A kind of brewer's yeast strain and its application

Abstract

This application relates to the fields of synthetic biology and microbial technology, specifically disclosing a *Saccharomyces cerevisiae* strain and its applications. The *Saccharomyces cerevisiae* strain PY-EDS8 was obtained through adaptive laboratory evolution after screening by ambient pressure room temperature plasma (ARTP) mutagenesis. The *Saccharomyces cerevisiae* strain PY-EDS8 of this application exhibits tolerance to high concentrations of succinic acid. The *Saccharomyces cerevisiae* strain obtained through this strain can efficiently produce succinic acid. This application demonstrates advantages such as stable production performance, high carbon conversion rate, and low byproduct production in succinic acid production, and has broad industrial application prospects.

Description

Technical Field

[0001] The present application relates to the fields of synthetic biology and microbial technology, and in particular to a brewer's yeast strain and its application, and further to a brewer's yeast strain tolerant to high concentration of succinic acid and its application. Background Art

[0002] Succinic acid (C4H6O4), also known as succinic acid, is a representative C4 organic acid fermentation product with important application value, mainly concentrated in the food, cosmetics, medicine, industry and agriculture industries. Polybutylene succinate (PBS) formed by the polymerization of succinic acid and its derivative 1,4-butanediol is currently recognized as the biodegradable plastic with the best comprehensive performance.

[0003] The traditional production method of succinic acid is chemical synthesis. In recent years, due to the excessive loss of non-renewable fossil resources and the increasingly serious environmental pollution problems, the market has gradually turned to a greener and more sustainable large-scale production method of succinic acid, that is, the production of succinic acid by microbial fermentation. Compared with traditional chemical synthesis, the biosynthesis of succinic acid is more environmentally friendly. It can not only use low-value renewable biomass resources as raw materials, but also fix carbon dioxide to reduce the greenhouse effect, showing a good development prospect. At present, the strains that have achieved industrial-scale succinic acid production are mainly concentrated in prokaryotes, such as succinic acid-producing Actinobacillus, succinic acid-producing Mannheimia, succinic acid-producing Anaerobic Spirillum, Corynebacterium glutamicum, Escherichia coli, etc. In the process of fermentation and production of succinic acid, as organic acids are continuously generated, the pH of the fermentation liquid continues to decrease. For bacteria that are sensitive to pH and osmotic pressure, it is necessary to add neutralizers (such as calcium carbonate, magnesium carbonate, and sodium hydroxide) to control the pH to 6.0-7.0 in order to maintain the vitality and fermentation efficiency of the strains during the production process. The large-scale use of neutralizing agents will convert succinic acid into succinate, requiring the subsequent separation and purification process to be re-acidified to obtain the succinic acid product. This not only increases production costs, but also generates a large amount of difficult-to-treat insoluble salt waste and high-salt wastewater, causing environmental pollution and limiting production capacity.

[0004] Compared with bacterial production strains, yeast naturally has strong acid resistance and high osmotic pressure resistance, so it can be fermented under low pH conditions. No or little neutralizer is added during the fermentation process, which can effectively reduce the cost of succinic acid separation. As a eukaryotic model microorganism, brewer's yeast has a clear genetic background, mature gene editing tools, simple nutritional requirements, tolerance to high-concentration substrates, and facultative anaerobicity, and is widely used in the production of various high-value-added chemicals.

[0005] Succinic acid is not a natural metabolic end product of Saccharomyces cerevisiae, and metabolic engineering is usually required to transform high-yield strains. Yan et al. constructed a rTCA branch in a pyruvate decarboxylase-deficient Saccharomyces cerevisiae strain to synthesize succinic acid. Under pH = 3.8, the strain produced 12.97 g / L succinic acid with a conversion rate of 0.13 g / g (BioresourceTechnology, 2014, 156: 232-239), which is obviously not up to the level of industrial production.

[0006] Therefore, there is an urgent need to develop a brewer's yeast strain that can tolerate high concentrations of succinic acid and efficiently produce succinic acid, which can effectively reduce the industrial production cost of succinic acid and solve environmental pollution problems. It has great industrial application value, but the currently disclosed brewer's yeast engineered bacteria do not have the above characteristics. Summary of the invention

[0007] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a brewer's yeast strain and its application. The brewer's yeast strain of the present application has the ability to tolerate high concentrations of succinic acid and can efficiently produce succinic acid. The present application has the advantages of stable production performance, high carbon conversion rate, and few by-products in the application of succinic acid production. The brewer's yeast strain of the present application that tolerates high concentrations of succinic acid and efficiently produces succinic acid has broad industrial application prospects.

[0008] To achieve the above purpose, the technical solution adopted by this application is:

[0009] The present application provides a brewer's yeast, which is brewer's yeast Saccharomyces cerevisiae PY-EDS8, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2023, with the deposit number GDMCC No: 63815, the classification number Saccharomyces cerevisiae, and the deposit address: Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The brewer's yeast Saccharomyces cerevisiae PY-EDS8 obtained in the present application has a typical ellipsoidal morphology of brewer's yeast, has the ability to tolerate high concentrations of succinic acid, and can grow rapidly in a liquid culture medium (pH=2.68) supplemented with 100 g / L succinic acid.

