28-30°C.

[0040] In one embodiment, the fermentation medium includes but is not limited to YPD medium.

[0041] In one embodiment, the fermentation process is also fed.

[0042] In one embodiment, the feed medium contains glucose, K + Mg 2+ 、Na + , Fe2+ .

[0043] In one embodiment, the feed medium contains glucose, potassium salts, magnesium salts, sodium salts, iron salts.

[0044] In one embodiment, the feed medium contains glucose, KH2PO4, MgSO4·7H2O, K2SO4, and Na2SO3.

[0045] In one embodiment, the feed medium contains glucose, KH2PO4, MgSO4·7H2O, K2SO4, Na2SO3 and FeSO4·7H2O.

[0046] In one embodiment, the engineered yeast Saccharomyces cerevisiae is cultured in a YPD medium at 200-220 rpm and 28-30° C. for 20-24 hours to prepare a seed solution.

[0047] In one embodiment, the seed liquid of the engineered yeast Saccharomyces cerevisiae is inoculated into a fermentation medium and cultured at 200-220 rpm and 28-30° C. for at least 72 h, or at least 96 h, or at least 100 h, or 100-120 h.

[0048] In one embodiment, the fermentation medium contains 40 g / L glucose, 15 g / L yeast powder, 30 g / L tryptone, and 1.5 g / L MgSO4.

[0049] In one embodiment, the pH is adjusted to 5.3-5.6 during the fermentation process.

[0050] In one embodiment, the pH is adjusted using 5 M NaOH.

[0051] In one embodiment, the method for preparing the seed liquid includes: inoculating the brewer's yeast engineered bacteria colony into the YPD culture medium, culturing at 200-220rpm, 28-30°C for 20-24h as the first-level seed liquid; inoculating the first-level seed liquid into the YPD culture medium at 3-5%, culturing at 200-220rpm, 28-30°C for 22-24h as the second-level seed liquid.

[0052] The present invention also provides application of the engineered yeast of saccharomyces cerevisiae or the method in preparing 7-dehydrocholesterol or a medicine containing 7-dehydrocholesterol.

[0053] Beneficial effects:

[0054] (1) The present invention uses Saccharomyces cerevisiae C800 as a host to obtain a recombinant Saccharomyces cerevisiae expressing the δ(24)-sterol reductase gene DHCR24 from Gallus gallus. The obtained recombinant Saccharomyces cerevisiae achieves the synthesis of 7-dehydrocholesterol. The accumulation of 7-dehydrocholesterol in Saccharomyces cerevisiae is further improved by knocking out the competing pathway genes ERG5 and ERG6, integrating two copies of the only exogenous gene DHCR24 for the synthesis of 7-dehydrocholesterol, and integrating a single copy of the key endogenous genes ERG2 and ERG3. On this basis, the non-pathway genes ROX1 and RPH1 are knocked out.

[0055] (2) The present invention verifies the up-regulation of key gene expression through qPCR, and further increases the accumulation of 7-dehydrocholesterol by increasing the rate-limiting step genes tHMG1, IDI1, ERG1, ERG11, DHCR24 and integrating the cell growth-promoting genes SET2 and HAP4.

[0056] (3) The present invention further improves the ability of Saccharomyces cerevisiae to synthesize 7-dehydrocholesterol from scratch by completing single-copy integration of TPS1 (to improve cell stress resistance), ERG25 (pathway gene), ERG26 (pathway gene), and ERG27 (pathway gene) at the GAL80 site of Saccharomyces cerevisiae, so that the 7-dehydrocholesterol production of the constructed engineered bacteria in YPD medium is 896.2 mg / L after shake flask fermentation for 100 hours, thereby obtaining a Saccharomyces cerevisiae strain with a high production of 7-dehydrocholesterol.

[0057] (4) The present invention also increases the yield of the strain at the fermentation tank level from 2.8 g / L to 4.51 g / L by optimizing the feeding conditions.

[0058] The present invention integrates animal heterologous metabolic pathway genes into the multi-copy sites of saccharomyces cerevisiae to achieve high production of 7-dehydrocholesterol, laying a foundation for the subsequent biosynthesis of steroidal compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the metabolism of heterologous synthesis of 7-dehydrocholesterol in Saccharomyces cerevisiae.

