Construction method and application of a fungus for synthesizing 24-epiergosterol

Abstract

The present invention provides a method for constructing a fungus that synthesizes 24-epiergosterol and its application. 24(28) The reductase DWF1 gene or its mutant gene is recombinantly expressed in ergosterol-producing fungi to construct a strain that synthesizes 24-epi-ergosterol; the reduction ability of DWF1 in fungi to a non-natural substrate 24(28)-dehydroergosterol is improved by directed evolution, and the yield of 24-epi-ergosterol is increased to 841.2 mg / L by combining with high-density fermentation; a synthetic biology strategy of enhancing esterification and hydrolysis to improve sterol balance is adopted to improve the metabolic flow of 24-epi-ergosterol in fungi, and the promoters of DWF1 and ACC1 genes are optimized and ERG5 is overexpressed to increase the proportion of 24-epi-ergosterol in sterols, and the yield of 24-epi-ergosterol is increased to 2.76 g / L by combining with high-density fermentation.

Description

Technical Field

[0001] The present invention relates to a construction method and application of a 24-epi-ergosterol synthesizing fungus. The present invention obtains a recombinant fungal strain producing 24-epi-ergosterol in fungi by gene editing, and relates to the technical field of synthetic biology and the field of enzyme engineering. Background Art

[0002] Brassinosteroids (BRs) are a class of natural steroid lactone / ketone substances with high physiological activity in plants and are considered to be the sixth type of plant hormones. They play a vital role in plant growth processes such as seed germination, root growth, and reproduction; at the same time, they can coordinate with other plant hormones to regulate the metabolism of plant oxidative free radicals, ethylene synthesis, and root gravity response, thereby improving the plant's resistance to harsh environments such as drought, high salt, and extreme temperatures. As the most biologically active compound among BRs, brassinolide (BL, also known as brassinolide) ( Figure 1 ) has great potential for agricultural and industrial applications.

[0003] However, since the content of brassinolide in plants is extremely low, even in pollen and seeds with higher content, the content is only 1-100ng / g (fresh weight), which is far from meeting the needs of research and agricultural applications; considering the structural complexity of BRs, predecessors have proposed and tried semi-synthetic methods based on natural sterols. In the attempt to semi-synthesize BL, crinoid sterol isolated from a rare invertebrate in the ocean (Crinoidea sp.) is considered to be the best synthetic precursor ( Figure 2)(MJ Thompson, et al, Steroids, 1981(38), 567). However, the scarcity of crinoid sterols is even greater than that of BL and is insufficient for industrial synthesis of BL. In addition, previous researchers have also tried to synthesize BL by using more readily available sterols as substrates, such as ergosterol or stigmasterol, to oxidize the side chain to generate steraldehydes, and then connect chiral accessories to construct a side chain with the same chirality as BL, especially the chirality of 24-C (Fung, S. and Siddall. JB, J. Am. Chem. Soc., 1980 (102), 6580; M. Sakakibara, et al, Heterocycles, 1982 (17), 301; TCMcMorris, et al, J. Chem. Soc., Perkin Trans, 1996 (1), 295; AL Hurski, et al, Org. Biomol. Chem., 2015 (13), 1446). However, due to the many synthetic steps, high cost, and high pollution, industrial production has not been achieved. Given the inaccessibility of natural BL, researchers have turned to the development of other BRs with relatively low activity but can be mass-produced. To date, two BRs have been widely used in agriculture, namely 24-epi-brassinolide (EBL) and 28-homo-brassinolide (HBL) ( Figure 1 ), whose semi-synthetic precursors are ergosterol (TCMcmorris, et al, J Org Chem, 1993 (58), 2338) and stigmasterol (TCMcmorris, et al, Phytochemistry, 1994 (36), 585) ( Figure 2 ).

[0004] In recent years, the development of synthetic biology has made it possible to use microbial cell factories to efficiently and cost-effectively produce complex natural products (such as BL). However, the biosynthetic pathway of BL has not been fully elucidated ( Figure 3 ), therefore, through synthetic biology, the transformation of fast-growing and low-cost microorganisms to enable them to synthesize sterols with the same side chains as crinoid sterols in large quantities for industrial production of BL is the most feasible and promising technical solution under the current circumstances.

[0005] Compared with EBL, one of the two BRs currently produced on a large scale, the structural difference of BL is only the chirality of the side chain 24-C, and this chiral structure is derived from ergosterol, the semi-synthetic precursor of EBL. Therefore, the present invention intends to transform the yeast ergosterol synthesis pathway through synthetic biology to synthesize 24-epiergosterol, which can be conveniently used to synthesize BL. The reaction that determines the chirality of 24-C in the yeast ergosterol synthesis pathway is the Δ 24(28) By comparing the substrate structures and chiral selectivity of enzymes in the steroid synthesis pathways of various plants and microorganisms, we found that the sterol Δ 24(28) The reductase DWF1 replacing ERG4 is most likely to cause yeast to switch from ergosterol synthesis to 24-epiergosterol synthesis; at the same time, due to the large structural differences between the two natural substrates, DWF1 needs to be modified to improve its catalytic activity towards non-natural substrates; secondly, a synthetic biology strategy of enhancing esterification and hydrolysis to improve sterol balance is adopted to increase the metabolic flow of yeast to 24-epiergosterol; finally, the expression levels of genes related to the synthesis pathway are further adjusted to achieve a satisfactory 24-epiergosterol yield. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a construction method and application of a 24-epiergosterol synthesizing strain.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] The present invention first provides a method for constructing a fungus for synthesizing 24-epiergosterol, which comprises the following steps: 24(28) The reductase DWF1 gene or its mutant gene is recombinantly expressed in the ergosterol-producing fungus to construct a strain that synthesizes 24-epiergosterol.

