Ura3 and leu2 defective saccharomyces cerevisiae engineering strain and application thereof in efficient synthesis of EGT

Abstract

The invention discloses a ura3 and leu2 defective saccharomyces cerevisiae engineering strain and an application thereof in efficient synthesis of EGT (Epidermal Glycine Transferase). A ura3 and leu2 double-defect strain SZA015 is constructed through a CRISPR / Cas9 technology, and the EGT yield is remarkably increased by combining promoter engineering, metabolic network regulation and culture medium optimization. The shake flask fermentation yield reaches 183.70 mg / L, the yield of a 10 L fermentation tank is 2.26 g / L, and efficient strains and process support are provided for EGT industrial production. Through targeted knockout of pho89 or sps100 genes, pH optimization and precursor addition strategies are combined, and EGT synthesis efficiency is remarkably improved. The fermentation yield of a shake flask reaches 196.30 mg / L, the yield of a 10 L fermentation tank is 2.79 g / L, the production efficiency is 23.2 mg / L / h, and efficient strains and process support are provided for EGT industrial production.

Description

Technical Field

[0001] The present invention relates to the fields of metabolic engineering and synthetic biology, and in particular to an engineered ura3- and leu2-deficient Saccharomyces cerevisiae strain and its application in the efficient synthesis of EGT. Background Art

[0002] Ergothioneine (EGT) is a sulfur-containing amino acid with strong antioxidant activity and has important application value in the fields of food, medicine and cosmetics. Its traditional production methods mainly rely on chemical synthesis or extraction from natural biological resources (such as mushrooms and fungi), which have problems such as complex processes, high costs and low yields. In recent years, microbial synthesis technology has become a research hotspot due to its green sustainability. Saccharomyces cerevisiae has been widely used in the biosynthesis of EGT due to its safety and mature metabolic engineering platform. However, existing technologies still face a bottleneck - uneven distribution of metabolic flux. In addition, traditional culture medium optimization strategies have failed to effectively balance precursor supply and metabolic pressure, resulting in limited improvement in EGT synthesis efficiency. Summary of the Invention

[0003] The present invention aims to develop an engineered strain of Saccharomyces cerevisiae deficient in ura3 and leu2 and its application in the efficient synthesis of EGT. Specifically, the invention provides a method for constructing an engineered strain of Saccharomyces cerevisiae deficient in ura3 and leu2 using CRISPR / Cas9 technology, as well as a technical solution for significantly improving ergothioneine (EGT) production through promoter engineering, metabolic network regulation, culture medium optimization, and fermentation process improvements.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] The present invention first provides a ura3 and leu2 double-deficient Saccharomyces cerevisiae engineered strain SZA015, by knocking out the ura3 and leu2 genes of Saccharomyces cerevisiae using CRISPR / Cas9 technology, and integrating two copies of TDH3p-EGT1-ADH2t-TEF1p-EGT2-CyC1t expression cassettes into the Saccharomyces cerevisiae genome to construct the Saccharomyces cerevisiae engineered strain SZA015, whose EGT production is 113.06 mg / L.

[0006] The present invention also provides an engineered yeast strain SZA033, which uses the engineered yeast strain SZA015 as the starting strain, knocks out the leu2 gene by CRISPR / Cas9 technology and replaces the ura3 promoter with a weak promoter P COG7 , constructed the Saccharomyces cerevisiae engineered strain SZA033, whose EGT production was 69.44 mg / L.

[0007] The present invention also provides a pho89-deficient Saccharomyces cerevisiae engineered strain SZA045, which is characterized in that the pho89 gene is knocked out using CRISPR / Cas9 technology using Saccharomyces cerevisiae engineering strain SZA045 as the starting strain, and the shake flask EGT yield is 89.09 mg / L.

[0008] The present invention also provides an sps100-deficient Saccharomyces cerevisiae engineered strain SZA046, which is characterized in that the sps100 gene is knocked out using CRISPR / Cas9 technology using Saccharomyces cerevisiae engineered strain SZA046 as the starting strain, and the shake flask EGT yield is 78.51 mg / L.

