Saccharomyces cerevisiae strain for producing L-malic acid, construction method and application thereof

Abstract

The present application belongs to the field of genetic engineering, and particularly relates to a L-malic acid producing Saccharomyces cerevisiae engineering strain and a construction method and application thereof. Specifically, the present application constructs a malic acid producing strain by introducing rTCA pathway, and improves the yield of malic acid by increasing precursor, multiple copies of key genes, and adjusting the supply of cofactors, which is 7.2 times higher than that of the initially constructed strain. On this basis, the malic acid producing strain is crossed with an acid-tolerant strain to improve the production efficiency of the strain and optimize the accumulation of by-products. In the shake flask fermentation for 24h, 12g / L of malic acid can be produced, the conversion rate is 0.69g / g, and the production rate is 0.5g / L / h. In the 1L fermenter, 65.3g / L of malic acid is obtained. Meanwhile, the foundation is laid for low pH fermentation for producing malic acid.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to an L-malic acid-producing Saccharomyces cerevisiae engineering strain and a construction method and application thereof. BACKGROUND

[0002] The statements in this section merely provide background information related to the present application and do not necessarily constitute prior art.

[0003] L-malic acid is an important platform compound and has a wide range of applications in the food and pharmaceutical industries. Malic acid is mainly synthesized by chemical method, enzymatic method and biological fermentation. The use of microbial cell factories to synthesize malic acid has the advantages of green and sustainability. The production strains for producing malic acid by fermentation mainly include filamentous fungi, Escherichia coli, yeast and the like. Filamentous fungi such as Aspergillus oryzae are natural malic acid-producing strains and have been used for industrial production, but the fermentation period of filamentous fungi is relatively long. Escherichia coli is the most widely used prokaryotic microbial cell factory and has also been engineered to synthesize malic acid. At present, the fermentation process of malic acid requires neutralization with alkali, and the use of a large amount of neutralizing agent brings inconvenience to the separation of the product and increases the production cost.

[0004] Saccharomyces cerevisiae is a very potential cell factory and has good acid tolerance. There are some examples of transforming Saccharomyces cerevisiae to produce malic acid in the prior art, such as Torulopsis glabrata CCTCC M202019 which is a yeast used for industrial production of pyruvic acid. After metabolic modification to produce malic acid, the main operations include overexpression of endogenous pyruvate carboxylase and malate dehydrogenase to construct rTCA pathway and overexpression of RoPYC, RoMDH and SpMAE1. In the final strain T.glabrata T.G-PMS, 8.5g / L of malic acid was produced, which was about 10 times higher than the initial strain. According to the investigation, there are still problems such as low yield of malic acid, limited growth of the strain itself or long fermentation time in the transformed Saccharomyces cerevisiae strains. SUMMARY

[0005] To overcome the shortcomings of the existing technology, this invention provides an engineered strain of *Saccharomyces cerevisiae* that produces L-malic acid, its construction method, and its applications. This study constructed a malic acid-producing strain by introducing the rTCA pathway and improved malic acid yield by increasing precursor substances, multiplying key genes, and regulating cofactor supply through metabolic engineering strategies, achieving a 7.2-fold increase compared to the initially constructed strain. Furthermore, crossbreeding the malic acid-producing strain with an acid-tolerant strain improved the strain's production efficiency and optimized byproduct accumulation. Fermentation in shake flasks for 24 hours yielded 12 g / L malic acid, with a conversion rate of 0.69 g / g and a production rate of 0.5 g / L / h. A 1 L fermenter yielded 65.3 g / L malic acid. This also lays the foundation for low-pH fermentation production of malic acid.

[0006] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0007] In a first aspect, the present invention provides an engineered strain of Saccharomyces cerevisiae that produces L-malic acid. The strain is obtained by constructing a malic acid production pathway, adding the precursor oxaloacetate, integrating malate dehydrogenase in multiple rounds, overexpressing phosphoenolpyruvate kinase, knocking out malate transport protein DIC1, and then overexpressing glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA.