[0009] As a preferred embodiment of the present invention, the ergosterol-producing fungus is an ergosterol-producing eukaryotic microorganism or an ERG4 gene knockout mutant thereof, preferably a eukaryotic microorganism of the family Saccharomycetaceae, Sclerotiniaceae, Cladosporiaceae, Hypocreaceae, Trichocomaceae, Aspergillaceae, Tricholomataceae or an ERG4 gene knockout mutant of the above eukaryotic microorganisms, particularly preferably Saccharomyces cerevisiae with the ERG4 gene knocked out.

[0010] As a preferred embodiment of the present invention, the method for obtaining the DWF1 mutant gene comprises the following steps:

[0011] Plant-derived sterols 24(28) The reductase DWF1 gene was used as a template and the concentration of 0.02-0.12 mM Mn 2+ Error-prone PCR was performed to construct a DWF1 mutant library;

[0012] The DWF1 mutant library and the linearized plasmid pRS42H were co-transformed into the ERG4 knockout ergosterol-producing fungal strain, and spread on a solid medium containing 50-200 μg / mL Hyg B; the growing strains were picked and cultured in a medium containing 50-200 μg / mL Hyg B and an ERG4 knockout strain growth inhibitor;

[0013] The strains that can grow were selected and inoculated into a culture medium containing 50-200 μg / mL Hyg B for cultivation, and liquid phase analysis was performed to obtain DWF1 mutants with improved catalytic activity.

[0014] Furthermore, the ERG4 knockout strain growth inhibitor is one or more of 0.01wt%-0.05wt% sodium dodecyl sulfate, 2μg / mL-20μg / mL nystatin, and 10μg / mL-100μg / mL fluconazole.

[0015] Furthermore, the DWF1 gene is derived from plants producing brassinolide compounds, preferably from plants of the Lamiaceae family, the Cruciferae family, and the Moraceae family, preferably from plants with a high content of brassinolide compounds, and particularly preferably from Ajuga reptans (Ar).

[0016] As a preferred embodiment of the present invention, the construction method further comprises: integrating the constructed ARE2, YEH1, and YEH2 expression cassette sequences SEQ ID NO.7 into the 16Ty3 site of the genome through CRISPR / Cas9 editing technology, thereby overexpressing the ARE2, YEH1, and YEH2 genes and enhancing the balance between esterification storage and hydrolysis release of sterols.

[0017] As a preferred embodiment of the present invention, the construction method further comprises: using CRISPR / Cas9 editing technology, recombinantly integrating SEQ ID NO.8 at the promoter of the ACC1 gene, replacing the ACC1 gene promoter, overexpressing the DWF1 gene or its mutant gene, and using CRISPR / Cas9 editing technology, integrating the constructed ERG5 expression cassette sequence SEQID NO.9 at the 15Ty2 site, overexpressing the ERG5 gene, thereby enhancing the synthesis of 24-epiergosterol and increasing the proportion of 24-epiergosterol in sterols.

[0018] Furthermore, the DWF1 gene encodes the amino acid sequence shown in SEQ ID NO.1.

[0019] The DWF1 mutant gene is a gene that can encode one of the following amino acid sequences:

[0020] (1) the amino acid sequence of SEQ ID NO.2 in the sequence listing;

[0021] (2) the amino acid sequence of SEQ ID NO.3 in the sequence listing;

[0022] (3) the amino acid sequence of SEQ ID NO.4 in the sequence listing;

[0023] (4) the amino acid sequence of SEQ ID NO.5 in the sequence listing;

[0024] (5) the amino acid sequence of SEQ ID NO.6 in the sequence listing;

[0025] Among them, the sterol Δ 24(28) The reductase was named ArV143G, and the valine at position 143 was mutated to glycine relative to the wild type; the amino acid sequence of SEQ ID NO.3 in the sequence list encoded the sterol Δ 24(28) The reductase was named ArM235T, and the methionine at position 235 was mutated to threonine relative to the wild type; the amino acid sequence of SEQ ID NO.4 in the sequence list encoded the sterol Δ 24(28) The reductase was named ArS306P, and the serine at position 306 was mutated to proline relative to the wild type; the amino acid sequence of SEQ ID NO.5 in the sequence list encoded the sterol Δ 24(28) The reductase is named ArY338H, and the tyrosine at position 338 is mutated to histidine relative to the wild type; the amino acid sequence of SEQ ID NO.6 in the sequence list encodes the sterol Δ 24(28)The reductase was named Ar207, and relative to the wild type, its valine at position 143 was mutated to glycine, its serine at position 306 was mutated to proline, and its tyrosine at position 338 was mutated to histidine;

[0026] Furthermore, the plant-derived sterol Δ 24(28) The reductase DWF1 gene is derived from Arabidopsis thaliana (At), Ajuga reptans (Ar), Brassica rapa (Br) or Cannabis sativa (Cs).

[0027] Furthermore, the plasmid is pRS42H plasmid.

