Recombinant saccharomyces cerevisiae for efficient synthesis of ergosterol and application thereof

Abstract

The application discloses a recombinant saccharomyces cerevisiae for efficiently synthesizing ergosterol and application thereof, and belongs to the technical field of genetic engineering and bioengineering. The application improves the synthesis level of the engineering strain ergosterol by overexpressing the key enzyme of the MVA pathway, regulating the expression and transcription factor of other enzyme genes of the pathway, and simultaneously regulating the modular expression of the ERGs enzyme gene of the squalene post-pathway and the transcription factor knockout. TOR1 and FOB1 In addition, the growth ability of the engineering strain is improved by regulating the life-span regulating factor of the saccharomyces cerevisiae engineering strain, Finally, the D013-5 strain with the ergosterol yield of 1007.52 mg / L is obtained by back-supplementing the defective type label and overexpressing the key precursor synthesis enzyme gene. The application provides a theoretical and technical reference for the metabolic engineering modification of saccharomyces cerevisiae for synthesizing long-pathway and multi-modification macromolecular lipid-soluble compounds.

Description

Technical Field

[0001] This invention relates to a recombinant brewing yeast for the efficient synthesis of ergosterol and its applications, belonging to the fields of genetic engineering and bioengineering technology. Background Technology

[0002] Utilizing engineered microorganisms to produce valuable chemical products is an attractive alternative to chemical synthesis or extraction of these compounds from plants and animals. Ergosterol is a major component of the cell membrane structure in eukaryotic organisms. In yeast, ergosterol is the main sterol responsible for structural features such as cell membrane fluidity, integrity, and permeability. Ergosterol is a provitamin of ergocalciferol and possesses antioxidant and anti-inflammatory properties. Under ultraviolet irradiation, it can be converted into vitamin D2, which can be used to treat rickets, osteoporosis, and tetany. In summary, ergosterol is a product of significant industrial value. Current reports indicate that in *Saccharomyces cerevisiae*, remodeling the Erg1p (squalene epoxidase) substrate channel significantly enhances the catalytic activity of squalene epoxidase, thereby significantly increasing the yield of the final product, ergosterol. V249H / L343A The mutant strain yielded 600 mg / L of ergosterol in shake flasks.

[0003] Traditional chemical synthesis methods have high requirements for reaction conditions, produce many byproducts, have low product yields, and cause serious pollution. Therefore, biosynthesis based on metabolic engineering and genetic engineering has a clear advantage in producing ergosterol. While breakthroughs have been achieved in synthesizing heterologous high-value compounds from Saccharomyces cerevisiae, the synthesis of macromolecular compounds, especially triterpenoids and steroidal compounds, involves long biosynthetic pathways. In unmodified engineered strains, the carbon flux is primarily used for the strain's growth, severely limiting further yield increases and failing to meet industrial-scale requirements.

[0004] Currently, measures to increase the yield of lipid-soluble compounds mainly include: engineering key enzymes, constructing organelle compartments, and modifying overall metabolic pathways (strengthening synthetic pathways, enhancing precursor pathways, and weakening competitive pathways). In yeast, many lipid-soluble compounds are stored in lipid droplets after synthesis. Therefore, many studies have focused on modifying and localizing lipid droplets to improve the synthesis level of exogenous lipid-soluble compounds. However, although ergosterol is a lipid-soluble compound, as an important component of the cell membrane, it is mainly stored within the cell membrane structure. Therefore, engineered strains that efficiently synthesize ergosterol exhibit a decline and death after 72 hours of fermentation, followed by a rapid decrease in ergosterol yield. Based on the problems observed in these studies, it is clear that the biosynthesis of ergosterol cannot be simply improved by controlling the storage space of lipid droplets. This invention addresses this by regulating the growth capacity of engineered strains, extending their growth cycle, and thus increasing the time for ergosterol accumulation during fermentation in engineered Saccharomyces cerevisiae strains, thereby increasing the yield of the target product, ergosterol. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention utilizes metabolic engineering and gene editing technologies to precisely regulate the expression of squalene synthesis pathway enzyme genes in engineered Saccharomyces cerevisiae strains, thereby balancing their growth and production processes and promoting the efficient synthesis of squalene, a precursor of ergosterol. Growth regulation extends the lifespan of the engineered strain, alleviating growth delays caused by the accumulation of the target product. Furthermore, it regulates the carbon flux and enzyme gene expression of the engineered strain, ultimately constructing an engineered strain capable of efficiently biosynthesizing ergosterol.

[0006] The first technical solution provided by this invention is a recombinant brewing yeast for efficient synthesis of ergosterol, wherein the recombinant yeast uses brewing yeast C800 as the chassis strain and integrates into the chassis strain. tHMG1 Gene, IDI1 Gene, ERG10 Gene, ERG13 Gene, ERG12 Gene, ERG8 Gene, ERG19 Gene, ZWF1 Gene, GND1 Gene, TKL1 Gene, TAL2 Gene, YHM2 Gene, CTP1 Gene, MPC1 Gene, MPC3 Gene, ERG1 Genes and ERG11 Gene.

[0007] In some embodiments, the tHMG1 Gene, IDI1 Gene, ERG10 Gene, ERG13 Gene, ERG12 Gene, ERG8 Gene, ERG19 Gene, ZWF1 Gene, GND1 Gene, TKL1 Gene, TAL2 Gene, YHM2 Gene, CTP1 Gene, MPC1 Gene, MPC3 Gene, ERG1 Genes and ERG11 The nucleotide sequences of the genes are shown in SEQ ID NO.1~17, respectively.

[0008] In some embodiments, the tHMG1 Genes and IDI1 Gene integration in Ty1 Site.

[0009] In some implementations, in YPL062W Site single copy integration ERG10 , ERG13 , ERG12 and ERG8 Gene.

[0010] In some implementations, in YJL064W Site single copy integration ERG19 Gene.