[0011] The present application provides a cerevisiae yeast strain, which is obtained by expressing malate dehydrogenase, fumarase, and fumarate reductase in cerevisiae Saccharomyces cerevisiae PY-EDS8, and knocking out glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase, and acetaldehyde dehydrogenase, to obtain the cerevisiae yeast strain Saccharomyces cerevisiae ST100-Suc3.

[0012] The malate dehydrogenase gene (MDH) is the malate dehydrogenase gene ScMDH3R from Saccharomyces cerevisiae with truncated C-terminal amino acids 341-343 (ΔSKL); the fumarase gene is the fumarase gene RoFUM from Rhizopus oryzae; and the fumarate reductase gene is the fumarate reductase gene TbFRD from Trypanosoma brucei.

[0013] In the present application, Saccharomyces cerevisiae ST00 (i.e., Saccharomyces cerevisiae PK2-1Ca) is used as the starting strain, which is first screened by mutagenesis using normal pressure room temperature plasma (ARTP), and then domesticated in a liquid culture medium supplemented with gradient concentrations of succinic acid.

[0014] Specifically:

[0015] (1) Atmospheric pressure room temperature plasma (ARTP) mutagenesis screening:

[0016] ① Strain activation and preparation of bacterial suspension: Saccharomyces cerevisiae ST00 was inoculated into liquid culture medium, cultured at 30°C, 220rpm for 48h, then transferred to fresh liquid culture medium at a 5% inoculation rate, and cultured at 30°C for 48h. The strains used in the following experiments were all second-generation bacterial liquid. The bacterial liquid was centrifuged and the supernatant was discarded. The bacteria were washed with PBS solution 3 times, and then resuspended with physiological saline. The OD value of the bacterial suspension was calculated. 600 Adjust to 1.0.

[0017] ②ARTP mutagenesis treatment: Take 10 μL of the prepared bacterial suspension and evenly spread it on the sterilized and cooled stainless steel slide, and place it in the ARTP breeding machine. The mutagenesis power is 120 W, the high-purity helium ventilation volume is 10 SLM, the treatment distance is 2 mm, and the treatment is 0, 20, 40, 60, 80, and 100 s respectively. After each mutagenesis, the stainless steel slide is placed in an EP tube containing 990 μL sterile saline, and oscillated and washed on a vortex for 1-2 min. The mutagenic bacteria are eluted and spread on a solid culture medium supplemented with 80 g / L succinic acid. It is cultured at 30°C for 48 h, and the grown colonies are selected.

[0018] ③ Screening of mutagenic strains: The preserved colonies after mutagenesis were inoculated into liquid culture medium, cultured at 30°C, 220rpm for 48h, then transferred to fresh liquid culture medium (pH=2.81) supplemented with 80g / L succinic acid, cultured at 30°C for 48h, and the OD was measured. 600 . Select OD 600 Colonies >10 were deposited.

[0019] (2) Adaptive evolution:

[0020] ① Inoculate the strain obtained after mutagenesis into liquid culture medium to obtain seed liquid;

[0021] ② The seed solution of step ① was transferred to a liquid culture medium supplemented with 85 g / L succinic acid at a 1-10% v / v inoculation rate, and long-term adaptive evolution culture was performed at 30°C and 220 rpm;

[0022] ③ After the Saccharomyces cerevisiae strain in the succinic acid liquid medium has gone through a long selection adaptation period, has obvious growth and has reached a stable period, the culture is transferred to a fresh liquid medium containing 85g / L succinic acid at a 1-10% v / v inoculation rate, and further adaptive culture is carried out at 30°C and 220rpm to shorten the hysteresis period of the strain;

[0023] ④ Repeat step ③ and repeatedly subculture the Saccharomyces cerevisiae strain in a fresh liquid culture medium containing 85 g / L succinic acid until the specific growth rate of the strain in the liquid culture medium containing 85 g / L succinic acid doubles, and screen out Saccharomyces cerevisiae mutants with higher tolerance to succinic acid concentrations for preservation.

[0024] ⑤ Repeat steps ①-④, gradually increase the concentration of exogenously added succinic acid to 90, 95, and 100 g / L, and finally screen and obtain Saccharomyces cerevisiae PY-EDS8 with the ability to tolerate 100 g / L succinic acid.

[0025] The present application provides a biological preparation, which includes the above-mentioned Saccharomyces cerevisiae PY-EDS8 or Saccharomyces cerevisiae ST100-Suc3.