[0008] The malic acid production pathway is constructed by overexpressing malic acid dehydrogenase and malic acid transporter MAE1.

[0009] The addition of the precursor oxaloacetate is achieved by overexpressing pyruvate carboxylase and phosphoenolpyruvate carboxylase (PPC) and knocking out glycerol-3-phosphate dehydrogenase.

[0010] In a specific embodiment of the present invention, the malate dehydrogenase is a malate dehydrogenase ScMDH3ΔSKL, an endogenous peroxidase localizing peptide from Saccharomyces cerevisiae, whose amino acid sequence is disclosed in the literature "Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export"; its amino acid sequence is shown in SEQ ID NO.1.

[0011] The malate transporter MAE1 was derived from SpMAE1 of Schizosaccharomyces cerevisiae, with GenBank number CAC37422.1.

[0012] The pyruvate carboxylases include PYC1 and PYC2, with GenBank numbers CAA96765.1 and CAA85182.1, respectively.

[0013] The phosphoenolpyruvate carboxylase PPC is a mutant PPC of phosphoenolpyruvate carboxylase derived from Escherichia coli. K620S In the amino acid sequence shown in GenBank:CAD6022922.1, K620 was mutated to S;

[0014] The glycerol-3-phosphate dehydrogenases include GPD1 and GPD2, with GenBank numbers KAG2509011.1 and KAG2511872.1, respectively.

[0015] The phosphoenolpyruvate kinase PCK has a GenBank number of AAC45394.1; the malate transport protein DIC1 has a GenBank number of QHB10491.1.

[0016] The glucose-6-phosphate dehydrogenase ZWF1 has the GenBank number CAA96146.1;

[0017] The transhydrogenase EcSthA has the GenBank number CAD6022916.1.

[0018] In a specific embodiment of the present invention, the ScMDH3ΔSKL gene is promoted by the UAS promoter. TEF-CIT-CLB -P GPD and Termination Sub-T CYC1 The regulation of the promoter sequence is disclosed in the literature Controlling Promoter Strength and Regulation in Saccharomyces cerevisiae Using Synthetic Hybrid Promoters, and the terminator is amplified from the published plasmid PIYC04 (addgene number: 64741).

[0019] In a specific embodiment of the present invention, the expression of the transporter protein is controlled by the promoter P. TEF1 and Termination T PGK1 All controls were amplified from the publicly available plasmid PJFE3 (PMID:36636342).

[0020] In a specific embodiment of the present invention, the multi-round integrated malate dehydrogenase integrates the expression cassette of the ScMDH3ΔSKL gene into the Delta region.

[0021] Secondly, the present invention provides the use of L-malic acid-producing engineered strains of *Saccharomyces cerevisiae* in any one or more of the following:

[0022] A1: Production of L-malic acid;

[0023] A2: Reagents or kits for preparing L-malic acid;

[0024] A3: Constructing a genetically engineered strain that produces high levels of L-malic acid;

[0025] A4: Production of downstream products of malic acid, such as succinic acid and fumaric acid.

[0026] In a specific embodiment of the present invention, in application A3, the method for constructing a genetically engineered strain that produces high levels of L-malic acid includes mating the L-malic acid-producing Saccharomyces cerevisiae engineered strain described in the first aspect with the acid-resistant strain MA-α to obtain a diploid strain, and then knocking out alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase GPD1 / GPD2.