[0060] Figure 2 This is the mass spectrum of 7-dehydrocholesterol of the engineered strain cultured in YPD.

[0061] Figure 3 This is the mass spectrum of 7-dehydrocholesterol standard.

[0062] Figure 4 This is a graph showing the 7-dehydrocholesterol production of the engineered strain under YPD culture.

[0063] Figure 5OD of 7-dehydrocholesterol producing strain of engineered strain in YPD culture 600 .

[0064] Figure 6 The graph shows the 7-dehydrocholesterol production of the engineered strain when cultured in a 5-L fermenter. DETAILED DESCRIPTION

[0065] 1. Technical terms

[0066] The terms "comprising", "including", "having", and "containing" used in the present invention mean "including but not limited to", "including but not limited to", "having but not limited to", and "containing but not limited to", and can be used interchangeably with corresponding phrases. Unless the context clearly indicates otherwise, the term "or" used in this article is used in this article to mean the term "and / or" and can be used interchangeably with it. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, the examples of suitable methods and materials described below are illustrative only and do not limit the present invention in any way.

[0067] The term "expression" refers to any step involved in the production of a protein or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0068] The term "integrative expression" refers to the expression of homologous or heterologous genes at the genomic level, which is the integration of foreign genes into the genome of the host organism so that they can be stably inherited and expressed.

[0069] The term "locus" refers to the specific location of a gene or a polynucleotide of a gene expression-related element on a chromosome.

[0070] (II) Culture medium

[0071] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0072] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L, amino acids (5 g / L uracil, 10 g / L tryptophan, 10 g / L leucine, 10 g / L histidine, and appropriate deletion of corresponding amino acids as needed).

[0073] YPD medium: peptone 20 g / L, yeast powder 10 g / L, glucose 40 g / L.

[0074] Fermentation medium: glucose 40 g / L, yeast powder 15 g / L, trypsin 30 g / L, MgSO4 1.5 g / L, defoamer 0.1‰, 5M NaOH was used to adjust the pH to 5.3-5.6 throughout the process.

[0075] (III) Preparation of competent cells of Saccharomyces cerevisiae: The competent cells of Saccharomyces cerevisiae were prepared using the Frozen-EZ Yeast Transformation II kit at 30°C with 10 mL of YPD medium to a medium magnitude (OD 600 =0.8-1.0). The following steps were carried out at room temperature.

[0076] 1. Centrifuge the cells at 3500 rpm for 5 min and remove the supernatant;

[0077] 2. Add 10 mL of EZ1 solution to wash the precipitate, centrifuge the precipitated cells again, and aspirate the supernatant;

[0078] 3. Add 1 mL of EZ2 solution to resuspend the pelleted cells.

[0079] (IV) Efficient transformation method of brewer’s yeast:

[0080] 1. Mix 50 μL competent cells with 0.2-1 μg DNA (volume less than 5 μL); add 500 μL EZ3 solution and mix thoroughly;

[0081] 2. Incubate at 30℃ for 45min. During the incubation, flick the tube or vortex 2-3 times to mix well.

[0082] 3. Take 50-150 μL of the transformation mixture and place it on the appropriate nutrient-deficient plate;

[0083] 4. Incubate the plates at 30°C for 3-5 days to grow transformants.

[0084] (V) HPLC determination of 7-dehydrocholesterol: Waters high performance liquid chromatography was used for determination. HPLC conditions: chromatographic column: InertSustain C18 250 mm × 4.6 mm column (particle size 5 μm); mobile phase B, methanol containing 1‰ trifluoroacetic acid; flow rate: 1 mL / min; column temperature: 30°C; injection volume: 10 μL; detector wavelength: 280 nm.