[0026] In the present application, the saccharomyces cerevisiae strain that efficiently produces succinic acid is obtained by implementing genome site-directed editing under the guidance of the CRISPR / Cas9 system, constructing the succinic acid synthesis pathway, knocking out the byproduct synthesis gene, and making the carbon metabolic flow flow more to the succinic acid synthesis pathway, and then cultivating and optimizing. The saccharomyces cerevisiae strain is specifically obtained by expressing the malate dehydrogenase gene (ScMDH3R), fumarase (RoFUM), and fumarate reductase (TbFRD) in saccharomyces cerevisiae PY-EDS8, and knocking out glycerol-3-phosphate dehydrogenase (GPD1), alcohol dehydrogenase (ADH1), and acetaldehyde dehydrogenase (ALD6), that is, the saccharomyces cerevisiae ST100-Suc3.

[0027] As a preferred embodiment of the method for constructing the Saccharomyces cerevisiae strain of the present application, the method for constructing the Saccharomyces cerevisiae strain comprises the following steps:

[0028] S1. Construction of gRNA expression vectors targeting glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase and acetaldehyde dehydrogenase;

[0029] S2, constructing a malate dehydrogenase recombinant expression vector, a fumarase recombinant expression vector, and a fumarate reductase recombinant expression vector, and obtaining a malate dehydrogenase expression cassette fragment, a fumarase expression cassette fragment, and a fumarate reductase expression cassette fragment by PCR nucleic acid amplification, respectively;

[0030] S3. Co-transform the gRNA expression vector obtained in step S1 and the malate dehydrogenase expression cassette fragment, the fumarase expression cassette fragment and the fumarate reductase expression cassette fragment obtained in step S2 into Saccharomyces cerevisiae PY-EDS8 competent cells to obtain the Saccharomyces cerevisiae strain Saccharomyces cerevisiae ST100-Suc3.

[0031] As a preferred embodiment of the method for constructing the Saccharomyces cerevisiae strain described in the present application, in the step S1, during the construction of the gRNA expression vector targeting glycerol-3-phosphate dehydrogenase, ethanol dehydrogenase and acetaldehyde dehydrogenase, primers as shown in the nucleotide sequences SEQ ID NOs: 2 to 9 are used for amplification.

[0032] As a preferred embodiment of the method for constructing the brewer's yeast strain described in the present application, in the step S2, the primers shown in the nucleotide sequences SEQ ID NOs: 10 to 21 are used for amplification during the construction of the malate dehydrogenase recombinant expression vector, the fumarase recombinant expression vector, and the fumarate reductase recombinant expression vector.

[0033] As a preferred embodiment of the method for constructing the Saccharomyces cerevisiae strain described in the present application, in the step S2, the amplification is performed using primers as shown in the nucleotide sequences SEQ ID NOs: 22 to 27.

[0034] The transcriptome expression difference analysis of the brewer's yeast Saccharomyces cerevisiae PY-EDS8 of the present application showed that compared with Saccharomyces cerevisiae ST00, the transcription levels of genes involved in gene expression such as replication, transcription, and translation, genes involved in energy generation and conversion, some genes involved in the TCA cycle, and genes involved in ion exchange secretion were increased by 2 to 40 times.

[0035] The present application provides the use of the above-mentioned Saccharomyces cerevisiae PY-EDS8 or Saccharomyces cerevisiae ST100-Suc3 in the production of succinic acid.

[0036] In the present application, after introducing the succinate synthesis pathway gene into the brewer's yeast Saccharomyces cerevisiae PY-EDS8, the central carbon metabolism pathway is optimized and redirected, and the brewer's yeast strain Saccharomyces cerevisiae ST100-Suc3 obtained has the ability to efficiently synthesize succinate.

[0037] The present application also provides a method for producing succinic acid, using the above-mentioned Saccharomyces cerevisiae ST100-Suc3 strain as a fermentation bacterium.

[0038] As a preferred embodiment of the method for producing succinic acid described in the present application, the method for producing succinic acid comprises the following steps:

[0039] 1) The above-mentioned yeast strain Saccharomyces cerevisiae ST100-Suc3 was cultured at a fermentation temperature of 25-35°C and a stirring speed of 180-220 rpm for 24-48 hours to obtain a seed solution;

[0040] 2) Fermenting the seed liquid obtained in step 1) in a fermenter at a fermentation temperature of 25-35° C., an aeration volume of 1.0-2.0 vvm, and a stirring speed of 400-800 rpm; during the fermentation process, the dissolved oxygen concentration is 20%-30%, and the glucose concentration is 10-20 g / L.

[0041] The brewer's yeast strain Saccharomyces cerevisiae ST100-Suc3 of the present application was fermented for 78 hours in a 7L bioreactor level fermentation, and the titer and conversion rate of succinic acid reached 76.5 g / L and 0.65 g / g respectively, with a final pH of 2.85. The strain has the potential for industrial application in the production of succinic acid by low pH fermentation.

[0042] Compared with the prior art, this application has the following beneficial effects:

[0043] 1. This application uses Saccharomyces cerevisiae ST00 as the starting strain, and obtains Saccharomyces cerevisiae PY-EDS8 through mutagenesis and adaptive evolution culture methods. Saccharomyces cerevisiae PY-EDS8 can tolerate high concentrations (100 g / L) of succinic acid, and the transcription levels of genes related to gene expression, energy metabolism, and ion transport are increased by 2-40 times.