[0027] The acid-tolerant strain MA-α is specifically obtained by knocking out the pyruvate decarboxylase isoenzyme PDC1 of Saccharomyces cerevisiae CEN.PK113-5D and changing its mating type to MATα. Preferably, to further improve its acid (malic acid) tolerance, before knocking out the pyruvate decarboxylase isoenzyme PDC1, Saccharomyces cerevisiae CEN.PK113-5D can be screened for higher malic acid tolerance using an adaptive laboratory evolution strategy. Specifically, the adaptive laboratory evolution strategy involves: culturing Saccharomyces cerevisiae CEN.PK113-5D in shake flasks with a medium containing 20 g / L malic acid, and growing to OD within 48 h. 600 If the concentration is 15-20, transfer the culture medium to a malic acid concentration of 25 g / L for further evolution; otherwise, continue to transfer the culture medium to a malic acid concentration of 20 g / L for further culture, and repeat the above operation.

[0028] The Genbank numbers of ADH1, GPD1 / GPD2, and PDC1 are KAF1902089.1, KAG2509011.1, KAG2511872.1, and CAA97575.1, respectively.

[0029] Thirdly, the present invention provides a genetically engineered strain for preparing high-yield L-malic acid production, as described in the second aspect.

[0030] Fourthly, the present invention provides a fermentation product / microbial agent obtained by fermentation of the above-mentioned engineered strain of Saccharomyces cerevisiae that produces L-malic acid or a genetically engineered strain that produces high levels of L-malic acid.

[0031] Fifthly, the present invention provides the use of the above-mentioned genetically engineered strains or ferments / microbial agents that produce high levels of L-malic acid in any one or more of the following:

[0032] A11: Production of L-malic acid;

[0033] A12: Reagents or kits for preparing L-malic acid;

[0034] A13: Used to degrade glucose;

[0035] A14: Reagents or kits for preparing glucose degradation products;

[0036] A15: Production of downstream products of malic acid, such as succinic acid and fumaric acid;

[0037] A16: Kit for preparing downstream products of malic acid.

[0038] In a sixth aspect, the present invention provides a method for producing L-malic acid, wherein the L-malic acid-producing Saccharomyces cerevisiae engineered strain described in the first aspect or the high-L-malic acid-producing genetically engineered strain described in the third aspect is fermented in a culture medium.

[0039] The culture medium is YPD medium; the YPD medium also contains 3-8 g / L CaCO3; preferably, the YPD medium also contains 5 g / L CaCO3.

[0040] In a seventh aspect, the present invention provides a method for constructing an engineered strain of *Saccharomyces cerevisiae* that produces L-malic acid or a genetically engineered strain that produces high levels of L-malic acid, comprising,

[0041] Starting with Saccharomyces cerevisiae BY-A0, malate dehydrogenase, malate transporter MAE1, pyruvate carboxylases PYC1 and PYC2, phosphoenolpyruvate carboxylase PPC, and oleyl-3-phosphate dehydrogenase GPD1 / 2 were sequentially overexpressed; then, ScMDH3ΔSKL was integrated into the Delta region with multiple copies; malate transporter DIC1 was knocked out while phosphoenolpyruvate kinase PCK was overexpressed; glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA were overexpressed to obtain an engineered Saccharomyces cerevisiae strain that produces L-malate.

[0042] Alternatively, an engineered strain of Saccharomyces cerevisiae that produces L-malic acid was crossbred with an acid-tolerant strain MA-α to obtain a diploid strain. Subsequently, alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase GPD1 / GPD2 were knocked out.

[0043] The acid-tolerant strain MA-α is specifically obtained by knocking out the pyruvate decarboxylase isoenzyme PDC1 of Saccharomyces cerevisiae CEN.PK113-5D and changing its mating type to MATα. Preferably, to further improve its acid (malic acid) tolerance, before knocking out the pyruvate decarboxylase isoenzyme PDC1, Saccharomyces cerevisiae CEN.PK113-5D can be screened for higher malic acid tolerance using an adaptive laboratory evolution strategy. Specifically, the adaptive laboratory evolution strategy involves: culturing Saccharomyces cerevisiae CEN.PK113-5D in shake flasks with a medium containing 20 g / L malic acid, continuously subculturing, and OD... 600 If the concentration reaches 10 or higher, transfer the culture medium to a malic acid concentration of 25 g / L for further evolution; otherwise, continue to transfer the culture medium to a malic acid concentration of 20 g / L for further culture. Repeat the above operation, gradually increasing the malic acid concentration until it reaches 40 g / L.

