Saccharomyces cerevisiae strain and use thereof
By gene editing and adaptive evolution of Saccharomyces cerevisiae strains, the problem of low efficiency in succinic acid production under high concentration conditions has been solved, achieving efficient and low-cost succinic acid production, which has promising industrial application prospects.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- KINGFA SCI & TECH CO LTD
- Filing Date
- 2025-10-23
- Publication Date
- 2026-04-30
AI Technical Summary
Existing Saccharomyces cerevisiae strains have low efficiency in producing succinic acid under high concentrations of succinic acid, resulting in high production costs and environmental pollution. Furthermore, the use of traditional neutralizing agents increases the difficulty of separation and purification and the pressure of waste disposal.
Saccharomyces cerevisiae PY-EDS8 was obtained through atmospheric pressure and room temperature plasma mutagenesis screening and adaptive evolution culture. Gene editing was performed using the CRISPR/Cas9 system to knock out the genes of glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase, and acetaldehyde dehydrogenase, and to express malate dehydrogenase, fumarate enzyme, and fumarate reductase, thus redirecting carbon metabolism to the succinic acid synthesis pathway.
This method enables the efficient production of brewing yeast under high concentration succinic acid conditions, reduces production costs, decreases byproducts, improves carbon conversion rate, and has low pH fermentation capability, thus possessing potential for industrial application.
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Abstract
Description
A strain of Saccharomyces cerevisiae and its application Technical Field
[0001] This application relates to the fields of synthetic biology and microbial technology, and in particular to a strain of Saccharomyces cerevisiae and its application, and more particularly to a strain of Saccharomyces cerevisiae that is tolerant to high concentrations of succinic acid and its application. Background Technology
[0002] Succinic acid (C4H6O4), also known as succinic acid, is a representative C4 organic acid fermentation product with significant applications, primarily in the food, cosmetics, pharmaceutical, and industrial sectors. Polybutylene succinate (PBS), formed by the polymerization of succinic acid and its derivative 1,4-butanediol, is currently recognized as the best-performing biodegradable plastic in terms of overall performance.
[0003] The traditional method for producing succinic acid is chemical synthesis. In recent years, due to the excessive depletion of non-renewable fossil resources and increasingly serious environmental pollution problems, the market has gradually shifted towards a greener and more sustainable large-scale production method for succinic acid: microbial fermentation. Compared with traditional chemical synthesis, the biosynthesis of succinic acid is more environmentally friendly. It can use low-value renewable biomass resources as raw materials and can also fix carbon dioxide to mitigate the greenhouse effect, showing promising development prospects. Currently, the strains used for industrial-scale succinic acid production are mainly prokaryotes, such as *Actinomyces succinate*, *Mannidaria succinate*, *Anaerobes succinate*, *Corynebacterium glutamicum*, and *Escherichia coli*. During the fermentation process of succinic acid production, as organic acids are continuously generated, the pH of the fermentation broth continuously decreases. For bacteria that are sensitive to pH and osmotic pressure, neutralizing agents (such as calcium carbonate, magnesium carbonate, and sodium hydroxide) need to be added to control the pH at 6.0-7.0 to maintain the activity and fermentation efficiency of the strains during production. The extensive use of neutralizing agents will convert succinic acid into succinate, requiring subsequent separation and purification processes to re-acidify in order 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 production capacity limitations.
[0004] Compared to bacterial production strains, yeast naturally possesses stronger acid and osmotic pressure resistance, enabling fermentation under low pH conditions. This allows for the use of little or no neutralizing agents during fermentation, effectively reducing the cost of succinic acid separation. As a eukaryotic model microorganism, *Saccharomyces cerevisiae* has a clear genetic background, mature gene-editing tools, simple nutritional requirements, tolerance to high substrate concentrations, and facultative anaerobic properties, making it 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 high-yielding strains are usually obtained through metabolic engineering. Yan et al. constructed an rTCA pathway to synthesize succinic acid in a pyruvate decarboxylase-deficient Saccharomyces cerevisiae strain. Under pH 3.8 conditions, the strain produced 12.97 g / L of succinic acid, with a conversion rate of 0.13 g / g (Yan et al. Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value, Bioresource Technology, 2014, 156: 232-239), which is clearly insufficient for industrial-scale production.
[0006] Therefore, there is an urgent need to develop a strain of Saccharomyces cerevisiae that can tolerate high concentrations of succinic acid and produce succinic acid efficiently. This would effectively reduce the cost of industrial production of succinic acid, solve environmental pollution problems, and have great industrial application value. However, the currently disclosed engineered strains of Saccharomyces cerevisiae do not possess the above characteristics. Summary of the Invention
[0007] The purpose of this application is to overcome the shortcomings of the prior art and provide a Saccharomyces cerevisiae strain and its application. The Saccharomyces cerevisiae strain of this application has the ability to tolerate high concentrations of succinic acid and can produce succinic acid efficiently. In the application of succinic acid production, this application has the advantages of stable production performance, high carbon conversion rate and few by-products. The Saccharomyces cerevisiae strain of this application that tolerates high concentrations of succinic acid and produces succinic acid efficiently has broad industrial application prospects.