[0028] Furthermore, the ergosterol-producing fungus is Saccharomyces cerevisiae.

[0029] The present invention also provides a 24-epi-ergosterol synthesizing fungus, and the strain is constructed by the above construction method.

[0030] The present invention also provides application of the above-mentioned 24-epiergosterol-synthesizing fungus in producing 24-epiergosterol.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The present invention provides a method for constructing a fungal strain for synthesizing 24-epi-ergosterol, and the obtained strain can increase the yield of 24-epi-ergosterol. The present invention also proposes a further optimized construction method, so that the obtained strain can enhance the synthesis ability of 24-epi-ergosterol while also increasing the proportion of 24-epi-ergosterol in sterols. The present invention establishes a method for synthesizing sterol Δ 24(28) A high-throughput screening method for the modification of the catalytic activity of reductase (DWF1) was used. Through this screening method, four points were screened: V143G, M235T, S306P, and Y338H. The best mutation combination Ar207 (ArDWF1 V143G / S306P / Y338H ), which significantly improved the synthesis efficiency of 24-epiergosterol. At the same time, with the help of gene editing, on the basis of further increasing the production of 24-epiergosterol, it also increased the proportion of 24-epiergosterol in sterols, providing a good foundation for the industrial fermentation production of 24-epiergosterol, and at the same time, providing a good precursor for the semi-synthesis of BL.

[0033] The present invention establishes a new sterol balance by overexpressing sterol acylase ARE2, hydrolase YEH1, YEH2, and enhancing esterification storage and hydrolysis release of sterols, thereby raising the sterol synthesis capacity in yeast to a new stage, which is twice that of strain YQE231.

[0034] The present invention utilizes homologous recombination technology to replace the promoters of Ar207 and ACC1 with stronger promoters, and at the same time, overexpresses ERG5 to increase the expression levels of Ar207, ACC1 and ERG5, thereby not only enhancing the synthesis capacity of 24-epiergosterol, but also increasing the proportion of 24-epiergosterol in sterols, thereby reducing the pressure of purification in the later stage.

[0035] The strain YQE734 constructed by the present invention increases the yield of 24-epiergosterol to 2.76 g / L through high-density fermentation, which is 3.28 times higher than that of the strain YQE231. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The structures of brassinolide, 24-epi-brassinolide, and 28-homo-brassinolide;

[0037] Figure 2 The synthesis diagrams of 24-epibrassinolide, 28-homobrassinolide and brassinolide from ergosterol, stigmasterol, crinosterol and 24-epibrassinolide respectively;

[0038] Figure 3 for the biosynthetic pathway of brassinolide in plants;

[0039] Figure 4 This is the synthetic pathway of 24-epiergosterol in Saccharomyces cerevisiae;

[0040] Figure 5 The growth curves of industrial strain CICC4746 and model strain BY4741;

[0041] Figure 6 is the standard curve of ergosterol;

[0042] Figure 7 The growth inhibition of bacteria with different ergosterol / 24-epi-ergosterol production under different SDS concentrations was used to determine the SDS concentration suitable for high-speed screening;

[0043] Figure 8 The relative enzyme activities of four mutants (ArV143G, ArM235T, ArS306P, ArY338H) obtained by high-level screening in the form of plasmids in the model strain;

[0044] Fig. 9The production of 24-epiergosterol after integration into the genome of the industrial strain after combining mutations;

[0045] Fig.10 This is a plasmid (pRS42H) map.

[0046] Fig.11 This is the plasmid map of pEB-Y1A2Y2.

[0047] Fig.12 pRS42H-P GAL1 -Ar207 plasmid map.

[0048] Fig.13 pRS42H-P CIT2 -ERG5 plasmid map.

[0049] Fig.14 This is a combination of the effects of overexpression of sterol acylase ARE2, hydrolase YEH1, YEH2, promoters replacing Ar207 and ACC1, and overexpression of ERG5 on the production of 24-epiergosterol and its precursors. YQE231 represents the starting strain, serving as the control group; YQE717 represents the strain overexpressing sterol acylase ARE2, hydrolase YEH1, and YEH2; YQE729 represents the strain with promoters replacing Ar207 and ACC1 based on YQE717; and YQE734 represents the strain overexpressing ERG5 based on YQE729.

[0050] Fig.15 This is the high-density fermentation diagram of YQE734. DETAILED DESCRIPTION

[0051] The present invention is further described and illustrated below in conjunction with specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0052] The culture medium of the present invention is as follows:

[0053] (1) YPD medium (1% yeast powder, 2% peptone, 2% glucose), solid medium with 2% agar powder added, sterilized at 115°C, for activation and pre-culture of Saccharomyces cerevisiae.

[0054] (2) YPD (HygB / G418) medium (1% yeast powder, 2% peptone, 2% glucose, 100 μg / mL HygB, 200 μg / mL G418), solid medium with 2% agar powder added, sterilized at 115°C, for screening KanMX markers.

[0055] (3) LB medium (1% sodium chloride, 1% tryptone, 0.5% yeast powder), solid medium with 2% agar powder added, sterilized at 121°C, used for activation and pre-culture of E. coli.

[0056] (4) LB (Amp) medium (1% sodium chloride, 1% tryptone, 0.5% yeast powder, 100 μg / mL ampicillin), solid medium with 2% agar powder added, sterilized at 121°C, for culturing Escherichia coli containing plasmids.