[0044] Furthermore, the genetically engineered strain that produces high levels of L-malic acid is Saccharomyces cerevisiae MAT-2-2, which has been deposited at the China Center for Type Culture Collection on June 7, 2024, with accession number CCTCC NO:M 20241183.

[0045] The above one or more technical solutions have the following beneficial effects:

[0046] This invention provides an engineered strain of *Saccharomyces cerevisiae* that produces L-malic acid. The malic acid biosynthesis pathway was constructed, the precursor oxaloacetate was added, and multiple copies of ScMDH3ΔSKL were integrated to increase its expression level. Subsequently, the malic acid transporter DIC1, which transports malic acid to mitochondria, was knocked out to weaken the malic acid consumption pathway. Finally, the expression of transhydrogenase increased intracellular NADH synthesis, further promoting malic acid synthesis, achieving a yield of 3.7 g / L, which is 7.2 times higher than the initial engineered strain.

[0047] This invention also constructs a high-yield L-malic acid genetically engineered strain using an L-malic acid-producing *Saccharomyces cerevisiae* engineered strain as the starting strain. The high-yield L-malic acid genetically engineered strain provided in this invention significantly increases malic acid production compared to the L-malic acid-producing *Saccharomyces cerevisiae* engineered strain, yielding 65.3 g / L of malic acid in a 1L fermenter using fed-batch fermentation. Furthermore, it is more acid-resistant, produces fewer byproducts, exhibits more stable cell activity, and requires a shorter fermentation time.

[0048] This invention broadens the thinking on metabolic engineering strategies for malic acid production by Saccharomyces cerevisiae, is a useful supplement to the research on malic acid biosynthesis, lays a certain foundation for the production of malic acid by low pH fermentation, provides a new strain for high-yield L-malic acid production, and has practical production significance.

[0049] Advantages of additional aspects of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0050] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0051] Figure 1 This is an example of the fermentation of different malic acid-producing strains in Example 1 of the present invention.

[0052] Figure 2 This is an example of the tolerance of strains to malic acid before and after mating in Example 2 of the present invention.

[0053] Figure 3 This shows the glucose consumption and metabolite accumulation of the mated strain in Example 2 of the present invention during 96 hours of fermentation.

[0054] Figure 4 This is a description of the accumulation of various metabolites and the growth status after GPD2 and GPD1 were knocked out in Example 2 of the present invention.

[0055] Figure 5 This is an example of the fermentation of strain MAT-2-2 in a 1L fermenter according to Example 2 of the present invention. Detailed Implementation

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0057] It should be noted that any aspects of this invention not described in detail are well known to those skilled in the art. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Experimental steps not described in detail are based on references to *Molecular Cloning: A Laboratory Manual* (edited by Michael R. Green and Joseph Sambrook, 4th edition), pathophysiological experiments, online databases, etc.

[0058] Unless otherwise specified, all materials and reagents used in the following embodiments were obtained commercially.

[0059] In this invention, the starting strain BY-A0 was previously constructed by the inventor's research group. It is a pyruvate decarboxylase (PDC) deficient strain with the following genotype: MATa; PDC1Δ::LEU2, PDC5Δ::URA3, PDC6Δ::TRP1,LEU2-3,112,URA3-52,TRP1-92,GAL,adaptive evolution in glucose; Specific information about BY-A0 is disclosed in the article [1] Wang Zhikun. Directed evolution and coenzyme engineering modification of pyruvate production by Saccharomyces cerevisiae BY5419 [D]. Shandong University, 2010. DOI:10.7666 / d.y1790924.