[0008] To achieve the above objectives, the technical solution adopted in this application is as follows:
[0009] This application provides a brewer's yeast strain, namely Saccharomyces cerevisiae PY-EDS8, which was deposited on September 14, 2023, at the Guangdong Provincial Microbiological Culture Collection Center with accession number GDMCC No: 63815 and classification number Saccharomyces cerevisiae. The deposit address is: Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China 510075.
[0010] The Saccharomyces cerevisiae PY-EDS8 obtained in this application exhibits the typical ellipsoidal morphology of Saccharomyces cerevisiae, has high tolerance to succinic acid concentrations, and can grow rapidly in liquid culture medium (pH=2.68) with 100 g / L succinic acid added.
[0011] This application provides a *Saccharomyces cerevisiae* strain, which is obtained by expressing malate dehydrogenase, fumarate, and fumarate reductase in *Saccharomyces cerevisiae* PY-EDS8, while simultaneously knocking out the glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase, and aldehyde dehydrogenase genes, resulting in the strain *Saccharomyces cerevisiae* ST100-Suc3. In some embodiments, the *Saccharomyces cerevisiae* ST100-Suc3 strain is obtained by overexpressing malate dehydrogenase, fumarate, and fumarate reductase genes in *Saccharomyces cerevisiae* PY-EDS8, while simultaneously knocking out the glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase, and aldehyde dehydrogenase genes.
[0012] The malate dehydrogenase gene (MDH) is the malate dehydrogenase gene ScMDH3R from Saccharomyces cerevisiae, which has a truncated C-terminal amino acid position 341-343 (ΔSKL); the fumarate enzyme gene is the fumarate enzyme gene RoFUM from Rhizopus oryzae; and the fumarate reductase gene is the fumarate reductase gene TbFRD from Trypanosoma brevicornu.
[0013] This application uses Saccharomyces cerevisiae ST00 (i.e. Saccharomyces cerevisiae PK2-1Ca) as the starting strain. It first screens the strain by ambient pressure room temperature plasma (ARTP) mutagenesis, and then domesticates it in a liquid medium with a gradient concentration of succinic acid to obtain Saccharomyces cerevisiae PY-EDS8.
[0014] The specific preparation process of the brewing yeast Saccharomyces cerevisiae PY-EDS8 is as follows:
[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 and cultured at 30℃ and 220 rpm for 48 h. Then, at a 5% inoculation rate, it was transferred to fresh liquid culture medium and cultured at 30℃ for 48 h. All strains used in the following experiments were second-generation bacterial suspensions. The bacterial suspension was centrifuged, the supernatant was discarded, and the bacterial cells were washed three times with PBS solution, then resuspended in physiological saline. The OD of the bacterial suspension was calculated. 600 Adjusted to 1.0.
[0017] ②ARTP mutagenesis treatment: Take 10 μL of the prepared bacterial suspension and spread it evenly on a sterilized and cooled stainless steel slide. Place the slide in an ARTP breeding machine with a mutagenesis power of 120W, a high-purity helium gas flow rate of 10 SLM, and a treatment distance of 2 mm. Treat for 0, 20, 40, 60, 80, and 100 seconds respectively. After each mutagenesis, place the stainless steel slide in an EP tube containing 990 μL of sterile physiological saline and vortex and wash for 1-2 minutes to wash off the mutagenic bacteria. Spread the mutagenic bacteria onto a solid culture medium supplemented with 80 g / L succinic acid and incubate at 30°C for 48 hours. Select the colonies that have grown.
[0018] ③ Screening of mutagenic strains: The colonies preserved after mutagenesis were inoculated into liquid culture medium and cultured at 30℃ and 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℃ for 48 h. The OD value was then measured. 600 Select OD 600 Colonies with a count >10 are preserved.
[0019] (2) Adaptive evolution:
[0020] ① The strains obtained after mutagenesis and screening were inoculated into liquid culture medium to obtain seed culture;
[0021] ② The seed culture from step ① was transferred to a liquid culture medium containing 85 g / L succinic acid at an inoculation rate of 1-10% v / v, and subjected to long-term adaptive evolution culture at 30℃ and 220 rpm.
[0022] ③ After the Saccharomyces cerevisiae strain in the succinic acid liquid medium has gone through the long selection and adaptation period, and has shown obvious growth and reached the stationary phase, the culture is transferred to fresh liquid medium containing 85 g / L succinic acid at an inoculation rate of 1-10% v / v. Further adaptation culture is carried out at 30℃ and 220 rpm to shorten the lag phase of the strain.
[0023] ④ Repeat step ③, and subculture the Saccharomyces cerevisiae strain multiple times in fresh liquid medium containing 85 g / L succinic acid until the specific growth rate of the strain in liquid medium containing 85 g / L succinic acid doubles. Screen for Saccharomyces cerevisiae mutant strains with higher succinic acid tolerance, and then preserve them.
[0024] ⑤ Repeat steps ①-④, gradually increasing the concentration of exogenously added succinic acid to 90, 95, and 100 g / L, and finally screen and obtain Saccharomyces cerevisiae PY-EDS8 which can tolerate 100 g / L succinic acid.
[0025] This application provides a biological agent comprising the above-mentioned Saccharomyces cerevisiae PY-EDS8 or Saccharomyces cerevisiae ST100-Suc3.