[0085] Detection method: First, prepare a 30% potassium hydroxide ethanol solution (ethanol solvent purity 90%) for use. For example, weigh 11.5g potassium hydroxide, measure 30mL ethanol and add 3mL sterile water, mix well and stir to dissolve, and the saponification solution is ready. Take out 0.25mL of fermentation liquid and place it in a crushing tube, add the prepared 30% potassium hydroxide ethanol solution to the bacteria, and shake and mix thoroughly. Place it in an 88℃ water bath for 4h, take it out and shake and mix several times during the period. After the reaction is completed, place the reaction solution in a 5mL centrifuge tube, add 750 microliters of sterile water to the centrifuge tube in advance, and then add anhydrous ether to make the volume to 3.5mL. Under normal circumstances, the layers are separated, with 2.5mL of the upper layer and 1mL of the lower layer. Take 1mL of the upper organic phase into the injection bottle, and quantify the liquid phase. At this time, the yield calculated by the liquid phase should be multiplied by 2.5×4 on this basis to obtain the final yield.

[0086] (VI) The strain information is shown in Table 1.

[0087] Table 1 Strains involved in the present invention

[0088]

[0089] Example 1 Construction of 7-dehydrocholesterol-producing recombinant Saccharomyces cerevisiae strains 7-DHC-A and 7-DHC-B

[0090] The δ(24)-sterol reductase gene DHCR24 (nucleotide sequence as shown in SEQ ID NO.3) and the C-8 sterol isomerase gene ERG2 (nucleotide sequence as shown in SEQ ID NO.4) are integrated at the position of ERG5 (nucleotide sequence as shown in SEQ ID NO.1); and the δ(24)-sterol reductase gene DHCR24 (nucleotide sequence as shown in SEQ ID NO.3) and the C-5 sterol desaturase gene ERG3 are integrated at the position of ERG6 (nucleotide sequence as shown in SEQ ID NO.2), and the specific steps are:

[0091] The promoter P was amplified using primers TEF1p-F / TEF1p-R. TEF1 (The nucleotide sequence is shown in SEQ ID NO.14), and the promoter P was amplified using primers TDH1p-F / TDH1p-R. TDH1 , using primers TDH3p-F / TDH3p-R to amplify promoter P TDH3 , primers TEF1t-F / TEF1t-R amplify terminator T TEF1The DHCR24 gene was amplified using primers DHCR24-F / DHCR24-R, the ERG2 gene was amplified using primers ERG2-F / ERG2-R, the ERG3 gene was amplified using primers ERG3-F / ERG3-R, and the upstream and downstream homology arms of the ERG5 / 6 site were amplified using primers ERG5 / 6-armup-F / ERG5 / 6-armup-R and ERG5 / 6-armdown-F / ERG5 / 6-armdown-R, respectively. The gene knockout plasmid ERG5sgRNA-ERG5sgRNA-ERG6sgRNA-ERG6sgRNA-cas9-URA3 was constructed using primers 4-1-ERG5-PAM-1-F / 4-1-ERG5-PAM-1-R / 4-2-ERG5-PAM-2-F / 4-2-ERG5-PAM-2-R / 4-3-ERG6-PAM-1-F / 4-3-ERG6-PAM-1-R / 4-4-ERG6-PAM-2-F / 1-4-R. The obtained engineered strain 7-DHC-B was fermented in YPD medium at 30°C and 220rpm for 96h. Liquid chromatography verified that the engineered strain 7-DHC-B could produce 7-dehydrocholesterol, such as Figure 4 The production of 7-DHC was 266.2 mg / L after 100 h of fermentation.

[0092] All primers and gene sequences are listed in Table 2 .

[0093] Table 2 Primers and sequences

[0094]

[0095] Example 2 Knockout of pathway suppressor genes

[0096] Based on the strain 7-DHC-B constructed in Example 1, the endogenous gene ROX1 of Saccharomyces cerevisiae (nucleotide sequence as SEQ ID NO.6) was knocked out, and the upstream and downstream homologous arms of the ROX1 site were amplified using primers ROX1-armup-F / ROX1-armup-R and ROX1-armdown-F / ROX1-armdown-R. The PCR product was recovered by ethanol precipitation, and the above fragments and vector pY26 were assembled using Gibson, and then the integration fragment was obtained from the constructed plasmid using primers ROX1-armup-F / ROX1-armdown-R. The gene knockout plasmid ROX1sgRNA-cas9-URA3 was constructed using primers 2-1-ROX1-PAM-1-R / 2-1-ROX1-PAM-1-F. About 30 μg of the integration fragment and about 1 μg of sgRNA were transformed into the Saccharomyces cerevisiae engineering strain 7-DHC-B constructed in Example 1 using the Saccharomyces cerevisiae efficient transformation method, and then spread on the screening YNB solid medium and cultured at 30°C for 3-5 days until colonies appeared. The correct clone was named 7-DHC-C. A single colony was picked and transferred to 5 mL YPD medium, cultured at 30°C for 17-24 hours, and then transferred to 25 mL YPD medium at a 1% inoculation amount. The yield of 7-DHC was 382.2 mg / L at 100 hours of fermentation.