[0060] Strain Preservation Information

[0061] Abbreviation of depositary institution: CCTCC

[0062] Name of depositary institution: China Center for Type Culture Collection

[0063] Address of the depositary institution: Collection Center of Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China. Date of deposit: June 7, 2024.

[0064] Collection Center Registration Number: CCTCC NO: M 20241183

[0065] Classification and nomenclature: Saccharomyces cerevisiae

[0066] Strain number: MAT-2-2

[0067] Materials used in this invention:

[0068] 1. Culture medium or solution:

[0069] (1) Culture medium: YPD, 40% glucose

[0070] (2) Preparation of standard solution

[0071] Malic acid and pyruvic acid mixed standard solution: Weigh 0.04 g of malic acid standard and 0.02 g of pyruvic acid, make up to 2 mL, and dilute stepwise to 5 concentration gradients. Use high performance liquid chromatography to prepare a standard curve.

[0072] Mixed standard solution of glucose, ethanol, glycerol and succinic acid: Weigh 0.044 g glucose monohydrate, 25.3 μL anhydrous ethanol, 15.8 μL glycerol and 0.02 g succinic acid standard, and dilute to 2 mL. Prepare a standard curve as described above.

[0073] 2. Experimental Methods

[0074] Shake-flask fermentation with brewing yeast:

[0075] After activating three different transformants twice from the plate, they were transferred to a 100 mL Erlenmeyer flask containing 50 mL of YPD medium and the initial OD was adjusted. 600 The incubation temperature was 30℃, the rotation speed was 200 r / min, and samples were taken every 24 hours to measure OD. 600 After centrifugation, the samples were stored at -20°C for subsequent metabolite analysis.

[0076] Pretreatment of fermentation products:

[0077] After thawing the fermentation sample, centrifuge it at 12000 r / min for 10 min, use a disposable syringe to draw up the supernatant, filter it through a 0.22 μm aqueous phase filter membrane for sterilization, and then inject it into a liquid chromatography vial for loading.

[0078] For fermentation samples with added CaCO3, concentrated hydrochloric acid was added and shaken before centrifugation to displace malate ions from calcium malate, and subsequent operations were the same as above.

[0079] High performance liquid chromatography:

[0080] All experiments were conducted using a Shimadzu high-performance liquid chromatograph.

[0081] Pyruvate liquid phase conditions:

[0082] Chromatographic column: Aminex HPX-87H, column oven temperature: 35℃, flow rate: 0.6mL / min, injection volume: 10μL, detector: diode array detector, mobile phase: 1.5mM dilute H2SO4.

[0083] Liquid phase conditions for glucose and organic acids:

[0084] Chromatographic column: Aminex HPX-87H, column oven temperature: 35℃, flow rate: 0.6mL / min, injection volume: 10μL, detector: differential refractive index detector, mobile phase: 5mM dilute H2SO4.

[0085] Before conducting sample analysis, a standard curve should be prepared. After different mixed standard solutions are tested using the corresponding methods, the linear relationship between peak area and component concentration should be plotted. The correlation coefficient should be above 0.990. Then, the liquid chromatography file of the sample is analyzed according to the obtained standard curve method file to obtain the concentration of each component in the sample.

[0086] Strains mating:

[0087] Two bacterial strains of different auxotrophic types and sexes were inoculated into YPD for 24 h of activation. In this experiment, the auxotrophic type of the malic acid-producing strain was labeled URA3, and the mating type was MATa; the auxotrophic type of the acid-tolerant strain was labeled HIS3, and the mating type was MATα. Both strains were then transferred and activated in YPD for 12 h. 50 μL of each strain was inoculated into 2 mL of YPD and cultured with shaking for 3 h. A small amount of the bacterial culture was streaked onto an SC-URA-HIS plate for isolation. The plates were incubated at 30℃ for 2-3 days until single colonies appeared. Different transformants were then selected for fermentation experiments.