[0011] In some implementations, in YNR063W Site single copy integration ZWF1 , GND1 , TKL1 and TAL2 Gene.

[0012] In some implementations, in VBA5 Site single copy integration YHM2 , CTP1 , MPC1 and MPC3 Gene.

[0013] In some implementations, in ROX1 Site single copy integration ERG1 and ERG11 Gene.

[0014] In some embodiments, simultaneously with the strains in the chassis DOS2 Site single copy integration ERG2 and ERG7 Gene, ERG2 and ERG7 The nucleotide sequences of the genes are shown in SEQ ID NO.18~19.

[0015] In some embodiments, simultaneously with the strains in the chassis NEM1 Site single copy integration ERG3 , ERG4 , ERG5 and ERG6 Gene, ERG3 , ERG4 , ERG5 and ERG6 The nucleotide sequences of the genes are shown in SEQ ID NO.20~23, respectively.

[0016] In some embodiments, the chassis strain is also knocked out. TOR1 Gene (Gene ID: 853529) FOB1 Gene (Gene ID: 851688) and / or GLK1 Gene (Gene ID: 850317).

[0017] In some embodiments, the glucose phosphorylation key enzyme gene of the chassis strain is... HXK1 The expression is weakened.

[0018] Furthermore, by HXK1 The promoter of the gene is replaced with P ADH2 Key enzyme genes for glucose phosphorylation HXK1 Expression weakening, promoter P ADH2 The nucleotide sequence is shown in SEQ ID NO.24.

[0019] In some embodiments, rDNA is simultaneously integrated into the chassis strain. tHMG1 and IDI1 Gene.

[0020] The second technical solution provided by the present invention is a method for biosynthesizing ergosterol, wherein the method involves culturing the recombinant brewing yeast described in the first technical solution in a fermentation system with glucose as the carbon source to synthesize ergosterol.

[0021] In some embodiments, the culture temperature is 30°C and the culture time is not less than 72 hours.

[0022] The third technical solution provided by the present invention is the application of the recombinant brewing yeast described in the first technical solution or the method described in the second technical solution in the preparation of ergosterol or ergosterol-containing products.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses engineered brewer's yeast C800 (CEN.PK2-1D: Using gal80::G418 as a host, efficient heterologous biosynthesis of ergosterol was achieved. The C800 chassis strain (CENPK2-1D:) was preserved in the laboratory. Based on gal80::G418, this invention enhances the production of squalene, a precursor of ergosterol, by regulating the expression of endogenous MVA pathway enzyme genes in *Saccharomyces cerevisiae*. Furthermore, it strengthens the efficient accumulation of squalene by optimizing the regeneration of NADPH and central carbon metabolism in *Saccharomyces cerevisiae*. By regulating the expression of endogenous ERG pathway enzyme genes in *Saccharomyces cerevisiae*, it achieves more efficient carbon flux synthesis of ergosterol. Through a combination of modularization and key enzyme engineering, it improves ergosterol biosynthesis at the shake-flask level. By regulating the growth cycle and lifespan of *Saccharomyces cerevisiae*, it extends the product accumulation time of the engineered strain, resulting in an engineered strain that accumulates ergosterol for a longer period than currently reported. This invention applies multi-strategy gene engineering technology to the regulation of ergosterol-synthesizing strains, providing insights for the efficient biosynthesis of other steroidal compounds. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the MVA pathway for enhancing squalene synthesis in chassis strains.

[0025] Figure 2 This is a schematic diagram illustrating the enhanced ergosterol synthesis in engineered strains via the push-pull ERGs pathway.

[0026] Figure 3 A schematic diagram illustrating the regulation of the growth lifespan of engineered strains to enhance ergosterol synthesis.

[0027] Figure 4 A schematic diagram illustrating the regulation of glucose utilization capacity in engineered strains to enhance ergosterol synthesis.

[0028] Figure 5 A schematic diagram illustrating how the key precursor synthesis pathway of engineered strains enhances ergosterol synthesis. Detailed Implementation

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] (a) Culture medium LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride. Add 20 g / L agar powder to prepare LB solid medium.

[0031] 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, with appropriate amino acid deletions as needed).

[0032] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L.

[0033] (II) Preparation and transformation of competent cells of Saccharomyces cerevisiae (1) Select a single colony of Saccharomyces cerevisiae and inoculate it into 5 mL of YPD medium. Incubate it in a 50 mL shake flask at 30 °C and 220 rpm for about 17 h to obtain the Saccharomyces cerevisiae seed culture.

[0034] (2) Measurement of OD 600 Value, take an appropriate amount of the above seed culture and add it to 50 mL of fresh YPD medium, so that OD 600 It is approximately equal to 0.3.

[0035] (3) Incubate at 30℃ and 220 rpm until OD 600 Between 1.2 and 1.5.

[0036] (4) Transfer to a 50 mL centrifuge tube and place on ice for 5 min.

[0037] (5) Centrifuge at 3500 rpm and 4℃ for 5 min and discard the supernatant.

[0038] (6) Wash with 25 mL of pre-cooled sterile water, centrifuge at 3500 rpm and 4℃ for 5 min, and discard the supernatant.

[0039] (7) Add 1 mL of 0.1 M lithium acetate and resuspend the bacterial cells in a 1.5 mL EP tube.

[0040] (8) Centrifuge at 4℃ and 3500 rpm for 2.5 min and discard the supernatant.

[0041] (9) Resuspend the bacterial cells in 100-400 µL of pre-cooled 0.1 M lithium acetate.

[0042] (10) Centrifuge at 3500 rpm for 2 min, discard the supernatant, and add 24 µL of 50% PEG3350 (m / V), 36 µL of 1 M lithium acetate, 25 µL of ssDNA (concentration of 2 mg / mL) and 50 µL of DNA fragment in sequence.

[0043] (11) Violent oscillation for 10 s.