[0057] The strain object for the construction method of the present invention can be any ergosterol-producing fungi, ergosterol is a component of fungal cell membranes in nature, and fungi basically produce ergosterol. Typically, saccharomyces cerevisiae can be used. In this embodiment, industrial saccharomyces cerevisiae CICC1746 is the main experimental object, but other saccharomyces cerevisiae or ergosterol-producing fungi can also be selected. The following industrial saccharomyces cerevisiae (strain number: CICC1746) was purchased from the China Industrial Microbiological Culture Collection Management Center, and the model strain BY4741 was purchased from the American ATCC Culture Collection Management Center.

[0058] The plasmid pRS42H used in the present invention is shown in the figure Fig.10 As shown;

[0059] The plasmid pEB-Y1A2Y2 used in the present invention is shown in the following figure: Fig.11 As shown;

[0060] The plasmid pRS42H-P used in the present invention GAL1 -Ar207 spectrum Fig.12 As shown;

[0061] The plasmid pRS42H-P used in the present invention CIT2 -ERG5 map Fig.13 As shown;

[0062] Example 1: Construction of ERG4 knockout yeast strains YQE101 and YQE102

[0063] The genome of industrial Saccharomyces cerevisiae CICC1746 was used as a template. The primers erg4Δ-UF (SEQ ID NO.10) / erg4Δ-UR (SEQ ID NO.11) were used to perform PCR amplification to obtain the 449 bp homology arm fragment erg4Δ-U upstream of the ERG4 site, and the primers erg4Δ-DF (SEQ ID NO.12) / erg4Δ-DR (SEQ ID NO.13) were used to amplify the 446 bp homology arm fragment erg4Δ-D downstream of the ERG4 site. The two amplified fragments were connected by overlap extension PCR to obtain the Kerg4 fragment. The Kerg4 fragment was transformed into the model strain BY4741 and the industrial Saccharomyces cerevisiae strain CICC1746 by lithium acetate / PEG3350 chemical transformation method. The CRISPR-Cas9 gene editing tool was used to perform double-strand break DNA cleavage at the ERG4 site under the guidance of ERG4-gRNA (SEQ ID NO.59). The Kerg4 fragment was transformed into the model strain BY4741 and the industrial Saccharomyces cerevisiae strain CICC1746 by containing 100 μg / mL Hyg B and 200 μg / mL G418 in YPD medium to screen the transformed positive clones to obtain ERG4 site knockout YQE101 (BY4741; erg4Δ) and YQE102 (CICC1746; erg4Δ);

[0064] Example 2: Construction of 24-epiergosterol-producing Saccharomyces cerevisiae strain YQE224

[0065] Using the ArDWF1 plasmid containing sterol Δ24(28) reductase from Ajuga reptans synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. as a template, PCR amplification was performed using primers VArDWF1-F (SEQ ID NO.16) / VArDWF1-R (SEQ ID NO.17) to obtain the ArDWF1 fragment; the ArDWF1 fragment was connected to the linearized pRS42H plasmid by seamless cloning to obtain the gene expression plasmid pRS42H-ArDWF1; using the plasmid pRS42H-ArDWF1 as a template, the primers Dor.-16Ty1-P TEF1 (SEQ ID NO.14) / Dor.-16Ty1-P ADH2(SEQ ID NO.15) was amplified by PCR to obtain the ArDWF1 expression cassette; the ArDWF1 expression cassette was transformed into YQE102 by lithium acetate / PEG3350 chemical transformation method; the CRISPR-Cas9 gene editing tool was used to perform double-strand break DNA cleavage at the 16Ty1 site under the guidance of 16Ty1-gRNA (SEQ ID NO.60), and YQE224 producing 24-epiergosterol was constructed. The 24-epiergosterol production reached 13.97 mg / L through YPD shake flask fermentation verification and liquid phase detection;

[0066] Example 3: Establishment of a high-throughput screening method for the modification of the catalytic activity of ArDWF1

[0067] After knocking out the ERG4 gene, yeast cells, such as YQE101 and YQE102, will accumulate 24(28)-dehydroergosterol as the final product of the sterol synthesis pathway due to the lack of ergosterol double bond reductase. Due to the lack of ergosterol, yeast cells will show certain growth defects in the growth environment, which is manifested as reduced tolerance to antibiotics, metal ions and SDS. Compared with ergosterol, 24-epiergosterol has only a difference in 24-C chirality in structure. Therefore, the growth condition can be coupled with the concentration of 24-epiergosterol according to the degree of reduced tolerance as a high-throughput screening method. To ensure the sensitivity and accuracy of the screening process, the factors that may affect the experiment are first optimized.

[0068] The first is the selection of strains. Since there are obvious differences in the growth of industrial strain CICC1746 and model strain BY4741 ( Figure 5 ), in order to facilitate the construction of the screening method, the ERG4 knockout strain YQE101 of the model bacteria BY4741 was selected as the host strain for high-level screening; secondly, in order to determine the optimal SDS screening concentration, the growth inhibition of strains producing different amounts of ergosterol / 24-epi-ergosterol (YQP1: BY4741 with plasmid pRS42H; YQP2: YQE101 with plasmid pRS42H; YQP3: YQE101 with plasmid pRS42H-AtDWF1, derived from Arabidopsis thaliana; YQP4: YQE101 with plasmid pRS42H-ArDWF1) under different SDS concentrations was determined. Figure 6 and Figure 7 As shown, 100 μg / mL HygB + 0.025% SDS in YPD was selected as the high screening condition.