[0026] The Saccharomyces cerevisiae strain that efficiently produces succinic acid in this application was obtained through site-specific genome editing guided by the CRISPR / Cas9 system. This involved constructing the succinic acid synthesis pathway, knocking out byproduct synthesis genes, and directing more carbon metabolism flow to the succinic acid synthesis pathway, followed by optimization through cultivation. Specifically, the Saccharomyces cerevisiae strain PY-EDS8 was obtained by expressing the malate dehydrogenase gene (ScMDH3R), fumarate enzyme (RoFUM), and fumarate reductase (TbFRD), while simultaneously knocking out the glycerol-3-phosphate dehydrogenase (GPD1), alcohol dehydrogenase (ADH1), and aldehyde dehydrogenase (ALD6) genes, resulting in the strain Saccharomyces cerevisiae ST100-Suc3.
[0027] In some embodiments of the method for constructing the *Saccharomyces cerevisiae* strain described in this application, the method includes the following steps:
[0028] S1. Construct gRNA expression vectors targeting the glycerol-3-phosphate dehydrogenase gene, alcohol dehydrogenase gene, and acetaldehyde dehydrogenase gene.
[0029] S2. Construct recombinant expression vectors for malate dehydrogenase, fumarate cassette, and fumarate reductase, and amplify them by PCR to obtain malate dehydrogenase, fumarate cassette, and fumarate reductase expression cassette fragments, respectively.
[0030] S3. The gRNA expression vector obtained in step S1 and the malate dehydrogenase expression cassette fragment, fumarate expression cassette fragment and fumarate reductase expression cassette fragment obtained in step S2 are co-transformed into Saccharomyces cerevisiae PY-EDS8 competent cells to obtain the Saccharomyces cerevisiae ST100-Suc3 strain.
[0031] In some embodiments of the method for constructing the Saccharomyces cerevisiae strain described in this application, in step S1, primers as shown in SEQ ID NO: 2-9 are used for amplification during the construction of the gRNA expression vector targeting the glycerol-3-phosphate dehydrogenase gene, the alcohol dehydrogenase gene, and the acetaldehyde dehydrogenase gene.
[0032] In some embodiments of the method for constructing the Saccharomyces cerevisiae strain described in this application, in step S2, primers as shown in SEQ ID NO: 10-21 are used for amplification during the construction of the malate dehydrogenase recombinant expression vector, fumarate recombinant expression vector, and fumarate reductase recombinant expression vector.
[0033] In some embodiments of the method for constructing the Saccharomyces cerevisiae strain described in this application, in step S2, the amplification is performed using primers as shown in nucleotide sequences SEQ ID NO: 22-27.
[0034] Transcriptome expression differential analysis of the *Saccharomyces cerevisiae* PY-EDS8 strain in this application showed that, compared with *Saccharomyces cerevisiae* ST00, the transcriptional levels of genes involved in replication, transcription, and translation, genes involved in energy production and conversion, some genes involved in the TCA cycle, and genes involved in ion exchange and secretion were increased by 2-14.2 times.
[0035] This application provides the application of the above-mentioned Saccharomyces cerevisiae PY-EDS8 or Saccharomyces cerevisiae ST100-Suc3 in the production of succinic acid.
[0036] This application describes a Saccharomyces cerevisiae strain, Saccharomyces cerevisiae ST100-Suc3, which, after introducing a succinic acid synthesis pathway gene into Saccharomyces cerevisiae PY-EDS8 and then optimizing and redirecting the central carbon metabolism pathway, possesses a highly efficient ability to synthesize succinic acid.
[0037] This application also provides a method for producing succinic acid, including using the above-mentioned Saccharomyces cerevisiae ST100-Suc3 as the fermentation strain.
[0038] In some embodiments of the method for producing succinic acid described in this application, the method includes the following steps:
[0039] 1) The above-mentioned Saccharomyces cerevisiae ST100-Suc3 strain was cultured at a fermentation temperature of 25-35℃ and a stirring speed of 180-220 rpm for 24-48 h to obtain seed culture; and
[0040] 2) The seed liquid obtained in step 1) is fermented in a fermenter at a fermentation temperature of 25-35℃, an aeration rate 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 Saccharomyces cerevisiae ST100-Suc3 strain of this application was fermented in a 7L bioreactor for 78 hours, 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. This strain has the potential for industrial application in the production of succinic acid through 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. Saccharomyces cerevisiae PY-EDS8 can tolerate high concentrations (100g / L) of succinic acid, and the transcription levels of gene expression-related genes, energy metabolism-related genes, and ion transport-related genes are increased by 2-14.2 times.
[0044] 2. This application constructs a succinic acid synthesis pathway in Saccharomyces cerevisiae PY-EDS8 and optimizes it by central carbon metabolism redirection to obtain the Saccharomyces cerevisiae ST100-Suc3 strain, which improves cell productivity and realizes efficient production of succinic acid in Saccharomyces cerevisiae cell factory under low pH conditions.
[0045] 3. Compared with traditional chemical synthesis methods and biosynthesis methods that require the addition of neutralizing agents, the method of reconstructing the biosynthetic pathway to produce succinic acid in the Saccharomyces cerevisiae ST100-Suc3 strain, which is tolerant to high concentrations of succinic acid, has the advantages of short cycle, low pollution, resource conservation, and low cost. Attached Figure Description
[0046] Figure 1 shows the succinic acid content in the fermentation broth of Saccharomyces cerevisiae PY-EDS8 at different fermentation time points in Example 2.