[0097] Table 3 Primer sequences

[0098]

[0099] Example 3 Increasing the copy number of rate-limiting genes

[0100] Since genes ERG1, ERG11, and DHCR24 are the key rate-limiting enzymes in the biosynthesis of 7-dehydrocholesterol, the endogenous genes ERG1 (nucleotide sequence shown in SEQ ID NO.12) and ERG11 (nucleotide sequence shown in SEQ ID NO.13) of Saccharomyces cerevisiae were overexpressed. Gene ERG1 was amplified from the genome of Saccharomyces cerevisiae using primers ERG1-F / ERG1-R, and gene ERG11 was amplified using primers ERG11-F / ERG11-R. Gene DHCR24 was amplified using primers DHCR24-F / DHCR24-R. The bidirectional promoter P was amplified using primers TEF1p-F and TEF1p-R. TEF1 (The nucleotide sequence is shown in SEQ ID NO.14). The bidirectional promoter P was amplified using primers PGK1p-F and PGK1p-R. PGK1 (The nucleotide sequence is shown in SEQ ID NO.15). The bidirectional promoter P was amplified using primers TDH1p-F and TDH1p-R. TDH1. Ty1-armup-F / Ty1-armup-R and Ty1-armdown-F / Ty1-armdown-R were used to amplify the upstream and downstream homologous arms of the Ty1 site respectively. The PCR product was recovered by ethanol precipitation, and the above fragments were assembled with the vector pY26 using Gibson, and then the primers Ty1-armup-F / Ty1-armdown-R were used to obtain the integrated fragment from the constructed plasmid. About 30 μg of the integrated fragment was transformed into the Saccharomyces cerevisiae engineering strain 7-DHC-C constructed in Example 2 using the Saccharomyces cerevisiae efficient transformation method, spread on the screening YNB solid culture medium, and cultured at 30°C for 3-5 days until colonies appeared. The correct clone was named 7-DHC-D. qPCR determination showed that the strain integrated 4 copies of the DHCR24, ERG1, and ERG11 genes. A single colony of the strain 7-DHC-D was picked and transferred to 5 mL of YPD medium, and then cultured at 30° C. for 17-24 hours and then transferred to 25 mL of YPD medium at a 1% inoculation rate. The yield of 7-DHC was 467.29 mg / L after fermentation for 100 hours.

[0101] Table 4 Primer sequences

[0102]

[0103] Example 4 Strengthening the supply of precursors

[0104] To promote the supply of precursor squalene, the endogenous genes tHMG1 (nucleotide sequence as shown in SEQ ID NO.7) and IDI1 (nucleotide sequence as shown in SEQ ID NO.8) of Saccharomyces cerevisiae were overexpressed. The gene tHMG1 was amplified from the genome of Saccharomyces cerevisiae using primers tHMG1-F / tHMG1-R, and the gene IDI1 was amplified using primers IDI1-F / IDI1-R. The bidirectional promoter P was amplified using primers GAL1 / 10p-F and GAL1 / 10p-R. GAL1 / 10(The nucleotide sequence is shown in SEQ ID NO.16). Ty3-armup-F / Ty3-armup-R and Ty3-armdown-F / Ty3-armdown-R were used to amplify the upstream and downstream homologous arms of the Ty3 site respectively. The PCR product was recovered by ethanol precipitation, and the above fragments and vector pY26 were assembled by Gibson, and then the integration fragment was obtained from the constructed plasmid using primers Ty3-armup-F / Ty3-armdown-R. About 30 μg of the integration fragment was transformed into the Saccharomyces cerevisiae engineering strain 7-DHC-D constructed in Example 3 using the Saccharomyces cerevisiae efficient transformation method, spread on the screening YNB solid culture medium, and cultured at 30°C for 3-5 days until colonies appeared. The correct clone was named 7-DHC-E. A single colony was picked and transferred to 5 mL of YPD medium. After culturing at 30°C for 17-24 h, it was transferred to 25 mL of YPD medium at a 1% inoculation rate. The yield of 7-DHC was 602.2 mg / L after 100 h of fermentation.