[0044] 2. This application constructs a succinic acid synthesis pathway in Saccharomyces cerevisiae PY-EDS8, and obtains the Saccharomyces cerevisiae ST100-Suc3 strain through central carbon metabolism redirection optimization, thereby improving cell production capacity and realizing efficient production of succinic acid by Saccharomyces cerevisiae cell factories under low pH conditions.

[0045] 3. Compared with the traditional chemical synthesis method and the biosynthesis method which requires the addition of a neutralizing agent, the method of producing succinic acid by reconstructing the biosynthetic pathway in the brewer's yeast strain Saccharomyces cerevisiae ST100-Suc3 which tolerates high concentrations of succinic acid has the characteristics of short cycle, low pollution, resource conservation and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The result diagram of the content of succinic acid in the fermentation broth of Saccharomyces cerevisiae PY-EDS8 at different fermentation time points in Example 2;

[0047] Figure 2 This is a comparison chart of bacterial growth before and after the strain mutation in Example 3;

[0048] Figure 3 This is the result diagram of the transcriptome data of Saccharomyces cerevisiae PY-EDS8 in Example 4;

[0049] Figure 4Metabolic diagram of the biosynthetic succinate pathway and byproduct synthesis pathway of Saccharomyces cerevisiae ST100-Suc3;

[0050] Figure 5 This is a diagram of the fermentation results of Saccharomyces cerevisiae ST100-Suc3 and Saccharomyces cerevisiae PY-EDS8 metabolizing glucose to synthesize succinic acid. DETAILED DESCRIPTION

[0051] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0052] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0053] The composition of the liquid culture medium is: glucose 20 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0054] The composition of the solid culture medium is: glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L, agar 20 g / L.

[0055] Example 1. Mutagenesis screening and adaptive evolution of succinic acid-tolerant strains of Saccharomyces cerevisiae

[0056] In this application, Saccharomyces cerevisiae ST00 (i.e., Saccharomyces cerevisiae PK2-1Ca) is used as the starting strain, and firstly, atmospheric pressure room temperature plasma (ARTP) mutagenesis screening is performed to obtain mutant strains, and then, succinic acid with a concentration gradient is exogenously added, and adaptive evolution culture is performed to screen for Saccharomyces cerevisiae mutant strains that tolerate higher concentrations of succinic acid and lower pH.

[0057] (1) Atmospheric pressure room temperature plasma (ARTP) mutagenesis screening:

[0058] ① Strain activation and preparation of bacterial suspension:

[0059] Saccharomyces cerevisiae ST00 was inoculated into liquid culture medium and cultured at 30°C, 220rpm for 48h. Then, it was transferred to fresh liquid culture medium at a 5% inoculation rate and cultured at 30°C for 48h. The strains used in the following experiments were all second-generation bacterial liquid. The bacterial liquid was centrifuged and the supernatant was discarded. The cells were washed three times with PBS solution and then resuspended with physiological saline. The OD value of the bacterial suspension was calculated. 600 Adjust to 1.0.

[0060] ②ARTP mutagenesis treatment:

[0061] Take 10 μL of the prepared bacterial suspension and evenly spread it on the sterilized and cooled stainless steel slide, and place it in the ARTP breeding machine. The mutagenesis power was 120 W, the high-purity helium ventilation volume was 10 SLM, the treatment distance was 2 mm, and the treatment was 0, 20, 40, 60, 80, and 100 s respectively. After each mutagenesis, the stainless steel slide was placed in an EP tube containing 990 μL sterile saline, and oscillated and washed on a vortex for 1-2 min. The mutagenic bacteria were eluted and spread on a solid culture medium supplemented with 80 g / L succinic acid. It was cultured at 30°C for 48 h, and the grown colonies were selected and preserved.

[0062] ③ Screening of mutagenic strains:

[0063] The preserved colonies after mutagenesis were inoculated into liquid culture medium and cultured at 30°C, 220 rpm for 48 h. Then, they were transferred to fresh liquid culture medium (pH = 2.81) supplemented with 80 g / L succinic acid and cultured at 30°C for 48 h. The OD 600 . Select OD 600 Colonies >10 were deposited.

[0064] (2) Adaptive evolution:

[0065] ① Inoculate the preserved mutant into 5 mL of liquid culture medium and culture at 30°C and 220 rpm for 12-24 h;

[0066] ② Cultivate until the stable phase, transfer the seed solution of step ① to 20 mL of liquid culture medium supplemented with 85 g / L succinic acid at a 1-10% v / v inoculation amount, and perform long-term adaptive evolution cultivation at 30°C and 220 rpm;

[0067] ③ After the Saccharomyces cerevisiae strain in the liquid medium containing succinic acid has gone through a long selection adaptation period, has obvious growth and has reached a stable period, the culture is transferred to a fresh liquid medium containing 85 g / L succinic acid at an inoculation rate of 1-10% v / v for further adaptive culture to shorten the hysteresis period of the strain. The culture conditions are the same as step ②;

[0068] ④ Repeat step ③ and repeatedly subculture the Saccharomyces cerevisiae strain in a fresh liquid culture medium containing 85 g / L succinic acid until the specific growth rate of the strain in the liquid culture medium containing 85 g / L succinic acid doubles, and screen out Saccharomyces cerevisiae mutants with higher tolerance to succinic acid concentrations for preservation.