[0009] The present invention also provides an optimized culture medium for any one of the above-mentioned engineered strains of Saccharomyces cerevisiae: 10 mg / L ura3, 10 mg / L leu2, 1.0 g / L L-cysteine, 1.0 g / L L-methionine, 1.0 g / L L-histidine and 2% glucose are added to a YPD culture medium at pH 5.0.

[0010] The present invention also provides a fed-batch fermentation process for any of the above-mentioned engineered strains of Saccharomyces cerevisiae: the pH is dynamically controlled to 5.0 and the DO is 20-40% in a 10L fermentor; when the glucose in the fermentation broth is consumed (approximately 16-20 hours of fermentation), 80% glucose mother liquor is added to maintain the glucose concentration at 2.1-2.6 g / L, and the EGT yield reaches 2.79 g / L.

[0011] The present invention aims to develop a synergistic strategy of gene editing and metabolic engineering to construct a stable Saccharomyces cerevisiae strain with high EGT production, while solving the problems of biomass reduction and culture medium cost caused by ura3 deficiency, and optimizing the fermentation process to achieve industrial production.

[0012] The present invention found that ura3 deficiency can drive metabolic reprogramming, prompting carbon flow to shift to EGT synthesis (SZA015 strain production reached 113.06 mg / L); weak promoters (such as P COG7 , strain SZA033) partially restored ura3 expression to balance growth and product synthesis (SZA033 strain OD 600Dynamic optimization of the culture medium (such as limited uracil supplementation and precursor amino acid addition) significantly increased EGT production (SZA045 reached 196.30 mg / L after optimization), which is the highest level of the current brewer's yeast system; SZA015 fed-batch fermentation yield reached 2.26 g / L, cell dry weight (DCW) 28.89 g / L, SZA015 fed-batch fermentation final EGT production reached 2.79 g / L, cell dry weight 41.38 g / L, OD 600 was 162.6, and ethanol accumulation was <1g / L, which has the potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Construction of leu2 and ura3 gene knockout strains. A: Colony PCR results for leu2 knockout; Lanes 1-5: Colony PCR results for positive clones; M: 1 kb ladder. B: Colony PCR results for ura3 knockout; Lanes 1-2, 4-6: Colony PCR results for positive clones; Lane 3: Colony PCR results for negative clones; M: D2000 plus. C: Construction of ura3 and leu2-deficient strains; D: Construction of ura3 and leu2-deficient EGT-producing strains.

[0014] Figure 2 The PCR product of the colony after ura3 promoter replacement and its synthetic EGT result. ura3 ::P TEF1p Lane 1: Colony PCR results of positive clones; Lanes 2-10: Colony PCR results of negative clones; M: 1 kb ladder. B: P ura3 ::P ATP14 Lanes 1-7, 9-10: Colony PCR results of positive clones; Lane 8: Colony PCR results of negative clones; M: 1k bladder. C: P ura3 ::P COX9 Lanes 1-10: Colony PCR results of positive clones; M: 1kb ladder. D: P ura3 ::P HXT1 ; Lanes 1-10: Colony PCR results of positive clones; M: 1kb ladder. E: P ura3 ::P PDA1 Lanes 1-2, 6-10: Colony PCR results of positive clones; Lanes 3-5: Colony PCR results of negative clones; M: 1 kb ladder. F: P ura3 ::P COG7Lanes 1-10: Colony PCR results of positive clones; M: 1kb ladder. G: EGT synthesis results of the corresponding engineered strain after ura3 promoter replacement

[0015] Figure 3 The results of fermentation using optimized SZA015 medium. A: Fermentation results using different concentrations of ura3 and leu2; B: Fermentation results using 10 mg / L ura3 as the substrate.

[0016] Figure 4 Figure 1: Fed-batch fermentation results of engineered strain SZA015 in a 10-L fermentor. A: Batch fermentation results of engineered strain SZA015 in a 10-L bioreactor; B: Residual glucose and ethanol content during batch fermentation of engineered strain SZA015. All data are mean ± SD of three biological replicates (n = 3).