[0069] Example 4: Construction and high-throughput screening of mutant libraries

[0070] Using the ArDWF1 coding gene as a template, primers Ep-ArDWF1-F (SEQ ID NO.18) / Ep-ArDWF1-R (SEQ ID NO.19) were used to generate the 5-mercaptoethanol in 0.07 mM Mn 2+ Error-prone PCR was performed under the conditions of RT-PCR to construct a mutant library of ArDWF1; at the same time, the plasmid pRS42H was linearized using restriction endonucleases Hind III and BamH I, and the ArDWF1 mutant library and the linearized pRS42H were co-transformed into YQE101 at a mol ratio of 6:1 by a lithium acetate / PEG3350 chemical transformation method. The homologous recombinase of the ERG4 knockout strain YQE101 connected the ArDWF1 mutant library and the linearized pRS42H into a complete plasmid, and the positive clones were screened on a plate containing 100 μg / mL hygromycin B (Hyg B) and 0.01% sodium dodecyl sulfate (SDS) to obtain a Saccharomyces cerevisiae strain with a successful reconstruction of the ArDWF1 mutant plasmid; the Saccharomyces cerevisiae strain carrying the successfully reconstructed ArDWF1 mutant plasmid was picked up to 2 mL of a plate containing 100 μg / mL Hyg B and 0.025% SDS. YPD (glucose 2% w / v, yeast extract 1% w / v, peptone 2% w / v)) 24-well plate, culture for 3 days; pick the bacterial concentration (OD 600 ) were inoculated into 5 mL YPD test tubes containing 100 μg / mL Hyg B, cultured for 4 days, and then screened in liquid phase. Because the mutants were expressed in the model strain as free plasmids, antibiotics needed to be added to the culture medium to maintain the plasmids, which would have a certain effect on the growth of the bacteria. Therefore, the ratio of 24-epiergosterol to 24(28)-dehydroergosterol was defined as the relative DWF1 activity and used as the main comparison object. Figure 8 As shown, four mutants (ArV143G, ArM235T, ArS306P, ArY338H) were finally obtained whose relative DWF1 activities were significantly improved compared with the wild-type ArDWF1.

[0071] Example 5: Combination mutations of ArDWF1

[0072] Using the recombinant plasmid pRS42H-ArDWF1 as a template, the four mutation points V143G, M235T, S306P, and Y338H, which had significantly improved the relative DWF1 activity compared with the wild-type ArDWF1, were subjected to combined mutation; the primers corresponding to V143G were Ar-V143G-F (SEQ ID NO.20) / Ar-V143G-R (SEQ ID NO.21), the primers corresponding to M235T were Ar-M235T-F (SEQ ID NO.22) / Ar-M235T-R (SEQ ID NO.23), the primers corresponding to S306P were Ar-S306P-F (SEQ ID NO.24) / Ar-S306P-R (SEQ ID NO.25), and the primers corresponding to Y338H were Ar-Y338H-F (SEQ ID NO.26) / Ar-Y338H-R (SEQ ID NO.27). NO.27); using the combined mutated plasmid as a template, the primer Dor.-16Ty1-P TEF1 (SEQ ID NO.14) / Dor.-16Ty1-P ADH2 (SEQ ID NO.15) was amplified by PCR to obtain the ArDWF1 combined mutation expression cassette, which was transformed into the ERG4 knockout industrial yeast YQE102 by lithium acetate / PEG3350 chemical transformation method, and integrated into the genome with the help of CRISPR-cas9 gene editing tools to obtain strain YQE231. Fig. 9 As shown, the best combination mutant Ar207 (V143G / S306P / Y338H) was determined through YPD shake flask fermentation verification and liquid phase detection, and its 24-epiergosterol production reached 46.72 mg / L, which was 334.4% higher than that of the wild type ArDWF1.

[0073] Example 6: Fermentation, post-treatment and liquid phase detection

[0074] The successfully edited transformants were streaked on YPD plates and cultured at 30°C for 48 h. A single clone was picked and inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 96 h. After the culture was completed, 500 μL of fermentation broth was collected and centrifuged to remove the supernatant. 600 μL of ethanol-KOH solution (25% [w / v] KOH was added to 50% ethanol) was added. After sufficient vortexing, the solution was saponified in a boiling water bath for 1 h. The solution was cooled to room temperature, 400 μL of water and 800 μL of petroleum ether were added. After sufficient vortexing, 500 μL of the upper petroleum ether extract was vacuum-dried, 500 μL of anhydrous ethanol was added to re-dissolve, and the solution was filtered through a 0.22 μm PVDF filter membrane into the insert tube in the liquid phase detection bottle as the detection sample. The metabolites in the samples were analyzed by Agilent 1160 high performance liquid chromatography. The chromatographic column model was Thermo C-18 column (ODS Hypersil, 4.6×250 mm, 5 μm), the detection wavelength was 280 nm, the analytical column was maintained at a constant 30°C, the mobile phase was: methanol: acetonitrile = 80:20, and the solution prepared with ergosterol standard was used to prepare the standard curve for quantitative analysis.