[0044] (12) Incubate at 30℃ for 50 min.

[0045] (13) In a water bath at 42℃ for 20 min.

[0046] (14) Centrifuge at 3500 rpm for 2.5 min and discard the supernatant.

[0047] (15) Resuspend the bacterial cells in 500-600 µL of ddH2O.

[0048] (16) Spread 50-80 µL of bacterial culture onto the corresponding YNB medium and incubate at 30℃ for 3-5 days.

[0049] (III) Shake-flask fermentation of ergosterol Shake-flask fermentation conditions: Single colonies were taken and cultured in 5 mL YPD medium in a 50 mL shake flask for 20-24 h at 30℃ and 220 rpm as the seed solution. The fermentation experiment used a 1% seed solution, cultured in 250 mL shake flasks with 25 mL YPD medium at 30℃ for 72 h. The fermentation time of the engineered strain after lifespan regulation was extended to 168 h, and the synthesis level of ergosterol was detected by sampling at different fermentation times; when the engineered strain after lifespan regulation was further modified for fermentation verification, the fermentation time was extended to 120 h.

[0050] (iv) Extraction and detection of ergosterol Sample processing method: After fermentation, the OD of the fermentation broth was measured. 600 First, prepare a 30% (w / w) potassium hydroxide-ethanol saponification solution (ethanol solvent purity 90%). Place the washed bacterial cells into a 2 mL saponification tube, add an appropriate amount of saponification solution, and saponify in an 80℃ water bath for 4 hours, shaking several times during the process. After saponification is complete, cool to room temperature, pour into a clean 5 mL centrifuge tube, add 1 mL of diethyl ether and 1 mL of deionized water, shake vigorously for 2 minutes, allow to stand for separation, and add the upper organic phase to a sample vial.

[0051] High performance liquid chromatography: Detector: UV detector; Column: Diamonsil C18 (5 μm, 250 mm × 4.6 mm); Mobile phase: pure methanol; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 210 nm.

[0052] (v) Detection of squalene Squalene extraction: Collect 500 μL of fermentation broth cells into a 2 mL grinding and disruption tube. Add 1.5 mL of acetone and 0.5 mm glass beads to the disruption tube, and tighten the cap. Place the tube in a homogenizer and perform four cycles of agitation. Centrifuge at 12000 rpm for 5 min to separate the cell fragments, glass beads, and acetone into layers. Filter a suitable amount of the supernatant through a syringe filter and transfer it to a HPLC vial for analysis by high-performance liquid chromatography (HPLC).

[0053] The liquid chromatography instrument used was a Waters UPLC with a UV detector. A C18 column (5 μm, 4.6 × 250 mm, Thermo Fisher Scientific, MA, USA) was used. Chromatographic conditions were: isocratic elution, phase B was acetonitrile (0.1% trifluoroacetic acid); detection wavelength 195 nm; column temperature 40 °C; 15 min.

[0054] (vi) The strain information is shown in Table 1.

[0055] Table 1. Strains involved in this invention

[0056] Example 1: Construction of Chassis Strains for High Squalene Production through Multi-Strategy Modification (1) Construction of strain D001 In chassis strain C800 Ty1 Site multicopy integration tHMG1 and IDI1 (The nucleotide sequence is shown in SEQ ID NO. 1~2). Using the Saccharomyces cerevisiae genome as a template, the gene was amplified using primers Z01-tHMG1-F1 and Z01-tHMG1-R1. tHMG1 Primers Z01-Gal10-F1 and Z01-Gal10-R1 amplified the bidirectional promoter P. GAL10 / 1 The gene was amplified using primers Z01-IDI1-F1 and Z01-IDI1-R1. IDI1 Using Pct125 (disclosed in patent publication number CN113403334A) as a template, the Pct125 vector integration fragment was amplified using primers Z01-KJ-R1 and Z01-KJ-F1, and the bidirectional promoter P was integrated into the fragment. GAL10 / 1 ,Gene tHMG1 ,Gene IDI1 The Pct125 vector integration fragment was used to construct plasmid Z001 using Gibson assembly.

[0057] Using plasmid Z001 as a template, the Ty1armup-tHMG1-PGAL10 / 1-IDI1-HISdeg-Ty1armdown integrated fragment was amplified using primers Z01-F and Z01-R. 5 μg of the integrated fragment Ty1armup-tHMG1-PGAL10 / 1-IDI1-HISdeg-Ty1armdown was transformed into the Saccharomyces cerevisiae engineered strain C800 using a high-efficiency transformation method. The transformed fragment was plated on YNB solid medium for screening for histidine deficiency and incubated at 30°C for 3 days. 5 days, until colonies appear.

[0058] like Figure 1 As shown, a series of single colonies were selected and fermented in YPD shake flasks for 96 h, and strain D001-5 with a squalene yield of 472.87 mg / L was finally obtained.

[0059] (2) Construction of strain D002 In D001 YPL062W Site single copy integration ERG10 , ERG13 , ERG12 and ERG8 (The nucleotide sequence is shown in SEQ ID NO. 3~6). Using the Saccharomyces cerevisiae genome as a template, the following sequences were amplified using primers 002-erg10-f1 and 002-erg10-r1: ERG10 Amplification was performed using primers 002-erg13-f1 and 002-erg13-r1. ERG13 Amplification was performed using primers 002-erg12-f1 and 002-erg12-r1. ERG12 Amplification was achieved using primers 002-erg8-f1 and 002-erg8-r1. ERG8 Amplification was achieved using primers 002-ypup-f1 and 002-ypup-r1. YPL062W The upstream homologous arm of the site is 500 bp; it was amplified using primers 002-ypdn-f1 and 002-ypdn-r1. YPL062W The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers 002-pgk-f1 and 002-pgk-r1. PGK1 Terminator T was amplified using primers 002-ter22-f1 and 002-ter22-r1. Ter22 Terminator T was amplified using primers 002-ttef-f1 and 002-ttef-r1. TEF The bidirectional promoter P was amplified using primers 002-gal1-10-f1 and 002-gal1-10-r1. GAL10 / 1The bidirectional promoter P was amplified using primers 002-ptdh1-f1 and 002-ptdh1-r1. PTDH1 Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers 002-kj-f1 and 002-kj-r1. ERG10 , ERG13 , ERG12, ERG8, YPL062W 500 bp upstream homology arm of the site YPL062W 500 bp downstream homologous arm of the site, promoter P PGK1 Termination of sub-T Ter22 Termination of sub-T TEF P GAL10 / 1 Bidirectional promoter P PTDH1 The pY26 vector fragment was used to construct plasmid Z002 using Gibson assembly.