[0017] Figure 5 The effects of Δpho89 and Δsps100 on EGT production in engineered strain SZA033. A: Relative expression of pho89 and sps100; B: Colony PCR verification of Δpho89; Lanes 1-10: Colony PCR results of positive clones; M: 1 kb bladder. C: Colony PCR verification of Δsps100; Lanes 1-10: Colony PCR results of positive clones; M: 1 kb ladder. D: Strain modification based on transcriptome downregulation data.

[0018] Figure 6 These are the results of shake flask fermentation of engineered strain SZA045 in culture media with different pH (A) and amino acid additions (B).

[0019] Figure 7 Figure 1: Fed-batch fermentation results of engineered yeast SZA045 in a 10-L fermentor. A: Fed-batch fermentation results of engineered strain SZA045 in a 10-L bioreactor; B: Residual glucose and ethanol content during fed-batch fermentation of engineered strain SZA045. All data are mean ± SD of three biological replicates (n = 3). DETAILED DESCRIPTION

[0020] Example 1 Culture medium and solution preparation

[0021] 1.1 Culture medium formulation

[0022] (1) Fermentation medium (two formulations):

[0023] Formula A: peptone 20 g / L, yeast extract 10 g / L, anhydrous glucose 20 g / L (sterilized separately), vitamin solution 12 mL / L, trace elements 10 mL / L, L-cysteine 5.0 g / L, L-histidine 5.0 g / L, L-methionine 5.0 g / L;

[0024] Formula B: peptone 20 g / L, yeast extract 10 g / L, anhydrous glucose 40 g / L (sterilized separately), vitamin solution 12 mL / L, trace elements 10 mL / L.

[0025] (2) Feed medium (two formulations):

[0026] Formula C: anhydrous glucose 800 g / L, ammonium sulfate 8 g / L, trace element mixture 30 mL / L, vitamin solution 30 mL / L, L-cysteine 5.0 g / L, L-histidine 5.0 g / L, L-methionine 5.0 g / L;

[0027] Formula D: anhydrous glucose 800 g / L, ammonium sulfate 8 g / L, trace element mixture 30 mL / L, vitamin solution 30 mL / L, L-cysteine 0.5 g / L, L-histidine 0.5 g / L, L-methionine 0.5 g / L.

[0028] (3) LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L;

[0029] (4) LB solid medium: add 20 g / L agar to LB liquid medium;

[0030] (5) YPD medium: peptone 20 g / L, yeast extract 10 g / L, anhydrous glucose 20 g / L (sterilized separately);

[0031] (6) YPD solid medium: add 20 g / L agar to YPD medium;

[0032] (7) YPDG solid medium: YPD solid medium plus geneticin (G418, 250 μg / mL);

[0033] (8) YPDN solid medium: YPD solid medium plus nourseothricin (NAT, 120 μg / mL);

[0034] (9) YPDGN solid medium: G418 (250 μg / mL) and NAT (120 μg / mL) were added to YPD solid medium.

[0035] 1.2 Solution preparation

[0036] (1) TF1 solution: 1 M potassium acetate, 1 M potassium chloride, 0.5 M calcium chloride, 1 M manganese chloride, 50% glycerol (v / v), pH 5.8 (adjusted with 0.2 M acetic acid), filter sterilize and store at 4°C;

[0037] (2) TF2 solution: 1 M MOPS, 1 M KCl, 0.5 M CaCl2, 50% glycerol (v / v), pH 6.8 (adjusted with 1 M KOH), filter sterilize and store at 4°C;

[0038] (3) 50×TAE buffer: Tris 242 g / L, NaEDTA-2H2O 37.2 g / L, acetic acid 57.1% (V / V), diluted 50-fold before use;

[0039] (4) Trace element mixture (1000×): disodium EDTA dihydrate 15.0 g / L, ZnSO4·7H2O 4.5 g / L, MnCl2·4H2O 1.0 g / L, CoCl2·6H2O 0.3 g / L, CuSO4·5H2O 0.3 g / L, Na2MoO4·2H2O 0.4 g / L, CaCl2·2H2O 4.5 g / L, FeSO4·7H2O 3.0 g / L, H3BO3 1.0 g / L, KI 0.1 g / L, sterilized at 121°C for 20 min;

[0040] (5) Vitamin solution (1000×): biotin 0.05 g / L, calcium pantothenate 1.0 g / L, niacin 1.0 g / L, inositol 2.5 g / L, vitamin B1 1.0 g / L, pyridoxine 1.0 g / L.