[0075] Example 7: A method for constructing ARE2, YEH1, and YEH2 overexpression strains

[0076] The plasmid pEB-Y1A2Y2 used to construct the ARE2, YEH1, and YEH2 overexpression strains was constructed as follows:

[0077] Using plasmid pRS42H as template and primers pEB-T ADH2 -R (SEQ ID NO. 28) and pEB-P TEF1 -F (SEQ ID NO.29) was amplified to obtain P TEF1 -HindIII-BamHI-T ADH2 fragment; using the genome of Saccharomyces cerevisiae CICC1746 as template, primers pEB-P TDH3 -F (SEQ ID NO.30) and pEB-P TDH3 -R (SEQ ID NO.31) amplified to obtain P TDH3 -XhoI-AflII fragment, using primer pEB-T CYC1 -F (SEQ ID NO.32) and pEB-T CYC1 -R (SEQ ID NO.33) amplified to obtain XhoI-AflII-T CYC1 fragment, using primer pEB-P FBA1 -F (SEQ ID NO.34) and pEB-P FBA1 -R (SEQ ID NO.35) amplified to obtain P FBA1-EcoRI-BlnI fragment, using primer pEB-T PGK1 -F (SEQ ID NO.36) and pEB-T PGK1 -R (SEQ ID NO.64) amplified to obtain EcoRI-BlnI-T PGK1 Fragments with homologous sequences were fused using overlap extension PCR and -Blunt Simple Cloning Vectors Kit, construct pEB

[0078] The YEH1 fragment was amplified using the genome of Saccharomyces cerevisiae CICC1746 as a template using primers VYEH1-F (SEQ ID NO.37) and VYEH1-R (SEQ ID NO.38). TDH3 -ARE2 (SEQ ID NO. 39) and ARE2-T CYC1 (SEQ ID NO.40) was amplified to obtain the ARE2 fragment, and primer P PBA1 -YEH2 (SEQ ID NO. 41) and YEH2-T PGK1 The YEH2 fragment (SEQ ID NO.42) was amplified and then recombined by enzyme digestion to construct pEB-Y1A2Y2.

[0079] The specific steps for constructing ARE2, YEH1, and YEH2 overexpression strains are as follows:

[0080] (1) Using primer Dor.-16Ty3-1-T with a 50 bp homology arm ADH2 (SEQ ID NO.43) / Dor.-16Ty3-1-T PGK1 (SEQ ID NO.44) was amplified by PCR to obtain the expression cassettes of ARE2, YEH1, and YEH2;

[0081] (2) transforming the ARE2, YEH1, and YEH2 expression cassettes into Saccharomyces cerevisiae strains YQE231 and YQE102 using a lithium acetate / PEG3350 chemical transformation method;

[0082] (3) Using the CRISPR-Cas9 gene editing tool, double-strand breaks were made at the 16Ty3 site under the guidance of 16Ty3-gRNA (SEQ ID NO. 61), and the DNA was integrated into the Saccharomyces cerevisiae genome. The HygB / G418 plates were coated and positive clones were screened.

[0083] (4) Single colonies were selected from the plates, cultured overnight in shake flasks, and the genome was extracted. PCR was performed to verify whether the ARE2, YEH1, and YEH2 expression cassettes were integrated into the 16Ty3 site on the genome.

[0084] (5) Based on the PCR verification results, positive clones were screened and YQE717 (YQE231; 16Ty3::T ADH2 -YEH1-P TEF1 -P TDH3 -ARE2-T CYC1 -P FBA1 -YEH2-T PGK1 ) and YQE103 (YQE102; 16Ty3::T ADH2 -YEH1-P TEF1 -P TDH3 -ARE2-T CYC1 -P FBA1 -YEH2-T PGK1 ).

[0085] (6) Through fermentation, post-processing and liquid phase detection, such as Fig.14 As shown, YQE717 had a significant increase in total sterol production and 24-epiergosterol production. At the shake flask level, the total sterol production and 24-epiergosterol production reached 220.07 mg / L and 71.04 mg / L, respectively, which were 2.09 times and 1.53 times that of strain YQE231, respectively.

[0086] Example 8: Determination of transcription levels of ARE2, YEH1 and YEH2.

[0087] The starting strain (YQE231) and the overexpression strain (YQE717) were cultured in liquid YPD medium. During the shake flask fermentation process, 1 mL of the fermentation broth was taken at 12 hours, 24 hours, 36 hours, 48 ​​hours and 72 hours respectively.

[0088] The total RNA of each sample was extracted using the RNA extraction kit TRIzol (Invitrogen). The genomic DNA was removed and reverse transcribed to obtain cDNA using a reverse transcription kit (TOYOBO). The experimental operation was carried out according to the kit instructions.

[0089] Using ACT1 gene as internal reference, real-time fluorescence quantitative PCR was performed on ARE2, YEH1, and YEH2 to obtain amplification curves and record CT values. -△△CT The relative transcription levels of ARE2, YEH1, and YEH2 were calculated.

[0090] Example 9: Strains replacing the promoters of ACC1 and ArDWF1 mutant Ar207.

[0091] Plasmid pRS42H-P for replacing the promoter of ACC1 and ArDWF1 mutant Ar207 GAL1 -Ar207, the steps are as follows:

[0092] Using pRS42H-Ar207 as a template, primers R-pRS42H-207-F (SEQ ID NO.45) and R-pRS42H-207-R (SEQ ID NO.46) were used to reversely amplify the promoter-removed fragment. Using the CICC1746 genome as a template, primers P GAL10-GAL1 -F (SEQ ID NO.47) and P GAL10-GAL1 -R (SEQ ID NO.48) amplified to obtain P GAL10 -P GAL1 By recombination, pRS42H-P GAL1 -Ar207.