[0060] Using plasmid Z002 as a template, the integrated fragment YPL062Warmup-P was amplified using primers Z002-F1 and Z002-R1. PGK1 -ERG10-T ter22 -ERG13-P GAL10 / 1 -ERG12-T tef -ERG8-P TDH1 -YPL062Warmdown, use 500-700ng to knock it out. YPL062W The plasmid (20nt-tgatgaggtctcctcttgcc) and 5 μg of the integrated fragment YPL062Warmup-P PGK1 -ERG10-T ter22 -ERG13-P GAL10 / 1 -ERG12-T tef -ERG8-P TDH1 YPL062Warmdown was transformed into the Saccharomyces cerevisiae engineered strain D001-5 in step (1) using a high-efficiency transformation method. It was then plated on YNB solid medium for screening for uracil defects and cultured at 30°C for 3 days. Five days, until colonies appear, then the correctly verified clone is named D002. Figure 1 As shown, the squalene yield of YPD after 96 h of shake-flask fermentation was 610.56 mg / L.

[0061] (3) Construction of strain D003 In D002 YJL064W Site single copy integration ERG19 (The nucleotide sequence is shown in SEQ ID NO.7). Using the Saccharomyces cerevisiae genome as a template, the nucleotide sequence was amplified using primers 003-erg19-f1 and 003-erg19-r1. ERG19Amplification was achieved using primers 003-yjup-f1 and 003-yjup-r1. YJL064W The upstream homologous arm of the site is 500 bp; it was amplified using primers 003-yjdn-f1 and 003-yjdn-r1. YJL064W The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers 002-ptef-f1 and 002-ptef-r1. TEF1 Terminator T was amplified using primers 003-ter22-f1 and 003-ter22-r1. Ter22 The pY26 vector fragment was amplified using primers 003-kj-f1 and 003-kj-r1. ERG19 , YJL064W 500 bp upstream homology arm of the site YJL064W 500 bp downstream homologous arm of the site, promoter P TEF1 Termination of sub-T Ter22 The pY26 vector fragment was used to construct plasmid Z003 using Gibson assembly.

[0062] Using plasmid Z003 as a template, the integrated fragment YJL064Warmup-P was amplified using primers Z003-F1 and Z003-R1. TEF1 -ERG19-T ter22 -YJL064Warmdown, use 500-700ng to knock it out. YJL064W The plasmid (20nt-gaattctgtagcaaacgctg) and 5 μg of the integrated fragment YJL064Warmup-P TEF1 -ERG19-T ter22 YJL064Warmdown was transformed into the engineered Saccharomyces cerevisiae strain D002 in step (2) using a high-efficiency transformation method. The transformed strain was then plated onto YNB solid medium for screening for uracil defects and cultured at 30°C for 3 days. Five days, until colonies appear, then the correctly verified clone is named D003. Figure 1 As shown, the squalene yield of YPD after 96 h of shake-flask fermentation was 790.49 mg / L.

[0063] (4) Construction of strain D004 In D003 YNR063W Site single copy integration ZWF1 , GND1 , TKL1 and TAL2 (The nucleotide sequence is shown in SEQ ID NO. 8~11). Using the Saccharomyces cerevisiae genome as a template, the following sequences were amplified using primers 09-ZWF1-F1 and 09-ZWF1-R1: ZWF1Amplification was performed using primers 09-GND1-F1 and 09-GND1-R1. GND1 Amplification was performed using primers 09-TKL1-F1 and 09-TKL1-R1. TKL1 Amplification was achieved using primers 09-TAL1-F1 and 09-TAL1-R1. TAL1 Amplification was achieved using primers 09-YNR-UP-F1 and 09-YNR-UP-R1. YNR063W The upstream homologous arm of the site is 500 bp; it was amplified using primers 09-YNR-DOWN-F1 and 09-YNR-DOWN-R1. YNR063W The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers 09-Ppgk-F1 and 09-Ppgk-R1. PGK1 The terminator T was amplified using primers 09-Tter22-F1 and 09-Tter22-R1. Ter22 Terminator T was amplified using primers 09-Ttef-F1 and 09-Ttef-R1. TEF The bidirectional promoter P was amplified using primers 09-gal1-10-F1 and 09-gal1-10-R1. GAL10 / 1 The bidirectional promoter P was amplified using primers 09-Ptdh1-F1 and 09-Ptdh1-R1. PTDH1 Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers 09-KJ-F1 and 09-KJ-R1. ZWF1 , GND1 , TKL1 , TAL2 YNR063W 500 bp upstream homology arm of the site YNR063W 500 bp downstream homologous arm of the site, promoter P PGK1 Termination of sub-T Ter22 Termination of sub-T TEF P GAL10 / 1 Bidirectional promoter P PTDH1 The plasmid Z004 was constructed by Gibson assembly of the pY26 vector fragment and the pY26 vector fragment.