[0069] ⑤ Repeat steps ①-④, gradually increase the concentration of exogenously added succinic acid to 90, 95, and 100 g / L, and finally screen and obtain Saccharomyces cerevisiae PY-EDS8 that can tolerate 100 g / L succinic acid. This adaptive evolution process lasted 236 days.

[0070] The brewer's yeast Saccharomyces cerevisiae PY-EDS8 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2023, with the deposit number GDMCC No: 63815, the classification number Saccharomyces cerevisiae, and the deposit address: Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0071] The genomic DNA of Saccharomyces cerevisiae PY-EDS8 was extracted and the ITS sequence was determined. The nucleotide sequence (SEQ ID NO: 1) is:

[0072]

[0073] Example 2: Verification of the cause of succinic acid tolerance in Saccharomyces cerevisiae PY-EDS8

[0074] It was verified that the succinate tolerance of Saccharomyces cerevisiae PY-EDS8 originated from its physiological adaptability to succinate rather than the degradation of succinate.

[0075] 1. Inoculate Saccharomyces cerevisiae PY-EDS8 into 5 mL of liquid culture medium and culture at 30°C and 220 rpm for 18-24 hours to obtain seed solution;

[0076] 2. When the strain reaches the stable stage, the seed solution in step 1 is heated to the initial OD 600 The cell culture medium was transferred to 10 mL of liquid culture medium supplemented with 100 g / L succinic acid at 0.05, and cultured at 30°C and 200 rpm for 48 h.

[0077] 3. During the fermentation process, the fermentation liquid was centrifuged, the supernatant was collected, and the content of succinic acid at each sampling point was detected ( Figure 1 ).

[0078] The results showed that the concentration of succinic acid in the culture medium remained stable during the fermentation process, indicating that Saccharomyces cerevisiae PY-EDS8 would not degrade succinic acid, and that the tolerance of Saccharomyces cerevisiae PY-EDS8 to high concentrations of succinic acid originated from its physiological adaptability to succinic acid.

[0079] Example 3. Comparison of Growth of Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8 in Succinic Acid Liquid Medium

[0080] 1. Inoculate the starting strains Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8 into 5 mL of liquid culture medium respectively, and culture at 30°C and 220 rpm for 18-24 hours to obtain seed solution;

[0081] 2. When the strain reaches the stable stage, the two seed solutions in step 1 are heated to the starting OD 600The culture medium was transferred to 20 mL of liquid culture medium with different concentrations of succinic acid added at a concentration of 0.05. The added concentrations of exogenous succinic acid (g / L) were: 0 (pH=6.8), 40 (pH=3.5), 60 (pH=3.0), 80 (pH=2.81), 90 (pH=2.75), and 100 (pH=2.68), and the culture was continued under the same conditions.

[0082] 3. Take samples every 12 hours during the culture process, and measure the strain OD at 72 hours. 600 , draw the bacterial growth curve ( Figure 2 ).

[0083] The results showed that in the liquid culture medium with succinic acid concentration higher than 40 g / L, the starting strain Saccharomyces cerevisiae ST00 hardly grew; while Saccharomyces cerevisiae PY-EDS8 could grow normally and accumulate biomass in the liquid culture medium with succinic acid, indicating that the tolerance of Saccharomyces cerevisiae PY-EDS8 to high concentrations of succinic acid was greatly improved.

[0084] Example 4. Gene expression difference analysis between Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8

[0085] The starting strains Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8 were inoculated into 120 mL of liquid culture medium containing 40 g / L succinic acid and cultured at 30 °C and 220 rpm until the early logarithmic growth phase of the strains. At this time, the OD of the two strains was 600 were 2.3 and 2.8 respectively; the bacteria were collected by centrifugation at 1000×g, and the wet weights of the two strains were 0.7g and 0.8g respectively; the total RNA of the strains was extracted using an RNA extraction kit, and the RNA library was constructed.

[0086] Transcriptome data showed that ( Figure 3), a total of 335 genes had significant differences in expression abundance. Among them, 232 genes were upregulated and 103 genes were downregulated. The functions of upregulated genes were mainly concentrated in gene expression, energy production and conversion, ion transport and secretion, and the expression of related genes increased by 2-40 times; the functions of downregulated genes were mainly concentrated in transposition and carbohydrate transport and metabolism, and the related genes were downregulated to 1 / 239-1 / 2. This shows that after adaptive evolution, Saccharomyces cerevisiae PY-EDS8 has higher gene expression stability, higher energy supply efficiency and stronger ion transport ability in the unfavorable growth environment of high acid and high osmotic pressure.