[0093] The construction of the strain replacing the promoter of ACC1 and the ArDWF1 mutant Ar207 is as follows:

[0094] (1) Using primer Dor.-P with 50 bp homology arm ACC1 -P GAL10 (SEQ ID NO.49) / Dor.-P ACC1 -T ADH2 (SEQ ID NO.50) was amplified by PCR to obtain P GAL10 -P GAL1 -Ar207 expression cassette;

[0095] (2) and convert the above P to GAL10 -P GAL1 -Transformation of Saccharomyces cerevisiae strain YQE103 with the Ar207 expression cassette;

[0096] (3) Using CRISPR-Cas9 gene editing tools, ACC1 -gRNA (SEQ ID NO.62) to the ACC1 promoter P ACC1 The DNA was cut by double-strand break at the locus and integrated into the Saccharomyces cerevisiae genome. The HygB / G418 plates were coated and positive clones were screened.

[0097] (4) Single colonies were selected from the plates, cultured overnight in shake flasks, and the genome was extracted and PCR was performed to verify P GAL10 -P GAL1 - Whether the Ar207 expression cassette is integrated into the ACC1 promoter on the genome ACC1 Location.

[0098] (5) Based on the PCR verification results, positive clones were screened and YQE729 was constructed.

[0099] (6) Through fermentation, post-processing and liquid phase detection, such as Fig.14 As shown, YQE729 had a significant improvement in 24-epiergosterol production. At the shake flask level, 24-epiergosterol reached 160.84 mg / L, which was 2.26 times that of YQE717.

[0100] Example 10: A method for constructing an ERG5 overexpression strain

[0101] Plasmid pRS42H-P used to construct ERG5 overexpression strain CIT2 -ERG5, the steps are as follows:

[0102] Using pRS42H-Ar207 as template, primers R-pRS42H-207-F and R-pRS42H-207-R were used to reversely amplify the promoter-removed fragment. Using the CICC1746 genome as template, primers P CIT2 -F (SEQ ID NO.51) and P CIT2 -R (SEQ ID NO.52) amplified to obtain P CIT2 By recombination, pRS42H-P CIT2 -Ar207.

[0103] pRS42H-P CIT2 -Ar207 as template, using primer RP CIT2 -R (SEQ ID NO.53) and RT ADH2 -F (SEQ ID NO.54) was reversely amplified to obtain the backbone fragment, and the CICC1746 genome was used as a template to amplify the ERG5 fragment using primers ERG5-F (SEQ ID NO.55) and ERG5-R (SEQ ID NO.56). By recombination, pRS42H-P was constructed. CIT2 -ERG5

[0104] The specific method for constructing the ERG5 overexpression strain is as follows:

[0105] (1) The ERG5 expression cassette was obtained by PCR amplification using primers Dor.-15Ty2-F (SEQ ID NO.57) / Dor.-15Ty2-R (SEQ ID NO.58) with 50 bp homology arms;

[0106] (2) transforming the ERG5 expression cassette into Saccharomyces cerevisiae strain by lithium acetate / PEG3350 chemical transformation method;

[0107] (3) Using the CRISPR-Cas9 gene editing tool, 15Ty2 was double-stranded and cut under the guidance of 15Ty2-gRNA (SEQ ID NO.63), integrated into the Saccharomyces cerevisiae YQE729 genome, coated on HygB / G418 plates, and positive clones were screened.

[0108] (4) Single colonies were selected from the plates, cultured overnight in shake flasks, and the genome was extracted. PCR was performed to verify whether the ERG5 expression cassette was integrated into the 15Ty2 site on the genome.

[0109] (5) Based on the PCR verification results, positive clones were screened and YQE734 was constructed.

[0110] (6) Through fermentation, post-processing and liquid phase detection, such as Fig.14 As shown, YQE734 significantly increased the proportion of 24-epiergosterol in total sterols. At the shake flask level, the proportion of 24-epiergosterol increased to 75.44% based on the yield of 171.31 mg / L, which was 1.27 times that of YQE729.

[0111] Example 11: Fermentation verification of modified strain fermenter

[0112] A single colony was inoculated into 5 mL YPD medium and cultured at 30°C and 220 rpm for 24 h. The culture was then inoculated into two 50 mL YPD shake flasks at a 2% inoculum (v / v). After 20 h of culture, the culture fluid in the two shake flasks was inoculated into a 2 L bioreactor (the fermentation medium formula was 10 g / L D-glucose, 10 g / L (NH 4 ) 2 SO 4 , 8g / L KH 2 PO 4 、3g / L MgSO 4 、0.72g / LZnSO 4 7H 2 O, 10mL / L trace metal solution and 12mL / L vitamin solution, the initial fermentation volume after inoculation is 1L).