[0105] Table 5 Primer sequences

[0106]

[0107] Example 5 Promote cell growth and increase bacterial density

[0108] In order to prolong cell life and increase bacterial activity, the endogenous genes SET2 (nucleotide sequence as shown in SEQ ID NO.9) and HAP4 (nucleotide sequence as shown in SEQ ID NO.10) of Saccharomyces cerevisiae were overexpressed, and RPH1 (nucleotide sequence as shown in SEQ ID NO.11) was knocked out. The specific steps are as follows:

[0109] Primers TDH1p-F and TDH1p-R were used to amplify the endogenous bidirectional promoter P of Saccharomyces cerevisiae. TDH1 (The nucleotide sequence is shown in SEQ ID NO.17). Primers TDH3p-F and TDH3p-R were used to amplify the endogenous bidirectional promoter P of Saccharomyces cerevisiae. TDH3 (The nucleotide sequence is shown in SEQ ID NO.18). The SET2 gene was amplified from the Saccharomyces cerevisiae genome using primers SET2-F / SET2-R, and the terminator T was amplified using primers TEF1t-F / TEF1t-R. TEF1. The gene HAP4 was amplified from the Saccharomyces cerevisiae genome using primers HAP4-F / HAP4-R. The upstream and downstream homology arms of the RPH1 locus were amplified using primers RPH1-armup-F / RPH1-armup-R and RPH1-armdown-F / RPH1-armdown-R, respectively. The PCR product was recovered by ethanol precipitation, and the above fragments were assembled with the vector pY26 using Gibson, and the integration fragment was obtained from the constructed plasmid using primers RPH1-armup-F / RPH1-armdown-R. The gene knockout plasmid RPH1sgRNA-cas9-URA3 was constructed using primers 3-1-RPH1-PAM-1-R / 3-1-RPH1-PAM-1-F. About 30 μg of the integration fragment and about 1 μg of sgRNA were transformed into the Saccharomyces cerevisiae engineering strain 7-DHC-E constructed in Example 4 using the Saccharomyces cerevisiae efficient transformation method, and then spread on the screening YNB solid medium and cultured at 30°C for 3-5 days until colonies appeared. The correct clone was named 7-DHC-F. A single colony was picked and transferred to 5 mL YPD medium, cultured at 30°C for 17-24 hours, and then transferred to 25 mL YPD medium at a 1% inoculation amount. Figure 4 As shown in the figure, the 7-dehydrocholesterol production was 821.5 mg / L after 100 h of fermentation, and the OD 600 The OD of the bacteria in Example 4 was 49.2. 600 , an increase of 11.5.

[0110] Table 6 Primer sequences

[0111]

[0112] Example 6 Improving cell stress resistance and increasing the copy number of pathway genes