[0047] Figure 2 shows a comparison of bacterial cell growth before and after the mutation of the strain in Example 3;
[0048] Figure 3 shows the transcriptome sequencing data of Saccharomyces cerevisiae PY-EDS8 in Example 4;
[0049] Figure 4 is a metabolic diagram of the biosynthetic pathway of succinic acid and the by-product synthesis pathway of Saccharomyces cerevisiae ST100-Suc3.
[0050] Figure 5 shows the fermentation results of Saccharomyces cerevisiae ST100-Suc3 and Saccharomyces cerevisiae PY-EDS8 metabolizing glucose to synthesize succinic acid. Detailed Implementation
[0051] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.
[0052] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.
[0053] The liquid culture medium consisted of 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.
[0054] The solid culture medium consists of: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and 20 g / L agar.
[0055] This application employs the classic CRISPR / Cas9 genome editing system to genetically modify *Saccharomyces cerevisiae*. This genome editing system consists of three parts: a Cas9 endonuclease expression vector, a single-stranded guide RNA expression vector, and gene expression cassette fragments. In this application, the gene expression cassette fragments are malate dehydrogenase expression cassette fragments, fumarate enzyme expression cassette fragments, and fumarate reductase expression cassette fragments.
[0056] The roles of each component in the genome editing system of this application are as follows:
[0057] (1) After the single-stranded guide RNA binds to the Cas9 endonuclease, it guides the Cas9 protein to the target genomic site (such as the nucleotide sequence encoding the glycerol-3-phosphate dehydrogenase gene mentioned in this application), and then the Cas9 protein cuts the DNA sequence at the target site, resulting in a DNA double-strand break.
[0058] (2) This DNA damage (i.e. DNA double-strand break) will induce the endogenous homologous recombination repair system of Saccharomyces cerevisiae. Under the action of this repair mechanism, the gene expression cassette fragment with the base complementary sequence at both ends of the target site can be successfully integrated into the target genomic site, thereby enabling the functional gene to be stably expressed in the strain.
[0059] Example 1: Mutagenesis and adaptive evolution of succinic acid-tolerant strains of Saccharomyces cerevisiae
[0060] This application uses Saccharomyces cerevisiae ST00 (i.e. Saccharomyces cerevisiae PK2-1Ca) as the starting strain. After obtaining mutant strains through ambient pressure room temperature plasma (ARTP) mutagenesis screening, mutant strains were obtained by adaptive evolution culture screening with exogenous addition of succinic acid at different concentrations and lower pH.
[0061] (1) Atmospheric pressure room temperature plasma (ARTP) mutagenesis screening:
[0062] ① Activation of bacterial strains and preparation of bacterial suspension:
[0063] Saccharomyces cerevisiae ST00 was inoculated into liquid culture medium and cultured at 30°C and 220 rpm for 48 h. Then, at a 5% inoculation rate, it was transferred to fresh liquid culture medium and cultured at 30°C for another 48 h. All strains used in the following experiments were second-generation bacterial suspensions. The bacterial suspension was centrifuged, the supernatant was discarded, and the bacterial cells were washed three times with PBS solution, then resuspended in physiological saline. The OD of the bacterial suspension was... 600 Adjusted to 1.0.
[0064] ②ARTP mutagenesis treatment:
[0065] Take 10 μL of the prepared bacterial suspension and spread it evenly on a sterilized and cooled stainless steel slide. Place the slide in an ARTP breeding machine with a mutagenesis power of 120 W, a high-purity helium gas flow rate of 10 SLM, and a treatment distance of 2 mm. Treat for 0, 20, 40, 60, 80, and 100 s respectively. After each mutagenesis, place the stainless steel slide in an EP tube containing 990 μL of sterile physiological saline and vortex for 1-2 min to wash off the mutagenized bacteria. Spread the mutagenized bacteria onto a solid culture medium supplemented with 80 g / L succinic acid and incubate at 30 °C for 48 h. Select the colonies that have grown and preserve them.
[0066] ③ Screening of mutant strains:
[0067] The colonies preserved after mutagenesis were inoculated into liquid culture medium and cultured at 30°C and 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 values were then measured.600 Select OD 600 Colonies with a count >10 are preserved.
[0068] (2) Adaptive evolution:
[0069] ①Inoculate the preserved mutant strain into 5 mL of liquid culture medium and culture at 30℃ and 220 rpm for 12-24 h;
[0070] ② After culturing to the stable period, the seed culture from step ① is transferred to 20 mL of liquid culture medium supplemented with 85 g / L succinic acid at an inoculation rate of 1-10% v / v, and subjected to long-term adaptive evolution culture at 30℃ and 220 rpm.
[0071] ③ After the Saccharomyces cerevisiae strain in the liquid succinic acid culture medium has gone through the long selection and adaptation period, and has shown obvious growth and reached the stationary phase, the culture is transferred to fresh liquid culture medium containing 85 g / L succinic acid at an inoculation rate of 1-10% v / v for further adaptation culture to shorten the lag phase of the strain. The culture conditions are the same as in step ②.