[0113] Fermentation tank parameter setting: temperature was set to 30°C, ammonia was added to control pH to maintain at 5.0, and dissolved oxygen was maintained at >25% saturation by adjusting the stirring rate (300 rpm to 950 rpm) and airflow rate (1 vvm to 3 vvm). After the carbon source in the initial culture medium was used up, a feed solution containing 500 g / L glucose and 12 mL / L vitamin solution was fed into the fermentation tank according to the pseudo-exponential feeding model; when the bacterial OD in the tank reached 600 When the fermentation temperature reaches about 200, anhydrous ethanol is added at a rate of 6 mL / h until the fermentation ends. The feed rate F in the pseudo-exponential feeding stage S Determined by the following equation:

[0114]

[0115] In the formula, X 0 、V 0 and S are the initial biomass density (gDCW / L), initial culture volume (L), and glucose concentration in the culture medium (g / L); Y X / S is the productivity of cell biomass to glucose (gDCW / g glucose); μ is the specific growth rate (h -1 ); m is the maintenance factor (g glucose / gDCW / h), and t is the time after the start of feeding (h). The specific growth rate was set to 0.12h -1 , Y X / S Set to 0.5 and m to 0.05.

[0116] After fermentation, HPLC was used to detect the 24-epiergosterol production of strain YQE734. The test results showed that ( Fig.15 ), the Ar207 strain YQE734 with combined mutations produced 2.76 g / L of 24-epiergosterol in a 2L fermenter, which was 3.28 times higher than that of the starting strain, laying a good foundation for the industrial production of 24-epiergosterol by this strain.

[0117] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for synthesizing 24-epiergosterol by using a fungus for synthesizing 24-epiergosterol, wherein the fungus for synthesizing 24-epiergosterol is synthesized by converting sterol Δ 24(28) The V143G, M235T, S306P, Y338H single point mutants and combined mutants of the reductase DWF1 gene were recombinantly expressed to produce ergosterol and knocked out ERG4 The gene is constructed in a yeast microorganism; the DWF1 gene is a gene encoding the amino acid sequence shown in SEQ ID NO. 1; the DWF1 mutant gene is a gene encoding any of the following amino acid sequences: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.

6.

2. A method for constructing a fungus that synthesizes 24-epiergosterol, characterized in that: Sterol Δ 24(28) The V143G, M235T, S306P, Y338H single point mutants and combined mutants of the reductase DWF1 gene were recombinantly expressed to produce ergosterol and knocked out ERG4 A strain synthesizing 24-epiergosterol is constructed in a yeast microorganism of the family of the DWF1 gene; the DWF1 gene is a gene encoding an amino acid sequence as shown in SEQ ID NO. 1; the DWF1 mutant gene is a gene encoding any of the following amino acid sequences: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6; The obtained strain was further edited by CRISPR / Cas9 technology and integrated into the 16Ty3 site of the genome. ARE2 , YEH1 , YEH2 Expression cassette sequence SEQ ID NO. 7, thereby overexpressing ARE2 , YEH1 , YEH2 Gene, enhancing the balance between esterification storage and hydrolysis release of sterols.

3. The construction method according to claim 2, characterized in that: The method for obtaining the DWF1 mutant gene comprises the following steps: Plant-derived sterols 24(28) The reductase DWF1 gene was used as a template and the concentration of 0.02-0.12 mM Mn 2+ Error-prone PCR was performed to construct a DWF1 mutant library; The DWF1 mutant library and the linearized plasmid pRS42H were co-transformed into the ERG4 knockout ergosterol-producing fungal strain, and spread on a solid medium containing 50-200 μg / mL Hyg B; the growing strains were picked and cultured in a medium containing 50-200 μg / mL Hyg B and an inhibitor of the growth of the ERG4 knockout strain; The growing strains were selected and inoculated into a culture medium containing 50-200 μg / mL Hyg B for culture. The cultured bacteria were subjected to saponification treatment, extraction and liquid phase analysis to screen out strains with improved enzyme activity. The plasmids of the strains with improved enzyme activity were extracted and sequenced to obtain the DWF1 mutant sequence with improved catalytic activity.

4. The construction method according to claim 2, characterized in that: The sterol Δ 24(28) The reductase DWF1 gene comes from plants that produce brassinolide compounds.

5. The construction method according to claim 4, characterized in that: The sterol Δ 24(28) The reductase DWF1 gene comes from plants of Lamiaceae, Cruciferae and Moraceae.

6. The construction method according to claim 5, characterized in that: The sterol Δ 24(28) The reductase DWF1 gene is derived from Ajuga prostrata ( Ajuga reptans , Ar).

7. The construction method according to claim 3, characterized in that: The ERG4 knockout strain growth inhibitor is one or more of 0.01wt%-0.05wt% sodium dodecyl sulfate, 2 μg / mL-20 μg / mL nystatin, and 10 μg / mL-100 μg / mL fluconazole.

8. The construction method according to claim 2, characterized in that: The construction method further comprises: the strain obtained by the method of claim 2 is edited by CRISPR / Cas9 technology. ACC1 SEQ ID NO. 8 was recombined and integrated into the promoter of the gene to replace the ACC1 gene promoter, overexpress the mutant gene of the DWF1 gene, and the constructed mutant gene was integrated into the 15Ty2 site through CRISPR / Cas9 editing technology. ERG5 Expression cassette sequence SEQ ID NO. 9, overexpression ERG5 Gene, thereby enhancing the synthesis of 24-epiergosterol and increasing the proportion of 24-epiergosterol in sterols.

9. A fungus for synthesizing 24-epiergosterol, characterized in that: The strain is constructed by the construction method described in any one of claims 2-8.

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