[0087] Example 5: Application of Saccharomyces cerevisiae PY-EDS8 in the efficient production of succinic acid

[0088] 1. Construction of Saccharomyces cerevisiae strains for synthesizing succinic acid:

[0089] (1) Construction of guide RNA (gRNA) expression vectors targeting byproducts glycerol-3-phosphate dehydrogenase (GPD1), alcohol dehydrogenase (ADH1), and acetaldehyde dehydrogenase (ALD6);

[0090] (2) Construction of malate dehydrogenase (ScMDH3R), fumarase (RoFUM), and fumarate reductase (TbFRD) recombinant expression vectors;

[0091] (3) The ScMDH3R expression cassette fragment, the RoFUM expression cassette fragment, and the TbFRD expression cassette fragment were obtained by PCR nucleic acid amplification;

[0092] (4) The guide RNA expression vector obtained in step (1) and the gene expression cassette fragment obtained in step (3) are co-transformed into Saccharomyces cerevisiae competent cells (Saccharomyces cerevisiae PY-EDS8) to obtain Saccharomyces cerevisiae ST100-Suc3 with the ability to efficiently produce succinic acid.

[0093] The guide RNA expression vector described in step (1) is obtained by the following method: using p426-SNR52p-gRNA.CAN.Y-SUP4t as a template, GPD1-1, GPD1-2, ADH1-1, ADH1-2, ALD6-1 and ALD6-2 fragments are amplified respectively, and GPD1-1 and GPD1-2, ADH1-1 and ADH1-2, ALD6-1 and ALD6-2 fragments are recombined respectively to obtain recombinant vectors with GPD1, ADH1 and ALD6 guide RNAs, and then the three recombinant vectors are used as templates to amplify fragments targeting GPD1, ADH1 and ALD6 respectively, and the fragments are connected to obtain guide RNA expression vectors for targeted knockout of glycerol-3-phosphate dehydrogenase (GPD1), alcohol dehydrogenase (ADH1), and acetaldehyde dehydrogenase (ALD6).

[0094] The primer sequences used are as follows:

[0095] GPD1-F: 5'-TCTACCAGCATTCAAGTGGCGTTTTAGAGCTAGAAATAG-3' (SEQ ID NO: 2);

[0096] GPD1-R: 5'-GCCACTTGAATGCTGGTAGAGATCATTTATCTTTCACTG-3' (SEQ ID NO: 3);

[0097] ADH1-F: 5'-GTTATCTTCTACGAATCCCAGTTTTAGAGCTAGAAATAG-3' (SEQ ID NO: 4);

[0098] ADH1-R: 5'-TGGGATTCGTAGAAGATAACGATCATTTATCTTTCACTG-3' (SEQ ID NO: 5);

[0099] ALD6-F: 5'-TTTGGAAGTGTGATCTTGACGTTTTAGAGCTAGAAATAG-3' (SEQ ID NO: 6);

[0100] ALD6-R: 5'-GTCAAGATCACACTTCCAAAGATCATTTATCTTTCACTG-3' (SEQ ID NO: 7);

[0101] Tong-F:5'-TAATAATGGTTTCTTAGTATGA-3' (SEQ ID NO: 8);

[0102] Tong-R: 5'-ACTAAGAAACATTATTATCAT-3' (SEQ ID NO: 9).

[0103] The malate dehydrogenase recombinant expression vector described in step (2) is obtained by the following method: using YEplac181 as a starting vector, introducing a malate dehydrogenase expression cassette, and obtaining the malate dehydrogenase recombinant expression vector YEplac181-PCCW12-ScMDH3R-TCYC1.

[0104] The primer sequences used are as follows:

[0105] ScMDH3R-F:5'-CTAGATATTGATATAGTATGGTCAAAGTCGCAATTC-3' (SEQ ID NO: 10);

[0106] ScMDH3R-R:5'-GAGTCGACGGGCCGCATAAGAGTCTAGGATGAAACTCTTGCC-3' (SEQ ID NO: 11);

[0107] CYC1t-F:5'-GAGTCGACGGGCCGCATCATGTAATTAGTTATG-3' (SEQ ID NO: 12);

[0108] CCW12p-R: 5'-CTAGATATTGATATAGTGTTTAAGCGAATGACAGAAGATTA-3' (SEQ ID NO: 13).

[0109] The fumarase recombinant expression vector described in step (2) is obtained by the following method: using YEplac181 as a starting vector, introducing a fumarase expression cassette, and obtaining the fumarase recombinant expression vector YEplac181-PCCW12-RoFUM-TCYC1.