[0072] ④ Repeat step ③, and subculture the Saccharomyces cerevisiae strain multiple times in fresh liquid medium containing 85 g / L succinic acid until the specific growth rate of the strain in liquid medium containing 85 g / L succinic acid doubles. Screen for Saccharomyces cerevisiae mutant strains with higher succinic acid tolerance, and then preserve them.
[0073] ⑤ Repeat steps ①-④, gradually increasing the concentration of exogenously added succinic acid to 90, 95, and 100 g / L, and finally screened and obtained the Saccharomyces cerevisiae PY-EDS8 which has the ability to tolerate 100 g / L succinic acid. This adaptive evolution process took 236 days.
[0074] The brewing yeast Saccharomyces cerevisiae PY-EDS8 was deposited on September 14, 2023, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 63815, classification number Saccharomyces cerevisiae, and deposit address: Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510075, China.
[0075] Genomic DNA was extracted from *Saccharomyces cerevisiae* PY-EDS8, and the ITS (Internally Transcribed Spacer) sequence was determined. Its nucleotide sequence (SEQ ID NO: 1) is as follows:
[0076] Example 2: Verification of the reasons for succinic acid tolerance in Saccharomyces cerevisiae PY-EDS8
[0077] This study verified that the succinic acid tolerance of Saccharomyces cerevisiae PY-EDS8 is due to physiological adaptation to succinic acid rather than degradation of succinic acid.
[0078] 1. Inoculate Saccharomyces cerevisiae PY-EDS8 into 5 mL of liquid culture medium and culture at 30℃ and 220 rpm for 18-24 h to obtain seed culture;
[0079] 2. Once the strain has reached the stationary phase, adjust the seed culture from step 1 according to the initial OD value. 600 The sample was transferred to 10 mL of liquid culture medium containing 100 g / L succinic acid at a concentration of 0.05 and cultured at 30 °C and 200 rpm for 48 h.
[0080] 3. During the fermentation process, the fermentation broth was centrifuged, the supernatant was collected, and the succinic acid content at each sampling point was detected (Figure 1).
[0081] 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 does not degrade succinic acid. The tolerance of Saccharomyces cerevisiae PY-EDS8 to high concentrations of succinic acid is due to its physiological adaptation to succinic acid.
[0082] Example 3: Comparison of the growth of Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8 in succinic acid liquid medium.
[0083] 1. The starting strains of Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8 were inoculated into 5 mL of liquid culture medium and cultured at 30℃ and 220 rpm for 18-24 h to obtain seed culture.
[0084] 2. Once the strain has reached the stationary phase, combine the two seed cultures from step 1 according to their initial OD values. 6000.05 μL of the exogenous succinic acid was transferred to 20 mL of liquid culture medium containing different concentrations of succinic acid. The concentrations (g / L) of exogenous succinic acid 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). The culture was continued under the same conditions.
[0085] 3. Samples were taken every 12 hours during the culture process, and the OD of the strain was measured after 72 hours. 600 Plot the cell growth curve (Figure 2).
[0086] The results showed that the starting strain *Saccharomyces cerevisiae* ST00 hardly grew in liquid medium with succinic acid concentrations above 40 g / L; while *Saccharomyces cerevisiae* PY-EDS8 grew normally and accumulated biomass in liquid medium with succinic acid, indicating that *Saccharomyces cerevisiae* PY-EDS8 had a significantly improved tolerance to high concentrations of succinic acid.
[0087] Example 4: Differential gene expression analysis between Saccharomyces cerevisiae ST00 and Saccharomyces cerevisiae PY-EDS8
[0088] The transcriptome sequencing data in this application were obtained from a commercial sequencing service. The transcriptome sequencing method included the following steps: (1) The starting strains *Saccharomyces cerevisiae* ST00 and *Saccharomyces cerevisiae* PY-EDS8 were inoculated into 120 mL of liquid 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 The values were 2.3 and 2.8 respectively; the bacterial cells were collected by centrifugation at 1000×g, and the wet weights of the two strains were 0.7g and 0.8g respectively; (2) the total RNA of the strains was extracted using an RNA extraction kit, and after the quality was tested and found to be qualified, an RNA library was constructed using the Illumina platform; (3) the sequencing data were processed by standard quality control and comparison procedures, and the transcriptome expression profile was obtained using conventional differential expression analysis methods.
[0089] Transcriptome data showed significant differences in expression abundance among 335 genes. Of these, 232 genes were upregulated and 103 were downregulated (Figure 3 shows the names of several genes with the most significant differences in expression abundance (upregulation or downregulation)). The functions of upregulated genes were mainly concentrated in gene expression, energy production and conversion, and ion transport and secretion, with expression levels increasing by 2-14.2-fold (YOR396W). Upregulated genes are located to the right of the vertical dashed line and above the horizontal dashed line in Figure 3, including YOR396W. The functions of downregulated genes were mainly concentrated in transposition and carbohydrate transport and metabolism, with expression levels downregulated to 1 / 239 (YJR026W)-1 / 2. Downregulated genes are located to the left of the vertical dashed line and above the horizontal dashed line in Figure 3, including YJR026W. This indicates that through adaptive evolution, Saccharomyces cerevisiae PY-EDS8 possesses higher gene expression stability, higher energy supply efficiency, and stronger ion transport capacity in the unfavorable growth environment of high acidity and high osmotic pressure.