[0113] In order to improve the stress resistance of cells, the endogenous gene TPS1 (a key gene for trehalose synthesis, the nucleotide sequence is shown in SEQ ID NO.19), ERG25 (a nucleotide sequence is shown in SEQ ID NO.20), ERG26 (a nucleotide sequence is shown in SEQ ID NO.21), ERG27 (a nucleotide sequence is shown in SEQ ID NO.22) of Saccharomyces cerevisiae was overexpressed, and GAL80 (a nucleotide sequence is shown in SEQ ID NO.23) was knocked out. The bidirectional promoter PGAL7p (a nucleotide sequence is shown in SEQ ID NO.24) was amplified with primers GAL7p-F and GAL7p-R. The bidirectional promoter PTDH3 (a nucleotide sequence is shown in SEQ ID NO.18) was amplified with primers TDH3p-F and TDH3p-R. The bidirectional promoter PTEF1 (a nucleotide sequence is shown in SEQ ID NO.14) was amplified with primers TEF1p-F and TEFp-R. The bidirectional promoter PPGK1 (a nucleotide sequence is shown in SEQ ID NO.15) was amplified with primers PGK1p-F and PGKp-R. The gene TPS1 (nucleotide sequence such as SEQ ID NO.19) was amplified from the genome of Saccharomyces cerevisiae using primers TPS1-F / TPS1-R, the gene ERG25 (SEQ ID NO.20) was amplified from the genome of Saccharomyces cerevisiae using primers ERG25-F / ERG25-R, the gene ERG26 (SEQ ID NO.26) was amplified from the genome of Saccharomyces cerevisiae using primers ERG26-F / ERG26-R, the gene ERG27 (SEQ ID NO.22) was amplified from the genome of Saccharomyces cerevisiae using primers ERG27-F / ERG27-R, and the gene URA3 (screening tag) was amplified from the genome of Saccharomyces cerevisiae using primers URA3-F / URA3-R. GAL80 used primers GAL80-armup-F / GAL80-armup-R and GAL80-armdown-F / GAL80-armdown-R to amplify the upstream and downstream homology arms of the GAL80 site, respectively. The PCR product was recovered by ethanol precipitation, and the above fragments were assembled with the vector pY26 using Gibson. Primers GAL80-armup-F / GAL80-armdown-R were used to obtain the integration fragment from the constructed plasmid. About 30 μg of the integration fragment was transformed into the Saccharomyces cerevisiae engineered strain 7-DHC-F constructed in Example 5 using the Saccharomyces cerevisiae efficient transformation method, spread on the screening YNB solid culture medium, and cultured at 30°C for 3-5 days until colonies appeared. The correct clone was named 7-DHC-G. A single colony was picked and transferred to 5 mL of YPD culture medium. After culturing at 30°C for 17-24 hours, it was transferred to 25 mL of YPD culture medium at a 1% inoculation amount, as shown in FIG. Figure 4 As shown in the figure, the 7-dehydrocholesterol production was 896.2 mg / L after 100 h of fermentation, and the OD600 It is 53.8.

[0114] Table 7 Primer sequences

[0115]

[0116]

[0117] Example 7 Fermentation of 7-dehydrocholesterol by strain 7-DHC-G in a 5-L fermenter

[0118] 5L tank culture medium (2.5L): glucose 40g / L, yeast powder 15g / L, trypsin 30g / L, MgSO4 1.5g / L, defoamer 0.1‰, and pH was adjusted to 5.3-5.6 with 5M NaOH throughout the process.

[0119] Feed medium 1 (1.5 L): glucose 600 g / L, KH2PO4 18 g / L, MgSO4·7H2O 10.24 g / L, K2SO4 7 g / L, Na2SO3 0.56 g / L (sterilized at 115°C for 20 min).

[0120] Preparation of fermentation tank seed solution: Pick a few fresh yeast colonies with an inoculation loop and inoculate them into a shake flask containing 20 mL YPD medium, and culture at 220 rpm and 30°C for 20-24 h as the first-level seed solution. Inoculate 5% of the first-level seed solution into a 500 mL shake flask containing 200 mL YPD, and culture at 220 rpm and 30°C for 22-24 h as the second-level seed solution.

[0121] During the fermentation process, the dissolved oxygen was 30%, the residual sugar in the culture medium was controlled to 2 g / L by feeding, and the ethanol was controlled below 10 g / L. During 93-96 hours of fermentation, the accumulation of 7-dehydrocholesterol reached 2.8 g / L, and then the bacteria autolyzed and the accumulation decreased.

[0122] Example 8 Optimization of fermentation production of 7-dehydrocholesterol by strain 7-DHC-G in a 5-L fermenter

[0123] The specific implementation method is the same as Example 7, except that FeSO4·7H2O is also fed and its concentration in the tank is controlled to be 0.4 g / L. During the fermentation process, the dissolved oxygen is 30%, the residual sugar is detected every 2 hours and controlled to be 0 g / L, and the ethanol is controlled below 5 g / L. The accumulation of 7-dehydrocholesterol can reach 4.51 g / L at 144 hours of fermentation.