[0064] Using plasmid Z004 as a template, the integration fragment YNR063Warmup-P was amplified using primers Z9-F and Z9-R. PGK1 -ZWF1-T ter22 -GND1-P GAL10 / 1 -TKL1-T tef -TAL2-P TDH1 -YNR063Warmdown, use 500-700ng to knock down YNR063WThe plasmid (20nt-gcgaagataacgctcctacg) and 5 μg of the integrated fragment YNR063Warmup-P PGK1 -ZWF1-T ter22 -GND1-P GAL10 / 1 -TKL1-T tef -TAL2-P TDH1 YNR063Warmdown was transformed into the engineered Saccharomyces cerevisiae strain D003 in step (3) using a high-efficiency transformation method. It was then plated on YNB solid medium for screening for uracil defects and cultured at 30°C for 3 days. Five days, until colonies appear, then the correctly verified clone is named D004. Figure 1 As shown, the squalene yield of YPD after 96 h of shake-flask fermentation was 987.10 mg / L.

[0065] (5) Construction of strain D005 In D004 VBA5 Site single copy integration YHM2 , CTP1 , MPC1 and MPC3 (The nucleotide sequence is shown in SEQ ID NO. 12~15). Using the Saccharomyces cerevisiae genome as a template, the following sequences were amplified using primers 014-YHM2-F1 and 014-YHM2-R1: YHM2 Amplification was performed using primers 014-CTP1-F1 and 014-CTP1-R1. CTP1 Amplification was performed using primers 014-MPC1-F1 and 014-MPC1-R1. MPC1 Amplification was performed using primers 014-MPC3-F1 and 014-MPC3-R1. MPC3 Amplification was achieved using primers 014-VBA5-UP-F1 and 014-VBA5-UP-R1. VBA5 The upstream homologous arm of the site is 500 bp; it was amplified using primers 014-VBA5-DOWN-F1 and 014-VBA5-DOWN-R1. VBA5 The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers 014-PGK1-F1 and 014-PGK1-R1. PGK1 Terminator T was amplified using primers 014-ter22-F1 and 014-ter22-R1. Ter22 Terminator T was amplified using primers 014-Ttef-F1 and 014-Ttef-R1. TEF The bidirectional promoter P was amplified using primers 014-Pgal1-10-F1 and 014-Pgal1-10-R1. GAL10 / 1The bidirectional promoter P was amplified using primers 014-Ptdh1-F1 and 014-Ptdh1-R1. PTDH1 Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers 014-KJ-F1 and 014-KJ-R1. YHM2 , CTP1 , MPC1 , MPC3, VBA5 500 bp upstream homology arm of the site VBA5 500 bp downstream homologous arm of the site, promoter P PGK1 Termination of sub-T Ter22 Termination of sub-T TEF P GAL10 / 1 Bidirectional promoter P PTDH1 The pY26 vector fragment was used to construct plasmid Z005 using Gibson assembly.

[0066] Using plasmid Z005 as a template, the integration fragment VBA5armup-P was amplified using primers Z14-F and Z14-R. PGK1 -YHM2-T ter22 -CTP1-P GAL10 / 1 -MPC1-T tef -MPC3-P TDH1 -VBA5armdown, using 500-700ng to knock out the YNR063W plasmid (20nt-gcaaatccaagggaaaaccc) and 5 μg of the integration fragment VBA5armup-P PGK1 -YHM2-T ter22 -CTP1-P GAL10 / 1 -MPC1-T tef -MPC3-P TDH1 -VBA5armdown, transformed into the Saccharomyces cerevisiae engineered strain D004 in step (4) using the efficient transformation method, and plated on YNB solid medium for screening for defective uracil, and cultured at 30℃ for 3 days. Five days, until colonies appear, then the correctly verified clone is named D005. Figure 1 As shown, the squalene yield of YPD after 96 h of shake-flask fermentation was 1151.56 mg / L.

[0067] Table 2 Primer sequences involved in Example 1

[0068] Example 2: Push-pull metabolic flux enhances ergosterol synthesis The expression of ERGs enzyme genes in the post-squalene pathway was enhanced, thus improving the ergosterol synthesis pathway and further strengthening the ergosterol synthesis level of the engineered strain.

[0069] (1) Construction of strain D006 In D005 ROX1 Site single copy integration ERG1 and ERG11 (The nucleotide sequence is shown in SEQ ID NO. 16~17). Using the Saccharomyces cerevisiae genome as a template, the following sequences were amplified using primers E1-ERG1-F1 and E1-ERG1-R1: ERG1 Amplification was performed using primers E1-ERG11-F1 and E1-ERG11-R1. ERG11 Amplification was achieved using primers E1-ROXUP-F1 and E1-ROXUP-R1. ROX1 The upstream homologous arm of the site is 500 bp; it was amplified using primers E1-ROXDN-F1 and E1-ROXDN-R1. ROX1 The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers E1-PGK1-F1 and E1-PGK1-R1. PGK1 Terminator T was amplified using primers E1-ter22-F1 and E1-ter22-R1. Ter22 The bidirectional promoter P was amplified using primers E1-PTEF-F1 and E1-PTEF-R1. TEF1 Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers E1-KJ-F1 and E1-KJ-R1. ERG1 , ERG11 , ROX1 500 bp upstream homology arm of the site ROX1 500 bp downstream homologous arm of the site, promoter P PGK1 Termination of sub-T Ter22 promoter P TEF1 The pY26 vector fragment was used to construct plasmid Z006 using Gibson assembly.

[0070] Using plasmid Z006 as a template, the integration fragment ROX1armup-P was amplified using primers E1-F1 and E1-R1. PGK1 -ERG11-T ter22 -ERG1-P TEF1 -ROX1armdown, use 500-700ng to knock it down. ROX1 The plasmid (20nt-caacaacagcagtcaaacac) and 5 μg of the integration fragment ROX1armup-P PGK1 -ERG11-T ter22 -ERG1-PTEF1 -ROX1armdown, using the high-efficiency transformation method of Saccharomyces cerevisiae, was transformed into the Saccharomyces cerevisiae engineered strain D005 in step (5) of Example 1, and spread on YNB solid medium for screening for defective uracil, and cultured at 30°C for 3 days. Five days, until colonies appear, then the correctly verified clone is named D006. Figure 2 As shown, the yield of ergosterol in YPD after 72 h of shake-flask fermentation was 215.09 mg / L.