[0090] Example 5: Application of Saccharomyces cerevisiae PY-EDS8 in the efficient production of succinic acid
[0091] 1. Construction of a Saccharomyces cerevisiae strain for synthesizing succinic acid:
[0092] (1) Construct guide RNA (gRNA) expression vectors for the genes targeting byproducts glycerol-3-phosphate dehydrogenase (GPD1), alcohol dehydrogenase (ADH1), and acetaldehyde dehydrogenase (ALD6).
[0093] (2) Construct recombinant expression vectors for malate dehydrogenase (ScMDH3R), fumarate enzyme (RoFUM), and fumarate reductase (TbFRD);
[0094] (3) The SCMDH3R expression cassette fragment (SEQ ID NO: 28), RoFUM expression cassette fragment (SEQ ID NO: 29), and TbFRD expression cassette fragment (SEQ ID NO: 30) were obtained by PCR nucleic acid amplification.
[0095] (4) The guide RNA expression vector obtained in step (1) and the gene expression cassette fragment obtained in step (3) were co-transformed into competent cells of Saccharomyces cerevisiae (Saccharomyces cerevisiae PY-EDS8) to obtain Saccharomyces cerevisiae ST100-Suc3 with efficient succinic acid production capacity.
[0096] The guide RNA expression vectors described in step (1) were obtained as follows: GPD1-1, GPD1-2, ADH1-1, ADH1-2, ALD6-1, and ALD6-2 fragments were amplified using p426-SNR52p-gRNA.CAN.Y-SUP4t (Addgene, catalog number #43803, USA) as a template. The GPD1-1 and GPD1-2, ADH1-1 and ADH1-2, and ALD6-1 and ALD6-2 fragments were then recombined to obtain recombinant vectors carrying guide RNAs for GPD1, ADH1, and ALD6. Then, fragments targeting GPD1, ADH1, and ALD6 were amplified using these three recombinant vectors as templates, and the fragments were ligated to obtain guide RNA expression vectors targeting the knockout of the glycerol-3-phosphate dehydrogenase (GPD1), alcohol dehydrogenase (ADH1), and aldehyde dehydrogenase (ALD6) genes.
[0097] The primer sequences used are as follows:
[0098] GPD1-F: 5'-TCTACCAGCATTCAAGTGGCGTTTTAGAGCTAGAAATAG-3' (SEQ ID NO: 2);
[0099] GPD1-R: 5'-GCCACTTGAATGCTGGTAGAGATCATTTATCTTTCACTG-3' (SEQ ID NO: 3);
[0100] ADH1-F: 5'-GTTATCTTCTACGAATCCCAGTTTTAGAGCTAGAAATAG-3' (SEQ ID NO: 4);
[0101] ADH1-R: 5'-TGGGATTCGTAGAAGATAACGATCATTTATCTTTCACTG-3' (SEQ ID NO: 5);
[0102] ALD6-F: 5'-TTTGGAAGTGTGATCTTGACGTTTTAGAGCTAGAAATAG-3' (SEQ ID NO: 6);
[0103] ALD6-R: 5'-GTCAAGATCACACTTCCAAAGATCATTTATCTTTCACTG-3' (SEQ ID NO: 7);
[0104] Tong-F:5'-TAATAATGGTTTCTTAGTATGA-3' (SEQ ID NO: 8);
[0105] Tong-R: 5'-ACTAAGAAACATTATTATCAT-3' (SEQ ID NO: 9).
[0106] The recombinant expression vector for malate dehydrogenase described in step (2) was obtained by the following method: YEplac181 (Sangon Biotech (Shanghai) Co., Ltd., catalog number A339062, China) was used as the starting vector, and malate dehydrogenase expression cassette was introduced to obtain the recombinant expression vector for malate dehydrogenase YEplac181-PCCW12-ScMDH3R-TCYC1.
[0107] The primer sequences used are as follows:
[0108] ScMDH3R-F: 5'-CTAGATATTGATATAGTATGGTCAAAGTCGCAATTC-3' (SEQ ID NO: 10);
[0109] ScMDH3R-R:5'-GAGTCGACGGGCCGCATAAGAGTCTAGGATGAAACTCTTGCC-3' (SEQ ID NO: 11);
[0110] CYC1t-F:5'-GAGTCGACGGGCCGCATCATGTAATTAGTTATG-3' (SEQ ID NO: 12);
[0111] CCW12p-R: 5'-CTAGATATTGATATAGTGTTTAAGCGAATGACAGAAGATTA-3' (SEQ ID NO: 13).
[0112] The fumarate recombinant expression vector described in step (2) is obtained by the following method: YEplac181 is used as the starting vector, and a fumarate expression cassette is introduced to obtain the fumarate recombinant expression vector YEplac181-PCCW12-RoFUM-TCYC1.
[0113] The primer sequences used are as follows:
[0114] RoFUM-F:5'-AACACTATATCAATATCTAGATGTCATCCGCTTCTG-3' (SEQ ID NO: 14);
[0115] RoFUM-R:5'-ATGATGCGGCCCGTCGACTCTTAATTCTTTAGCTGAAATCATATCTTC-3' (SEQ ID NO: 15);
[0116] CYC1t-F:5'-GAGTCGACGGGCCGCATCATGTAATTAGTTATG-3' (SEQ ID NO: 16);
[0117] CCW12p-R: 5'-CTAGATATTGATATAGTGTTTAAGCGAATGACAGAAGATTA-3' (SEQ ID NO: 17).