[0088] Feed-in yeast fermentation:

[0089] Primary seeds were cultured in 30 mL culture flasks using 5 mL of YPD medium at 30 °C and 220 rpm for 20 h. Then, at a 1% inoculum size, they were transferred to 300 mL Erlenmeyer flasks for secondary activation and cultured at 30 °C and 220 rpm for 20 h. OD... 600 Approximately 20, using a 5% inoculum and a 60 mL syringe, was added to the fermenter, and samples were retained every 24 hours for fermentation component determination.

[0090] The conditions for fed-batch fermentation were set as follows: rotation speed 500 rpm, aeration rate 1 vvm. YPD was used as the fermentation medium, with an initial sugar concentration of 60 g / L glucose. The glucose was replenished daily based on the previous day's consumption. The initial CaCO3 concentration was 20 g / L, and 10 g / L was added on the third day of fermentation.

[0091] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0092] Example 1

[0093] This embodiment discloses the engineered strain of Saccharomyces cerevisiae that produces L-malic acid, BY-MAD2-2-3, as well as its construction method and application.

[0094] 1. Introduction and optimization of malic acid biosynthesis pathway in Saccharomyces cerevisiae

[0095] The malate dehydrogenase ScMDH3ΔSKL, a truncated peroxidase localizing peptide derived from Saccharomyces cerevisiae, and SpMAE1 from Schizosaccharomyces pombe were overexpressed in the originating strain BY-A0 and subjected to UAS. TEF-CIT-CLB -P GPDThe promoter and TCYC1 terminator or PTEF1 promoter and TPGK1 terminator were integrated into chromosomes II and VIII using CRISPR-Cas9 technology. The sgRNA sequences are shown in target sequences 1 and 2. Subsequently, to increase the accumulation of precursor substances, PYC1 / 2 were inserted into the positions of the glycerol-3-phosphate dehydrogenase genes GPD1 / 2, respectively. The sgRNA sequences are shown in target sequences 3 and 4. The expression of PYC1 and PYC2 genes was controlled using the PTEF1 promoter.

[0096] The mutant PPC, which uses the strong promoter PENO2 to control phosphoenolpyruvate carboxylase derived from E. coli, is used. K620S The scMDH3ΔSKL gene expression cassette was integrated into chromosome VII of the genome. The sgRNA sequence is shown in target sequence 5. To increase malate dehydrogenase activity, the scMDH3ΔSKL gene expression cassette was integrated into the Delta region in two rounds. The sgRNA sequence is shown in target sequence 6. Saccharomyces cerevisiae chromosomal DNA contains at least 100 Delta repeat sequences, enabling multi-copy integration. Based on the multi-round integration, to balance the rTCA pathway, phosphoenolpyruvate kinase (PCK), which can synthesize ATP, was introduced. pckA from Anaerobiospirillum succiniciproducens was overexpressed. The sgRNA sequence is shown in target sequence 7. To reduce malate consumption, the malate-to-mitochondrial transport protein DIC1 was knocked out. The sgRNA was designed and positioned within the DIC1 gene, specifically at the target sequence 8 and the homologous arm of DIC1, guiding the Cas9 nuclease portion to cleave. At the resulting gap, homologous recombination was performed using a homologous arm of approximately 160 bp. A stop codon was introduced into the homologous arm to prematurely terminate translation, thus knocking out the gene. Subsequently, the soluble pyridine nucleotide transhydrogenase SthA from *E. coli* was overexpressed and integrated into chromosome XI via upstream and downstream homologous arms; the specific location is shown in target sequence 9. Prior to expressing EcsthA, the strong promoter UAS was also replaced. TEF-CIT-CLB -P GPD The gene ZWF1, encoding glucose-6-phosphate-1-dehydrogenase, a key enzyme in the PPP pathway that produces NADPH, was overexpressed. The specific substitution location is shown in target sequence 10. The sgRNA sequences used in the construction strains are shown in Table 1-2, the homologous arm amplification primers are shown in Table 1-3, and the strains or intermediate strains used and their genotypes are shown in Table 1-1.