[0124] Example 9 strains in shake flask horizontal fermentation

[0125] The genetically engineered bacteria constructed in Examples 1 to 6 are also suitable for the following fermentation method:

[0126] Preparation of shake flask seed solution: Pick a few fresh yeast colonies with an inoculation loop and inoculate them into a shake flask containing 10 mL of YPD. Cultivate at 200-220 rpm and 28-30°C for 20-24 h.

[0127] 250mL shake flask horizontal fermentation: the liquid volume of YPD medium in the shake flask is 25mL, the inoculation amount is 350μL, 200-220rpm, 28-30℃, and culture for 96-120h.

[0128] Comparative Example:

[0129] Taking strain 7-DHC-5 in publication number CN114703077A as a control, shake flask fermentation was carried out according to the method of Example 6 and fermentation was carried out according to the method of Example 8. The results showed that the strain had a yield of 650 mg / L at the shake flask level and a yield of 2 g / L at the fermenter level.

[0130] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An engineered yeast strain of Saccharomyces cerevisiae for synthesizing 7-dehydrocholesterol, characterized in that: Based on the starting strain, the δ(24)-sterol reductase gene DHCR24 and the gene ERG2 or gene ERG3 from Gallus gallus were integrated and expressed.

2. The brewer's yeast engineering bacterium according to claim 1, characterized in that The engineered yeast of cerevisiae also has one or more of the following improvements: (1) Reduce the expression of genes ERG5 and ERG6; (2) reduce the expression of the gene ROX1; (3) Overexpression of one or more of the following endogenous genes of Saccharomyces cerevisiae: ERG2, ERG3, tHMG1, IDI1, ERG1, ERG11, SET2, HAP4, TPS1, ERG25, ERG26, ERG27.

3. The engineered yeast of claim 1 or 2, characterized in that: Multiple copies of the gene DHCR24 were integrated.

4. The engineered yeast of any one of claims 1 to 3, characterized in that: Genes tHMG1 and IDI1 were integrated at the Ty3 locus.

5. The engineered yeast of any one of claims 1 to 4, characterized in that: Genes SET2 and HAP4 were integrated at the RPH1 locus.

6. The engineered yeast of any one of claims 1 to 4, characterized in that: Genes TPS1, ERG25, ERG26 and ERG27 were integrated at the GAL80 locus.

7. The engineered yeast of any one of claims 1 to 6, characterized in that: The starting strain includes but is not limited to Saccharomyces cerevisiae C800.

8. A microbial preparation containing the engineered yeast of Saccharomyces cerevisiae according to any one of claims 1 to 7.

9. A method for preparing 7-dehydrocholesterol, characterized in that: The engineered yeast of any one of claims 1 to 7 is cultured in a culture medium.

10. The method according to claim 9, characterized in that The brewer's yeast engineered bacteria is inoculated into a fermentation medium and fermented at 28-30°C.

11. The method according to claim 9 or 10, characterized in that: The fermentation process is also fed; the feed medium contains glucose, K + Mg 2+ 、Na + .

12. The method according to claim 11, characterized in that The feed medium also contained iron salts.

13. The method according to any one of claims 9 to 12, characterized in that: The seed liquid of the engineered yeast of saccharomyces cerevisiae is inoculated into a fermentation medium. The preparation method of the seed liquid comprises: inoculating the colony of the engineered yeast of saccharomyces cerevisiae into a YPD medium, culturing at 200-220 rpm and 28-30° C. for 20-24 hours; Optionally, 3-5% of the first-level seed solution is inoculated into a YPD medium and cultured at 200-220 rpm and 28-30° C. for 22-24 h as a second-level seed solution.

14. Use of the engineered yeast of Saccharomyces cerevisiae according to any one of claims 1 to 7, or the microbial preparation according to claim 8, or the method according to any one of claims 9 to 13 in the preparation of 7-dehydrocholesterol or a drug containing 7-dehydrocholesterol.

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