[0041] Example 2 Construction of microbial engineering strains

[0042] 2.1 Heat shock transformation of Escherichia coli (for related plasmid construction and amplification)

[0043] (1) Inoculate E. coli DH10B into LB solid medium and culture at 37°C overnight; pick a single colony and transfer it to LB liquid medium and culture at 37°C with shaking until OD 600 =0.4-0.5;

[0044] (2) After ice bath, centrifuge (3500 rpm, 10 min), wash the cells with TF1 and TF2 solutions, respectively, and store at -80°C after aliquoting.

[0045] (3) Take 100 μL of competent cells, add 1 μL of plasmid or 10 μL of recombinant product, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, recover and culture for 45 minutes, spread on LB plates containing antibiotics, and culture at 37°C for 16-24 hours.

[0046] 2.2 CRISPR-Cas9 gene editing in Saccharomyces cerevisiae (construction of engineered bacteria)

[0047] (1) Transforming the Cas9 plasmid into Saccharomyces cerevisiae competent cells (preparation method see Example 3);

[0048] (2) Co-transform the donor DNA and sgRNA plasmid into the Cas9-positive strain, culture with shaking at 30°C, and then spread on YPDGN plates to screen for positive clones;

[0049] (3) Gene knockout / knock-in was verified by colony PCR and resistance plate analysis, and strains that lost the plasmid were screened for fermentation; and strains that grew on YPDG but not YPDN were used for the next round of genetic manipulation.

[0050] Example 3 Preparation and transformation of competent cells of Saccharomyces cerevisiae

[0051] 3.1 Preparation of competent cells (6 mL system)

[0052] (1) Streak the Saccharomyces cerevisiae strain onto YPD solid medium and culture at 30°C for 2-3 days. Pick a single colony and inoculate it into 2 mL YPD liquid medium and culture it at 30°C and 220 rpm overnight.

[0053] (2) Transfer 0.25 mL of bacterial solution to 10 mL of YPD medium and culture until OD 600 =0.8-1.0;

[0054] (3) Centrifuge (500 × g, 3 min) and discard the supernatant. Add 1.2-1.5 mL of EZ solution 1 to resuspend the cells. Centrifuge again and add 0.12 mL of EZ solution 2. Aliquot into 1.5 mL centrifuge tubes and store at -80°C.

[0055] 3.2 Competent transformation

[0056] (1) Place competent cells on ice for 5 min, add 1 μL of plasmid or 10 μL of recombinant product, vortex to mix, and incubate at 30°C.

[0057] (2) Vortex once every 20 minutes for a total of 5 times;

[0058] (3) After centrifugation, apply YPD solid medium containing antibiotics and culture at 30°C for 2-3 days.

[0059] Example 4 EGT detection method

[0060] (1) Sample pretreatment: The fermentation broth was ground with liquid nitrogen, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane.

[0061] (2) LC-MS identification: Agilent TC-C18 column, mobile phase A (0.1% formic acid in water), B (0.1% formic acid in acetonitrile), gradient elution (0-47 min, 2%-95% B); mass spectrometry parameters: ESI source, spray voltage 3.5 kV, ion source temperature 300°C;

[0062] (3) HPLC quantification: isocratic elution (2% B, 30 min), standard curve regression equation y = 546.88x-4.0573 (R 2 =0.9988).