[0110] The primer sequences used are as follows:

[0111] RoFUM-F:5'-AACACTATATCAATATCTAGATGTCATCCGCTTCTG-3' (SEQ ID NO: 14);

[0112] RoFUM-R:5'-ATGATGCGGCCCGTCGACTCTTAATTCTTTAGCTGAAATCATATCTTC-3' (SEQ IDNO: 15);

[0113] CYC1t-F:5'-GAGTCGACGGGCCGCATCATGTAATTAGTTATG-3' (SEQ ID NO: 16);

[0114] CCW12p-R: 5'-CTAGATATTGATATAGTGTTTAAGCGAATGACAGAAGATTA-3' (SEQ ID NO: 17).

[0115] The fumarate reductase recombinant expression vector described in step (2) is obtained by the following method: using YEplac181 as a starting vector, introducing a fumarate reductase expression cassette, and obtaining the fumarate reductase recombinant expression vector YEplac181-PCCW12-TbFRD-TCYC1.

[0116] The primer sequences used are as follows:

[0117] TbFRD-F:5'-CTAGATATTGATATAGTGTTATGGTTGATGGTAGATCTTCAGCTT-3' (SEQ IDNO: 18);

[0118] TbFRD-R:5'-GAGTCGACGGGCCGCATTTAGCTACCACTAGGTTCAGTTTCATC-3' (SEQ ID NO: 19);

[0119] CYC1t-F:5'-GAGTCGACGGGCCGCATCATGTAATTAGTTATG-3' (SEQ ID NO: 20);

[0120] CCW12p-R: 5'-CTAGATATTGATATAGTGTTTAAGCGAATGACAGAAGATTA-3' (SEQ ID NO: 21).

[0121] The expression cassette fragments described in step (3) are donor1, donor2 and donor3, which are amplified using the recombinant expression vector in step 2 as a template.

[0122] The primers used are as follows:

[0123] ScMDH3R-Donor-F:5'-AGATGTCTGCTGCTGCTGATAGATTAAACTTAACTTCCGGCTGGGTGAGCATATGTTCC-3' (SEQ ID NO: 22);

[0124] ScMDH3R-Donor-R:5'-GGCTTTTCGGCAGCCTTCAAAGAAACAGAAGAGGAACTTCTACGCCAAGCTTTTTGATT-3' (SEQ ID NO: 23);

[0125] RoFUM-Donor-F:5'-TCAAGCTATACCAAGCATACAATCAACTATCTCATATACAGAATTCGAGCTCAACCAGG-3' (SEQ ID NO: 24);

[0126] RoFUM-Donor-R:5'-ATCTTTGTATTCCAACTTACCGTGGGATTCGTAGAAGATAGCAAATTAAAGCCTTCGAG-3' (SEQ ID NO: 25);

[0127] TbFRD-Donor-F:5'-TCAGAATACAATGACTAAGCTACACTTTGACACTGCTGAAATACTAGCGTTGAATGTTA-3' (SEQ ID NO: 26);

[0128] TbFRD-Donor-R:5'-TGGTTTGACATACGAGCAACCAACCGGTCTATTCATTAACCATAGGGTAGGGGAATTTC-3' (SEQ ID NO: 27);

[0129] Step (4) is as follows: In the presence of the gene expression cassette fragment, the Cas9 system is activated after the guide RNA expression vector obtained in step 1 is transformed into a Saccharomyces cerevisiae competent cell, achieving site-specific editing of the yeast genome and obtaining Saccharomyces cerevisiae ST100-Suc3 that efficiently produces succinic acid. The metabolic diagram of the biosynthetic succinic acid pathway and the byproduct synthesis pathway is shown in Figure 2. Figure 4 shown.

[0130] The Cas9 system is a yeast gene editing Cas9 expression plasmid p414-TEF1p-Cas9-CYC1t.

[0131] The method for constructing an engineered yeast strain for efficiently producing succinic acid also includes the steps of activating and enriching the engineered bacteria obtained in step (4): activating, inoculating, culturing, inoculating, and fermenting the obtained engineered bacteria.

[0132] The solid culture medium used for activation is composed of 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone and 20 g / L agar.

[0133] The liquid culture medium used for inoculation, culture and fermentation is composed of 20 g / L glucose, 10 g / L yeast extract and 20 g / L peptone.

[0134] The inoculation, culture and fermentation conditions are: temperature 25-35°C, rotation speed 180-220rpm, and culture 24-48h.

[0135] 2. Engineering strains metabolize glucose to produce succinic acid in 7 L bioreactor

[0136] (1) Seed solution preparation

[0137] Saccharomyces cerevisiae ST100-Suc3 and Saccharomyces cerevisiae PY-EDS8 were streaked on solid plate culture medium, cultured in a constant temperature incubator at 30°C for 48 h, single colonies were picked and inoculated into 50 mL seed culture medium, and cultured at 30°C and 220 rpm for 48 h.

[0138] (2) 7 L bioreactor fermentation culture

[0139] The seed liquid prepared in step (1) was inoculated into the fermentation tank at a 5% inoculation rate, the volume of the fermentation medium was 3 L, the fermentation temperature was 30 ° C, the ventilation volume was 1.5 vvm, the initial stirring speed was 400 rpm, and the dissolved oxygen concentration was controlled at 20%-30% during the fermentation process, and the glucose concentration was controlled at 10-20 g / L.