[0071] (2) Construction of strain D007 In D006 DOS2 Site single copy integration ERG2 and ERG7 (The nucleotide sequence is shown in SEQ ID NO. 18~19). Using the Saccharomyces cerevisiae genome as a template, the following sequences were amplified using primers 011-ERG7-F1 and 011-ERG7-R1: ERG7 ; Amplification was performed using primers 011-ERG2-F1 and 011-ERG2-R1. ERG2 Amplification was performed using primers 011-DOS2UP-F1 and 011-DOS2UP-R1. DOS2 The upstream homologous arm of the site is 500 bp; it was amplified using primers 011-DOS2DN-F1 and 011-DOS2DN-R1. DOS2 The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers 011-PGK1-F1 and 011-PGK1-R1. PGK1 Terminator T was amplified using primers 011-TERT-F1 and 011-TERT-R1. Ter22 The bidirectional promoter P was amplified using primers 011-TEFp-F1 and 011-TEFp-R1. TEF1 Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers 011-KJ-F1 and 011-KJ-R1. ERG2 , ERG7 , DOS2 500 bp upstream homology arm of the site DOS2 500 bp downstream homologous arm of the site, promoter P PGK1 Termination of sub-T Ter22 promoter P TEF1 The pY26 vector fragment was used to construct plasmid Z007 using Gibson assembly.

[0072] Using plasmid Z007 as a template, the integration fragment DOS2armup-P was amplified using primers E2-F and E2-R. PGK1 -ERG2-T ter22 -ERG7-PTEF1 -DOS2armdown, use 500-700ng to knock down DOS2 The plasmid (20nt-caaaaaggaaaaggaaacgg) and 5 μg of the integration fragment DOS2armup-P PGK1 -ERG2-T ter22 -ERG7-P TEF1 -DOS2armdown, using the high-efficiency transformation method of Saccharomyces cerevisiae, was transformed into the Saccharomyces cerevisiae engineered strain D006 in step (1), and spread on YNB solid medium for screening for defective uracil, and cultured at 30℃ for 3 days. Five days, until colonies appear, then the correctly verified clone is named D007. Figure 2 As shown, the yield of ergosterol in YPD after 72 h of shake-flask fermentation was 314.75 mg / L.

[0073] (3) Construction of strain D008 In D007 NEM1 Site single copy integration ERG3 , ERG4 , ERG5 and ERG6 (The nucleotide sequence is shown in SEQ ID NO. 20~23). Using the Saccharomyces cerevisiae genome as a template, the following sequences were amplified using primers E3-ERG3-F1 and E3-ERG3-R1: ERG3 Amplification was performed using primers E3-ERG4-F1 and E3-ERG4-R1. ERG4 Amplification was performed using primers E3-ERG5-F1 and E3-ERG5-R1. ERG5 Amplification was performed using primers E3-ERG6-F1 and E3-ERG6-R1. ERG6 Amplification was performed using primers E3-NEM1UP-F1 and E3-NEM1UP-R1. NEM1 The upstream homologous arm of the site is 500 bp; it was amplified using primers E3-NEM1DN-F1 and E3-NEM1DN-R1. NEM1 The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers E3-PGK-F1 and E3-PGK-R1. PGK1 Terminator T was amplified using primers E3-TER22-F1 and E3-TER22-R1. Ter22 Terminator T was amplified using primers E3-TLEU-F1 and E3-TLEU-R1. LEU The bidirectional promoter P was amplified using primers E3-GAL-F1 and E3-GAL-R1. GAL10 / 1 The bidirectional promoter P was amplified using primers E3-PTEF-F1 and E3-PTEF-R1. TEF1Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers E3-KJ-F1 and E3-KJ-R1. ERG3 , ERG4 , ERG5 , ERG6 , NEM1 500 bp upstream homology arm of the site NEM1 500 bp downstream homologous arm of the site, promoter P PGK1 Termination of sub-T Ter22 Termination of sub-T LEU P GAL10 / 1 Bidirectional promoter P TEF1 The pY26 vector fragment was used to construct plasmid Z008 using Gibson assembly.

[0074] Using plasmid Z008 as a template, the integration fragment NEM1armup-P was amplified using primers NEM1-F1 and NEM1-R1. PGK1 -ERG6-T LEU -ERG5-P GAL10 / 1 -ERG4-T Ter22 -ERG3-P TEF1 -NEM1armdown, use 500-700ng to knock it down. NEM1 The plasmid (20nt-gaagtgaagtttggattgag) and 5 μg of the integrated fragment NEM1armup-P PGK1 -ERG6-T LEU -ERG5-P GAL10 / 1 -ERG4-T Ter22 -ERG3-P TEF1 -NEM1armdown, using the efficient transformation method of Saccharomyces cerevisiae, was transformed into the Saccharomyces cerevisiae engineered strain D007 in step (2), and spread on YNB solid medium for screening for defective uracil, and cultured at 30℃ for 3 days. Five days, until colonies appear, then the correctly verified clone is named D008. Figure 3 As shown, the yield of ergosterol in YPD after 72 h of shake-flask fermentation was 392.99 mg / L.

[0075] Table 3 Primer sequences designed in Example 2

[0076] Example 3: Extending the lifespan of engineered strains and enhancing their ability to accumulate ergosterol. (1) Construction of strain D009 Knock out based on D008 TOR1The site was amplified using the *Saccharomyces cerevisiae* genome as a template and primers E5-TOR1UP-F1 and E5-TOR1UP-R1. TOR1 The upstream homologous arm of the site is 500 bp; it was amplified using primers E5-TOR1DN-F1 and E5-TOR1DN-R1. TOR1 The downstream homologous arm of the site is 500 bp; using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers E5-KJ-F1 and E5-KJ-R1. TOR1 500 bp upstream homology arm of the site TOR1 The downstream homologous arm of the site (500 bp) and the pY26 vector fragment were assembled using Gibson fusion to construct plasmid Z009.