[0063] Example 5 Microbial engineering strain construction results

[0064] 5.1 Construction of ura3- and leu2-deficient and EGT-producing strains

[0065] The leu2 gene (encoding β-isopropylmalate dehydrogenase, NCBI Gene ID: ID: 850342) of Saccharomyces cerevisiae CEN.PK113-7D was knocked out using CRISPR / Cas9 technology to construct strain SZA012. 600 is 43.08;

[0066] Knockout ura3 (encoding orotidine-5'-phosphate decarboxylase, NCBI Gene ID: ID: 856692) gene, construct strain SZA012U, OD 600 is 12.99;

[0067] Construction of the Δleu2Δura3 double-deficient strain SZA012UL, OD 600 is 12.15( Figure 1 C).

[0068] Gene knockout verification: The change in gene fragment length after knockout was confirmed by colony PCR (Table 1), and the strains that lost the Cas9 / sgRNA plasmid were screened by resistance plates.

[0069] The EGT1 gene (SEQ ID NO.1) and the EGT2 gene (SEQ ID NO.2) were amplified and codon optimized and gene synthesized. The EGT1 and EGT2 genes were integrated into the Saccharomyces cerevisiae genome using CRISPR-Cas9 technology and the strong promoter P was used to express the EGT1 gene. TDH3 and P TEF1Drive expression. TDH3p-EGT1-ADH2t-TEF1p-EGT2-CyC1t expression cassettes were integrated into the 416d and 1021b sites, respectively, to obtain the EGT expression strain SZA013 with two copies of the expression cassettes. The EGT production was 39.16 mg / L ( Figure 1 D).

[0070] Using SZA013 as the starting strain, ura3 and leu2 deficient strains were constructed, and metabolic pressure was used to drive carbon flow to EGT synthesis. The yield of SZA015 (ura3 and leu2 double deficient) increased by 189% (39.16 mg / L→113.06 mg / L), and the OD 600 After knocking out the leu2 gene, the EGT production (39.98 mg / L) and biomass (OD 600 The EGT production of the engineered strain SZA016 with only the ura3 gene knocked out was 108.03 mg / L, and the OD 600 The result was 12.21, which was close to the result of SZA015. It was confirmed that ura3 gene knockout promoted EGT anabolism in engineered bacteria ( Figure 1 D) Overexpression of EGT1-EGT2 in the SZA015 strain using a 2μ plasmid (with the ura3 gene) can restore bacterial growth. 600 was 37.35, but the EGT yield dropped to 68.41 mg / L ( Figure 1 D).

[0071] The growth characteristics of the knockout strains were evaluated in the absence of the EGT biosynthesis pathway. The results showed that the OD of the strain SZA012, in which only the leu2 gene was knocked out, was 600 was 43.08, which was not significantly different from the growth of the original strain Saccharomyces cerevisiaeCEN.PK113-7D; the OD of the strain SZA012U with only ura3 knocked out was 600 The OD of the strain SZA012UL in which both leu2 and ura3 were knocked out decreased to 12.99. 600 It dropped to 12.15 ( Figure 1 C) These results indicate that ura3 knockout leads to a significant decrease in biomass, regardless of whether the EGT biosynthesis pathway is introduced. This suggests that ura3 deletion is the key factor leading to the significant decrease in biomass, while leu2 knockout alone has no significant effect on host growth.

[0072] Table 1 Gene knockout primers and fragment length changes

[0073]

[0074] 5.2ura3 promoter replacement and metabolic regulation

[0075] Using SZA014 as the starting strain, the ura3 promoter was replaced with promoters of different strengths (P TEF1 , SEQ ID NO.3, P ATP14 , SEQ ID NO.4, P COX9 , SEQ ID NO.5, P HXT1 , SEQ ID NO.6, P PDA1 , SEQ ID NO.7, P COG7 , SEQ ID NO.8), and engineered strains SZA028-SZA033 were constructed. The promoter insertion was verified by colony PCR (Table 2, Figure 2 AF) and EGT production evaluation, and the optimal strain SZA033 (P COG7 promoter), its EGT production reached 69.44 mg / L, OD 600 The promoter replacement strategy revealed weak promoters (P COG7 ) can balance growth and product synthesis, and the EGT yield is increased by 74% compared with SZA014 ( Figure 2 G).