[0140] 3. Detection of fermentation products

[0141] During the fermentation process, the fermentation broth was centrifuged at 12000rpm for 3min, 200 μL of the fermentation broth supernatant was taken, 800 μL of 5mM H2SO4 was added, and filtered into a clean liquid chromatography bottle with a 0.22μm sterile inorganic filter. The fermentation broth product was detected by high performance liquid chromatography (HPLC), chromatographic column: AminexHPX-87H; mobile phase: 5mM H2SO4; flow rate: 0.6mL / min; column temperature: 35℃; injection volume: 10 μL; detector: differential detector.

[0142] From the fermentation results ( Figure 5) It can be seen that Saccharomyces cerevisiae PY-EDS8 produces almost no succinic acid, and the yields of ethanol, acetic acid, and glycerol are 51.3 g / L, 12.5 g / L, and 5.6 g / L, respectively. However, after Saccharomyces cerevisiae ST100-Suc3 produced 15 g / L succinic acid in the initial 20 hours, the pH dropped to 3.9, and it was still able to continue to use glucose for growth and conversion, and finally produced 76.5 g / L succinic acid within 78 hours of fermentation, with a conversion rate of 0.65 g / g, and the yields of byproducts ethanol, acetic acid, and glycerol were 5.36 g / L, 2.3 g / L, and 0.4 g / L, respectively. The yield and conversion rate of succinic acid are the highest levels currently reported for Saccharomyces cerevisiae, and have broad prospects in industrial production.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A brewer's yeast, characterized in that: The brewer's yeast is Saccharomyces cerevisiae PY-EDS8, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2023, with the collection number GDMCC No: 63815, and the collection address: Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A Saccharomyces cerevisiae ST100-Suc3 strain, characterized in that: The Saccharomyces cerevisiae strain is obtained by overexpressing malate dehydrogenase, fumarase, and fumarate reductase in the Saccharomyces cerevisiae PY-EDS8 according to claim 1, and simultaneously knocking out glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase, and acetaldehyde dehydrogenase.

3. A biological agent, characterized in that: The biological agent comprises the Saccharomyces cerevisiae PY-EDS8 according to claim 1 or the Saccharomyces cerevisiae ST100-Suc3 according to claim 2.

4. A method for constructing a cerevisiae yeast strain as claimed in claim 2, characterized in that: The following steps are involved: S1. Construction of gRNA expression vectors targeting glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase and acetaldehyde dehydrogenase; S2, constructing a malate dehydrogenase recombinant expression vector, a fumarase recombinant expression vector, and a fumarate reductase recombinant expression vector, and obtaining a malate dehydrogenase expression cassette fragment, a fumarase expression cassette fragment, and a fumarate reductase expression cassette fragment by PCR nucleic acid amplification, respectively; S3. Co-transform the gRNA expression vector obtained in step S1 and the malate dehydrogenase expression cassette fragment, the fumarase expression cassette fragment and the fumarate reductase expression cassette fragment obtained in step S2 into Saccharomyces cerevisiae PY-EDS8 competent cells to obtain the Saccharomyces cerevisiae strain.

5. The method for constructing a saccharomyces cerevisiae strain according to claim 4, characterized in that: In the step S1, during the construction of the gRNA expression vector targeting glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase and acetaldehyde dehydrogenase, primers as shown in the nucleotide sequences SEQ ID NOs: 2 to 9 are used for amplification.

6. The method for constructing a saccharomyces cerevisiae strain according to claim 4, characterized in that: In the step S2, during the construction of the malate dehydrogenase recombinant expression vector, the fumarase recombinant expression vector, and the fumarate reductase recombinant expression vector, primers as shown in the nucleotide sequences SEQ ID NOs: 10 to 21 are used for amplification.

7. The method for constructing a saccharomyces cerevisiae strain according to claim 4, characterized in that: In the step S2, the amplification is performed using primers as shown in the nucleotide sequences SEQ ID NOs: 22-27.

8. Use of the saccharomyces cerevisiae according to claim 1 or the saccharomyces cerevisiae strain according to claim 2 in producing succinic acid.

9. A method for producing succinic acid, characterized in that: The saccharomyces cerevisiae strain as claimed in claim 2 is used as the fermentation bacteria.

10. The method according to claim 9, characterized in that The following steps are involved: 1) amplifying and culturing the brewer's yeast strain according to claim 2, and culturing for 24-48 hours at a fermentation temperature of 25-35° C. and a stirring speed of 180-220 rpm to obtain a seed solution; 2) Fermenting the seed liquid obtained in step 1) in a fermenter at a fermentation temperature of 25-35° C., an aeration volume of 1.0-2.0 vvm, and a stirring speed of 400-800 rpm; during the fermentation process, the dissolved oxygen concentration is 20%-30%, and the glucose concentration is 10-20 g / L.

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