[0097] Co-expression of ZWF1 and Ecsth in the strain significantly increased malic acid content to 3.7 g / L, an increase of 131%. The overall malic acid production of the strain is as follows: Figure 1 .

[0098] The primers, plasmids, and strains used in this embodiment are as follows:

[0099] Table 1-1 Strains used in Example 1

[0100]

[0101]

[0102] Table 1-2 Example 1 sgRNA Target Sequence

[0103]

[0104] Table 1-3 Primers used in Example 1

[0105]

[0106]

[0107]

[0108]

[0109] Example 2

[0110] This embodiment provides a genetically engineered strain that produces high levels of L-malic acid, its construction method, and its application in the fermentation production of malic acid.

[0111] To improve the metabolic activity and malic acid tolerance of the strain in Example 1, it was crossbred with a strain with increased malic acid tolerance obtained through adaptive evolution.

[0112] 1. Crossbreeding of malic acid-producing strains and malic acid-tolerant strains

[0113] The *Saccharomyces cerevisiae* CEN.PK113-5D strain underwent laboratory adaptation via a four-carbon dicarboxylic acid (CEN.PK113-5D strain was cultured in shake flasks in a medium containing 20 g / L malic acid, and continuously subcultured; OD...) 600 If the concentration reaches 10 or higher, the strain is transferred to a culture medium with a malic acid concentration of 25 g / L for further evolution; otherwise, it is transferred to a culture medium with a malic acid concentration of 20 g / L for further culture. The above operation is repeated, and the malic acid concentration is gradually increased until it reaches 40 g / L. The resulting strain has the pyruvate decarboxylase isoenzyme PDC1 knocked out, and the yeast strain whose mating type is changed to MATα (i.e., the acid-tolerant strain MA-α) is mated with the malic acid producing strain BY-MAD2-2-3 constructed in Example 1 to improve the metabolic efficiency and acid tolerance of the malic acid producing strain, and further increase the malic acid synthesis efficiency.

[0114] The resulting strain underwent malic acid tolerance testing compared to the strain before mating, and growth tests were performed in a medium containing 20 g / L malic acid. Figure 2 As shown, the MAT strain after mating grew well in a medium containing 20 g / L malic acid, while the strain before mating could hardly grow. The acid tolerance of the strain was significantly improved.

[0115] Next, it will undergo shake-flask fermentation, such as... Figure 3 The MAT strain shown obtained the highest malic acid titer of 4.4 g / L after 24 h of fermentation, and the metabolic rate of the diploid strain was significantly improved.

[0116] 2. Byproduct synthesis of weakened diploid strains

[0117] To preserve some of the cell's ethanol synthesis capacity while minimizing ethanol competition for carbon sources, the expression cassette of ScMDH3ΔSKL was integrated into the ADH1 gene. Homologous arms were designed to ensure that the insertion of ScMDH3ΔSKL simultaneously caused a frameshift mutation within the ADH1 gene, preventing it from functioning properly. After 24 hours of shake-flask fermentation, ethanol accumulation decreased to 1.7 g / L. However, this ethanol reduction also led to a decrease in biomass and malic acid accumulation (11% decrease), while glycerol accumulation significantly increased to 4.1 g / L. Although the glycerol synthesis pathway had been knocked out in the strain before mating, the other parent involved in mating still retained glycerol synthesis capacity. Therefore, using the sgRNA from Example 1 (which knocked out GPD1 / 2), ScMDH3ΔSKL was inserted into the GPD2 gene to enhance malic acid synthesis and weaken glycerol synthesis. Furthermore, PYC2 was inserted into the GPD1 gene to weaken the glycerol pathway while enhancing carboxylation. Simultaneous knockout of GPD1 and GPD2 significantly reduced glycerol accumulation and markedly increased malic acid production. Figure 4 As shown, the concentration reached 6 g / L. The sgRNA sequence used to construct this strain is shown in Table 2-2, the primers used to amplify the homologous arms are shown in Table 2-3, and the intermediate strain and its genotype are described in Table 2-1.