[0077] Using plasmid Z009 as a template, the integration fragment TOR1armup-TOR1armdown was amplified using primers TOR1-F1 and TOR1-R1, and then knocked out using 500-700 ng. TOR1 The plasmid (20nt-caggcagtgaatgacaacag) and 5 μg of the integrated fragment TOR1armup-TOR1armdown were transformed into the Saccharomyces cerevisiae engineered strain D008 in step (3) of Example 2 using the high-efficiency transformation method of Saccharomyces cerevisiae. The transformed strain was plated on YNB solid medium for screening for defective uracil and cultured at 30°C for 3 days. Fermentation continued for 5 days until colonies appeared. The correct clone was named D009. After 72 h of shake-flask fermentation with YPD, the ergosterol yield was 469.83 mg / L.

[0078] like Figure 3 As shown, after fermentation for 72, 96, 120, 144, and 168 h, the ergosterol yields of engineered strain D009 were 469.83, 531.54, 536.99, 538.32, and 540.10 mg / L, respectively, with OD values ​​of [missing data]. 600 The figures are 29.71, 32.22, 33.52, 32.94, and 30.99, respectively.

[0079] (2) Construction of strain D010 Knock out based on D009 FOB1 Site. Using the Saccharomyces cerevisiae genome as a template, the site was amplified using primers E7-fob1up-F1 and E7-fob1up-R1. FOB1 The upstream homologous arm of the site is 500 bp; it was amplified using primers E7-fob1DN-F1 and E7-fob1DN-R1. FOB1 The downstream homologous arm of the site is 500 bp; using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers E7-KJ-F1 and E7-KJ-R1. FOB1 500 bp upstream homology arm of the site FOB1 The downstream homologous arm of the site (500 bp) and the pY26 vector fragment were assembled using Gibson fusion to construct plasmid Z010.

[0080] Using plasmid Z010 as a template, the integration fragment FOB1armup-FOB1armdown was amplified using primers FOB1-F1 and FOB1-R1, and then knocked out with 500-700 ng. FOB1 The plasmid (20nt-aagaaaggcaacgtcacctg) and 5 μg of the integrated fragment FOB1armup-FOB1armdown were transformed into the Saccharomyces cerevisiae engineered strain D009 in step (1) using the high-efficiency transformation method of Saccharomyces cerevisiae. The transformed strain was then plated on YNB solid medium for screening for defective uracil and cultured at 30°C for 3 days. Fermentation was continued for 5 days until colonies appeared. The correct clone was named D010. After 72 h of shake-flask fermentation with YPD, the ergosterol yield was 504.17 mg / L.

[0081] like Figure 3 As shown, after fermentation for 72, 96, 120, 144, and 168 h, the yields of ergosterol were 504.17, 532.54, 603.05, 704.69, and 739.04 mg / L, respectively, and the OD600 values ​​were 25.61, 31.12, 32.61, 31.05, and 31.10, respectively.

[0082] Table 4 Primer sequences involved in Example 3

[0083] Example 4: Regulating glucose phosphorylation levels to enhance ergosterol synthesis (1) Genes of key enzymes for glucose phosphorylation HXK1 Expression weakening Knock out P from D010 HXK1 Replace with P ADH2 Using the Saccharomyces cerevisiae genome as a template, primers E11-HXK1UP-F1 and E11-HXK1UP-R1 were used to amplify the genome. HXK1 The upstream homologous arm of the site is 500 bp; it was amplified using primers E11-HXK1DN-F1 and E11-HXK1DN-R1. HXK1 The downstream homologous arm of the site is 500 bp; the promoter P was amplified using primers E11-PADH2-F1 and E11-PADH2-R1. ADH2(The nucleotide sequence is shown in SEQ ID NO.24). Using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers E11-KJ-F1 and E11-KJ-R1. HXK1 500 bp upstream homology arm of the site HXK1 500 bp downstream homologous arm of the site, promoter P ADH2 The pY26 vector fragment was used to construct plasmid Z011 using Gibson assembly.

[0084] Using plasmid Z011 as a template, the integration fragment HXK1armup-P was amplified using primers E11-F1 and E11-R1. ADH2 -HXK1armdown, use 500-700ng to knock it down. HXK1 The plasmid (20nt-ctaaggtaccaaaatccggg) and 5 μg of the integration fragment HXK1armup-P ADH2 -HXK1armdown, using the high-efficiency transformation method of Saccharomyces cerevisiae, was transformed into the Saccharomyces cerevisiae engineered strain D010 in step (2) of Example 3, and plated on YNB solid medium for screening for defective uracil, and cultured at 30°C for 3 days. Five days, until colonies appear, then the verified clone is named D011. Figure 4 As shown, the ergosterol yield of YPD after 120 h of shake-flask fermentation was 682.47 mg / L.

[0085] (2) Genes of key enzymes for glucose phosphorylation GLK1 Knockout Knock out based on D011 GLK1 The locus was amplified using the *Saccharomyces cerevisiae* genome as a template, and primers E10-GLK1UP-F1 and E10-GLK1UP-R1. GLK1 The upstream homologous arm of the site is 500 bp; it was amplified using primers E10-GLK1DN-F1 and E10-GLK1DN-R1. GLK1 The downstream homologous arm of the site is 500 bp; using the pY26 expression vector as a template, the pY26 vector fragment was amplified using primers E10-KJ-F1 and E10-KJ-R1. GLK1 500 bp upstream homology arm of the site GLK1 The downstream homologous arm of the site (500 bp) and the pY26 vector fragment were assembled using Gibson fusion to construct plasmid Z012.