[0076] Table 2 Changes in length of ura3 promoter replacement fragments

[0077]

[0078] 5.3 Construction of Δpho89 and Δsps100 engineered strains and verification of fermentation performance

[0079] Using SZA033 as the starting strain, the pho89 gene (NCBI Gene ID: 852599) was knocked out using CRISPR / Cas9 technology to construct strain SZA045 (Δpho89); the sps100 gene (NCBI GeneID: 856541) was knocked out to construct strain SZA046 (Δsps100). Pho89 and sps100 were knocked out to construct strain SZA053 (Δpho89Δsps100). The gene knockout was verified by colony PCR and electrophoresis. The EGT production of SZA045 reached 89.09 mg / L (OD 600 =40.00), which was 28.30% higher than that of the starting strain SZA033; the EGT yield of SZA046 was 78.51 mg / L (OD 600 =41.17), which was 13.06% higher than that of the original strain SZA033. The EGT production of the strain SZA053, which had both pho89 and sps100 genes knocked out, decreased to 52.92 mg / L ( Figure 5D), indicating that the double knockout induces metabolic imbalance.

[0080] Example 6 Optimization of EGT fermentation process of strain SZA015

[0081] 4.1 SZA015 shake flask culture medium optimization

[0082] (1) The engineered strain SZA015 was inoculated into YPD medium containing different concentrations of glucose (20-40 g / L), L-cysteine (0.5-5.0 g / L), L-methionine (0.5-5.0 g / L), and L-histidine (0.5-5.0 g / L) and cultured at 30°C and 220 rpm for 72 h;

[0083] (2) The EGT content was detected by HPLC, and the optimal formula was determined to be: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, L-cysteine 2 g / L, L-methionine 2 g / L, L-histidine 2 g / L, and the EGT yield reached 183.70 mg / L.

[0084] The best solution for optimizing the SZA015 culture medium obtained in this example is:

[0085] Ura3 / leu2 supplementation strategy: The optimal ura3+leu2 addition amount was determined to be 10 mg / L, and the EGT yield increased by 31.76% (148.97 mg / L, Figure 3 A). Optimization of precursor addition: Adding 2.0g / L L-cysteine, 2.0g / L L-methionine, and 2.0g / L L-histidine to 2% glucose medium, the EGT production reached 183.70mg / L, an increase of 23.31% compared with the original results ( Figure 3 B).

[0086] 4.2SZA015 fed-batch fermentation

[0087] (1) A 10-L bioreactor with an initial volume of 7 L was used, with a glucose concentration of 20 g / L, a temperature of 30°C, a dissolved oxygen (DO) of 20–40%, an agitation rate of ≤800 rpm, an aeration ratio of 1–3 VVM, and a dynamic pH control of 5.0.

[0088] (2) After 16 h of fermentation, 80% glucose solution was added to maintain a low sugar concentration;

[0089] (3) The EGT yield was monitored by HPLC, and the final yield reached 2.26 g / L.

[0090] The optimal process for the fed-batch fermentation of SZA015 in a 10 L fermenter in this example is:

[0091] A two-stage fermentation strategy was used: the initial stage maintained a low sugar (glucose) concentration (1.2-1.8 g / L), pH 5.0, and DO 20-40%; glucose was continuously added during the feeding stage (80% glucose solution was added after 16 hours of fermentation to maintain a low sugar concentration of 1.2-1.8 g / L). The final EGT production reached 2.26 g / L, the cell dry weight was 28.89 g / L, and the OD 600 117.8, providing key process parameters for industrial production. ( Figure 4 ).

[0092] Example 7 pH Optimization and Substrate Addition of Strain SZA045

[0093] (1) Determination of optimal fermentation pH

[0094] Strain SZA045 was inoculated into YPD medium at pH 5.0-6.8 and cultured at 30°C and 220 rpm for 72 h;

[0095] Conclusion: The optimal pH is 5.0, and the EGT yield reaches 94.58 mg / L (OD 600 =38.83), see Figure 6 A.