[0118] The fumarate reductase recombinant expression vector mentioned in step (2) is obtained by the following method: using YEplac181 as the starting vector, a fumarate reductase expression cassette is introduced to obtain the fumarate reductase recombinant expression vector YEplac181-PCCW12-TbFRD-TCYC1.
[0119] The primer sequences used are as follows:
[0120] TbFRD-F:5'-CTAGATATTGATATAGTGTTATGGTTGATGGTAGATCTTCAGCTT-3' (SEQ ID NO: 18);
[0121] TbFRD-R:5'-GAGTCGACGGGCCGCATTTAGCTACCACTAGGTTCAGTTTCATC-3' (SEQ ID NO: 19);
[0122] CYC1t-F:5'-GAGTCGACGGGCCGCATCATGTAATTAGTTATG-3' (SEQ ID NO: 20);
[0123] CCW12p-R: 5'-CTAGATATTGATATAGTGTTTAAGCGAATGACAGAAGATTA-3' (SEQ ID NO: 21).
[0124] The expression cassette fragments mentioned in step (3) are donor1, donor2 and donor3, which are amplified respectively using the recombinant expression vector in step 2 as a template.
[0125] The primers used are as follows:
[0126] ScMDH3R-Donor1-F:5'-AGATGTCTGCTGCTGCTGATAGATTAAACTTAACTTCCGGCTGGGTGAGCATATGTTCC-3' (SEQ ID NO: 22);
[0127] ScMDH3R-Donor1-R:5'-GGCTTTTCGGCAGCCTTCAAAGAAACAGAAGAGGAACTTCTACGCCAAGCTTTTTGATT-3' (SEQ ID NO: 23);
[0128] RoFUM-Donor2-F:5'-TCAAGCTATACCAAGCATACAATCAACTATCTCATATACAGAATTCGAGCTCAACCAGG-3' (SEQ ID NO: 24);
[0129] RoFUM-Donor2-R:5'-ATCTTTGTATTCCAACTTACCGTGGGATTCGTAGAAGATAGCAAATTAAAGCCTTCGAG-3' (SEQ ID NO: 25);
[0130] TbFRD-Donor3-F:5'-TCAGAATACAATGACTAAGCTACACTTTGACACTGCTGAAATACTAGCGTTGAATGTTA-3' (SEQ ID NO: 26);
[0131] TbFRD-Donor3-R:5'-TGGTTTGACATACGAGCAACCAACCGGTCTATTCATTAACCATAGGGTAGGGGAATTTC-3' (SEQ ID NO: 27);
[0132] 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 competent Saccharomyces cerevisiae cells, thereby achieving site-specific editing of the yeast genome and obtaining Saccharomyces cerevisiae ST100-Suc3, a Saccharomyces cerevisiae strain that efficiently produces succinic acid. The metabolic diagram of the succinic acid biosynthesis pathway and byproduct synthesis pathway is shown in Figure 4.
[0133] The Cas9 system described is the yeast gene editing Cas9 expression plasmid p414-TEF1p-Cas9-CYC1t (Addgene, catalog number #43802, USA).
[0134] The method for constructing the highly efficient brewing yeast engineered strain for producing succinic acid further includes the steps of activating and enriching the engineered strain obtained in step (4): activating, inoculating, culturing, inoculating, and fermenting the obtained engineered strain.
[0135] The solid culture medium used for activation consists of: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and 20 g / L agar.
[0136] The liquid culture medium used for inoculation, cultivation, and fermentation consists of: 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.
[0137] The inoculation, culture, and fermentation conditions are: temperature 25-35℃, rotation speed 180-220rpm, and culture time 24-48h.
[0138] 2. Engineered strains metabolize glucose and synthesize succinic acid in a 2.7L bioreactor
[0139] (1) Seed liquid preparation
[0140] Saccharomyces cerevisiae ST100-Suc3 and Saccharomyces cerevisiae PY-EDS8 were streaked on solid agar plates and cultured at 30°C for 48 h. Single colonies were picked and inoculated into 50 mL of seed culture medium and cultured at 30°C and 220 rpm for 48 h.
[0141] (2) Fermentation culture in a 7L bioreactor
[0142] The seed culture prepared in step (1) was inoculated into the fermenter at an inoculation rate of 5%. The fermentation medium volume was 3L, the fermentation temperature was 30℃, the aeration rate was 1.5vvm, the initial stirring speed was 400rpm, the dissolved oxygen concentration was controlled at 20%-30% during fermentation (this percentage is the relative value of dissolved oxygen in water compared with oxygen content in air, which is detected by a dissolved oxygen electrode, where air is calibrated to 100% and 5wt% sodium sulfite solution is calibrated to 0%), and the glucose concentration was controlled at 10-20g / L.