[0086] Using plasmid Z012 as a template, the integration fragment GLK1armup-GLK1armdown was amplified using primers E10-F1 and E10-R1, and then knocked out with 500-700 ng. GLK1The plasmid (20nt-gcattaaccaacgacaccgt) and 5 μg of the integrated fragment GLK1armup-GLK1armdown were transformed into the Saccharomyces cerevisiae engineered strain D011 in step (1) using the high-efficiency transformation method of Saccharomyces cerevisiae. The transformed strain was then plated on YNB solid medium for screening for defective uracil and cultured at 30°C for 3 days. Five days, until colonies appear, then the correctly verified clone is named D012. Figure 4 As shown, the yield of ergosterol in YPD after 120 h of shake-flask fermentation was 811.50 mg / L.

[0087] Table 5 shows the primer sequences involved in Example 4.

[0088] Example 5: Precursor supply for the MVA pathway reinforced by remedial defective tags rDNA multiple copy integration based on D012 tHMG1 and IDI1 Using plasmid Z001 as a template, the gene was amplified using primers E13-GENE-F1 and E13-GENE-R1. tHMG1 -P GAL10 / 1 - IDI1 The expression frame, using Pct22 (disclosed in patent publication number CN114752619B) as a template, amplifies the Pct22 vector integration fragment using primers E13-KJ-F1 and E13-KJ-R1, and then... t tHMG1 -P GAL10 / 1 - IDI1 The expression frame and the Pct22 vector integration fragment were used to construct plasmid Z013 using Gibson assembly.

[0089] Using plasmid Z013 as a template, the integrated fragment rDNAarmup-tHMG1-PGAL10 / 1-IDI1-URA3deg-rDNAarmdown was amplified using primers rDNA-F1 and rDNA-R1. 5 μg of the integrated fragment rDNAarmup-tHMG1-PGAL10 / 1-IDI1-URA3deg-rDNAarmdown was transformed into the engineered Saccharomyces cerevisiae strain D012 using a high-efficiency transformation method. The transformed fragment was plated on YNB solid medium for screening for uracil defects and incubated at 30°C for 3 days. 5 days, until colonies appear.

[0090] like Figure 5 As shown, a series of single colonies were selected and fermented in YPD shake flasks for 120 h, and strain D013-5 with an ergosterol yield of 1007.52 mg / L was finally obtained.

[0091] Table 6 shows the primer sequences involved in Example 5.

[0092] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant brewing yeast, characterized in that, The recombinant yeast uses Saccharomyces cerevisiae C800 as the chassis strain, and integrates into the chassis strain. tHMG1 Gene, IDI1 Gene, ERG10 Gene, ERG13 Gene, ERG12 Gene, ERG8 Gene, ERG19 Gene, ZWF1 Gene, GND1 Gene, TKL1 Gene, TAL2 Gene, YHM2 Gene, CTP1 Gene, MPC1 Gene, MPC3 Gene, ERG1 Genes and ERG11 Genes, the ones mentioned tHMG1 Gene, IDI1 Gene, ERG10 Gene, ERG13 Gene, ERG12 Gene, ERG8 Gene, ERG19 Gene, ZWF1 Gene, GND1 Gene, TKL1 Gene, TAL2 Gene, YHM2 Gene, CTP1 Gene, MPC1 Gene, MPC3 Gene, ERG1 Genes and ERG11 The nucleotide sequences of the genes are shown in SEQ ID NO.1~17.

2. The recombinant brewing yeast according to claim 1, characterized in that, The tHMG1 Genes and IDI1 Gene integration in Ty1 Site; in YPL062W Site single copy integration ERG10 , ERG13 , ERG12 and ERG8 Genes; in YJL064W Site single copy integration ERG19 Genes; in YNR063W Site single copy integration ZWF1 , GND1 , TKL1 and TAL2 Genes; in VBA5 Site single copy integration YHM2 , CTP1 , MPC1 and MPC3 Genes; in ROX1 Site single copy integration ERG1 and ERG11 Gene.

3. The recombinant brewing yeast according to claim 1, characterized in that, At the same time in the chassis strain DOS2 Site single copy integration ERG2 and ERG7 Gene, ERG2 and ERG7 The nucleotide sequences of the genes are shown in SEQ ID NO.18~19.

4. The recombinant brewing yeast according to claim 1, characterized in that, At the same time in the chassis strain NEM1 Site single copy integration ERG3 , ERG4 , ERG5 and ERG6 Gene, ERG3 , ERG4 , ERG5 and ERG6 The nucleotide sequences of the genes are shown in SEQ ID NO.20~23, respectively.

5. The recombinant brewing yeast according to claim 1, characterized in that, At the same time, the chassis strain was also knocked out TOR1 Gene, FOB1 Genes and / or GLK1 Gene.

6. The recombinant brewing yeast according to claim 1, characterized in that, The key enzyme gene for glucose phosphorylation in the chassis strain was extracted. HXK1 The expression is weakened.

7. The recombinant brewing yeast according to claim 6, characterized in that, By HXK1 The promoter of the gene is replaced with P ADH2 Key enzyme genes for glucose phosphorylation HXK1 Expression weakening, promoter P ADH2 The nucleotide sequence is shown in SEQ ID NO.

24.

8. The recombinant brewing yeast according to claim 1, characterized in that, Simultaneously, rDNA integration was performed in the chassis strain. tHMG1 and IDI1 Gene.

9. A method for biosynthesizing ergosterol, characterized in that, The method involves culturing and synthesizing ergosterol using the recombinant brewing yeast described in any one of claims 1 to 8 in a fermentation system with glucose as the carbon source.

10. The use of the recombinant brewing yeast according to any one of claims 1 to 8 or the method according to claim 9 in the preparation of ergosterol or ergosterol-containing products.

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