[0096] (2) Optimization of precursor types and addition amounts

[0097] The strain SZA045 was inoculated into YPD medium at pH 5.0, and different concentrations of ( Figure 6 B) L-cysteine, L-methionine, and L-histidine were cultured at 30°C and 220 rpm for 72 h.

[0098] Conclusion: Under pH 5.0, the addition of 2.0 g / L L-cysteine, L-methionine, and L-histidine to YPD medium increased EGT production to 196.30 mg / L ( Figure 6 B), OD 600 =35.97, which is the highest level of brewer's yeast shake flask fermentation at present.

[0099] Example 8 Fermentation tank process verification of strain SZA045

[0100] (1) The engineered Saccharomyces cerevisiae strain was amplified with seed solution (12 h for primary seed solution and 10 h for secondary seed solution) and then inoculated into a 10 L fermenter;

[0101] (2) Dynamically control the pH to 5.0, DO 20-40%, maintain the glucose concentration at 2.1-2.6 g / L, and continuously add glucose solution during the feeding phase (when the glucose in the fermentation broth is consumed (approximately 16-20 h of fermentation), add 80% glucose mother solution to maintain the glucose concentration at 2.1-2.6 g / L);

[0102] (3) The final EGT production of strain SZA045 in a 10L fermenter reached 2.79g / L, the cell dry weight was 41.38g / L, and the OD 600 =162.6.

[0103] Conclusion: The feeding strategy was as follows: the glucose concentration was maintained at 2.1-2.6 g / L in the initial stage, and the pH was dynamically adjusted to 5.0; glucose was continuously added during the feeding stage, and the final EGT production reached 2.79 g / L, the cell dry weight was 41.38 g / L, and the OD 600 162.6, ethanol accumulation <1g / L ( Figure 7 ).

Claims

1. A ura 3 and leu 2. Double-defective Saccharomyces cerevisiae engineered strain SZA015, characterized in that: Knockout of Saccharomyces cerevisiae using CRISPR / Cas9 technology ura 3 and leu 2 genes, and two copies TDH3p-EGT1-ADH2t-TEF1p-EGT2- CyC1t The expression cassette was integrated into the Saccharomyces cerevisiae genome to construct the Saccharomyces cerevisiae engineered strain SZA015.

2. An engineered yeast strain SZA033, characterized in that: The cerevisiae engineered strain SZA015 according to claim 1 was used as the starting strain and the CRISPR / Cas9 technology was used to knock out leu2 Genes and ura3 The promoter was replaced with a weak promoter P COG7 , constructed the Saccharomyces cerevisiae engineered strain SZA033.

3. A pho89-deficient Saccharomyces cerevisiae engineered strain SZA045, characterized in that: The Saccharomyces cerevisiae engineering SZA033 described in claim 2 was used as the starting strain, and CRISPR / Cas9 technology was used to knock out pho89 Gene, construction of Saccharomyces cerevisiae engineered strain SZA045.

4. An sps100-deficient Saccharomyces cerevisiae engineered strain SZA046, characterized in that: The cerevisiae engineering strain SZA033 described in claim 2 was used as the starting strain, and the sps100 gene was knocked out by CRISPR / Cas9 technology to construct the cerevisiae engineering strain SZA046.

5. An optimized culture medium for the engineered strain of Saccharomyces cerevisiae according to any one of claims 1 to 4, characterized in that: Add 10 mg / L of YPD medium at pH 5.0 ura 3. 10 mg / L leu2, 1.0 g / L L-cysteine, 1.0 g / L L-methionine, 1.0 g / L L-histidine, and 2% glucose.

6. A fed-batch fermentation process for the engineered strain of Saccharomyces cerevisiae according to any one of claims 1 to 4, characterized in that: The pH was dynamically adjusted to 5.0 and the DO was 20-40% in a 10 L fermenter. When the glucose in the fermentation broth was consumed, 80% glucose mother solution was added to maintain the glucose concentration at 2.1-2.6 g / L.

7. the application of the saccharomyces cerevisiae engineered strain as described in any one of claims 1 to 4 in synthesizing thioneine.

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