[0143] 3. Detection of fermentation products
[0144] During fermentation, the fermentation broth was centrifuged at 12,000 rpm for 3 min. 200 μL of the supernatant was collected and 800 μL of 5 mM H₂SO₄ was added. The mixture was then filtered through a 0.22 μm sterile inorganic filter into a clean HPLC vial. The fermentation products were analyzed using high-performance liquid chromatography (HPLC). The chromatographic parameters were: column: Aminex HPX-87H; mobile phase: 5 mM H₂SO₄; flow rate: 0.6 mL / min; column temperature: 35℃; injection volume: 10 μL; detector: differential detector.
[0145] The fermentation results (Figure 5) show that *Saccharomyces cerevisiae* PY-EDS8 produced almost no succinic acid, with yields of ethanol, acetic acid, and glycerol at 51.3 g / L, 12.5 g / L, and 5.6 g / L, respectively. In contrast, *Saccharomyces cerevisiae* ST100-Suc3 produced 15 g / L of succinic acid within the initial 20 hours. Even after the pH dropped to 3.9, it continued to utilize glucose for growth and conversion, ultimately producing 76.5 g / L of succinic acid within 78 hours of fermentation, a conversion rate of 0.65 g / g. The yields of byproducts ethanol, acetic acid, and glycerol were 5.36 g / L, 2.3 g / L, and 0.4 g / L, respectively. These succinic acid yields and conversion rates represent the highest levels reported for *Saccharomyces cerevisiae* to date, indicating broad prospects for industrial production.
[0146] The nucleotide sequences involved in this application include the following:
[0147] The nucleotide sequence of the malate dehydrogenase gene ScMDH3R expression cassette fragment (its structure is a base pairing sequence + functional gene expression cassette sequence + base pairing sequence):
[0148] The nucleotide sequence of the RoFUM expression cassette fragment of the fumarate gene (whose structure is a base pairing sequence + a functional gene expression cassette sequence + a base pairing sequence):
[0149] The nucleotide sequence of the fumarate reductase gene TbFRD expression cassette fragment (whose structure consists of a base pairing sequence + a functional gene expression cassette sequence + a base pairing sequence):
[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.
Claims
1. A brewing yeast, characterized in that, The brewing yeast strain is Saccharomyces cerevisiae PY-EDS8, which was deposited on September 14, 2023, at the Guangdong Provincial Microbiological Culture Collection Center, with accession number GDMCC No: 63815, located at Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China 510075.
2. A strain of Saccharomyces cerevisiae ST100-Suc3, characterized in that, The *Saccharomyces cerevisiae* strain is obtained by expressing malate dehydrogenase, fumarate, and fumarate reductase in *Saccharomyces cerevisiae* PY-EDS8 as described in claim 1, while simultaneously knocking out the glycerol-3-phosphate dehydrogenase gene, alcohol dehydrogenase gene, and acetaldehyde dehydrogenase gene.
3. A biological agent, characterized in that, The biological agent includes Saccharomyces cerevisiae PY-EDS8 as described in claim 1 or Saccharomyces cerevisiae ST100-Suc3 as described in claim 2.
4. A method for constructing a *Saccharomyces cerevisiae* strain as described in claim 2, characterized in that, Includes the following steps: S1. Construct gRNA expression vectors targeting the glycerol-3-phosphate dehydrogenase gene, alcohol dehydrogenase gene, and acetaldehyde dehydrogenase gene. S2. Construct recombinant expression vectors for malate dehydrogenase, fumarate cassette, and fumarate reductase, and amplify them by PCR to obtain malate dehydrogenase, fumarate cassette, and fumarate reductase expression cassette fragments, respectively. S3. The gRNA expression vector obtained in step S1 and the malate dehydrogenase expression cassette fragment, fumarate expression cassette fragment and fumarate reductase expression cassette fragment obtained in step S2 are co-transformed into Saccharomyces cerevisiae PY-EDS8 competent cells to obtain the Saccharomyces cerevisiae strain.
5. The method for constructing the *Saccharomyces cerevisiae* strain as described in claim 4, characterized in that, In step S1, the primers shown in SEQ ID NO: 2-9 are used for amplification during the construction of the gRNA expression vector targeting the glycerol-3-phosphate dehydrogenase gene, alcohol dehydrogenase gene, and acetaldehyde dehydrogenase gene.
6. The method for constructing the *Saccharomyces cerevisiae* strain as described in claim 4, characterized in that, In step S2, primers as shown in SEQ ID NO: 10-21 are used for amplification during the construction of malate dehydrogenase recombinant expression vector, fumarate recombinant expression vector, and fumarate reductase recombinant expression vector.
7. The method for constructing the *Saccharomyces cerevisiae* strain as described in claim 4, characterized in that, In step S2, the amplification is performed using primers as shown in nucleotide sequences SEQ ID NO: 22-27.
8. The application of the Saccharomyces cerevisiae as described in claim 1 or the Saccharomyces cerevisiae strain as described in claim 2 in the production of succinic acid.
9. A method for producing succinic acid, characterized in that, This includes using the Saccharomyces cerevisiae strain as described in claim 2 as the fermentation strain.
10. The method as described in claim 9, characterized in that, Further steps include: 1) The Saccharomyces cerevisiae strain described in claim 2 is cultured at a fermentation temperature of 25-35℃ and a stirring speed of 180-220rpm for 24-48h to obtain seed culture; and 2) The seed liquid obtained in step 1) is fermented in a fermenter at a fermentation temperature of 25-35℃, an aeration rate 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.