[0118] Fed-batch fermentation of 3 strains of MAT-2-2

[0119] Fed-batch fermentation was carried out with an initial glucose concentration of 60 g / L. Glucose concentration was measured every 24 hours, and feeding was adjusted based on the previous day's glucose consumption. 20 g / L CaCO3 was added at the beginning of fermentation to slowly release CO3. 2- This replenishes the substrate HCO3 in the carboxylation reaction process. - Add 10g / L CaCO3 during the middle stage of fermentation to prevent the CaCO3 from being completely consumed during fermentation. The results are as follows... Figure 5The entire fermentation process consumed 146.2 g / L of glucose, and the final malic acid yield reached 65.3 g / L, with a productivity of 0.54 g / L / h and a conversion rate of 0.45 g / g glucose. This shows that strain MAT-2-2 can achieve a higher malic acid yield after scale-up fermentation in a fermenter.

[0120] This embodiment improves the acid resistance and metabolic capacity of the malic acid producing strain through strain mating and a series of gene modifications, while shortening the fermentation cycle and increasing the yield of malic acid and the conversion rate of glucose.

[0121] The primers, plasmids, and strains used in this embodiment are as follows:

[0122] Table 2-1 Strains used in Example 2

[0123]

[0124]

[0125] Table 2-2 Example 2 sgRNA Target Sequence

[0126]

[0127] Table 2-3 Primers used in Example 2

[0128]

[0129]

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered strain of *Saccharomyces cerevisiae* that produces L-malic acid, characterized in that, Starting with Saccharomyces cerevisiae BY-A0, a malic acid production pathway was constructed by adding the precursor oxaloacetate, integrating malic acid dehydrogenase in multiple rounds, overexpressing phosphoenolpyruvate kinase PCK, knocking out malic acid transport protein DIC1, and then overexpressing glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA. The malic acid production pathway is constructed by overexpressing malic acid dehydrogenase and malic acid transporter MAE1. The addition of the precursor oxaloacetate is achieved by overexpressing pyruvate carboxylase and phosphoenolpyruvate carboxylase PPC, and knocking out glycerol-3-phosphate dehydrogenase. The malate dehydrogenase is a malate dehydrogenase ScMDH3ΔSKL, which is an endogenous peroxidase localizing peptide of Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.

1. The malate transporter MAE1 was derived from SpMAE1 of Schizosaccharomyces cerevisiae, with GenBank number CAC37422.

1. The pyruvate carboxylases include PYC1 and PYC2, with GenBank numbers CAA96765.1 and CAA85182.1, respectively. The phosphoenolpyruvate carboxylase PPC is a mutant PPC of phosphoenolpyruvate carboxylase derived from Escherichia coli. K620S In the amino acid sequence shown in GenBank: CAD6022922.1, K620 was mutated to S; The glycerol-3-phosphate dehydrogenases include GPD1 and GPD2, with GenBank numbers KAG2509011.1 and KAG2511872.1, respectively. The phosphoenolpyruvate kinase PCK has a GenBank number of AAC45394.1; The malate transport protein DIC1 has the GenBank number QHB10491.1; The glucose-6-phosphate dehydrogenase ZWF1 has the GenBank number CAA96146.1; The transhydrogenase EcSthA has the GenBank number CAD6022916.1; The multi-round integrated malate dehydrogenase will ScMDH3ΔSKL Gene expression cassettes are integrated into Delta The district has achieved this.

2. The use of the L-malic acid-producing Saccharomyces cerevisiae engineered strain according to claim 1 in any one or more of the following: A1: Production of L-malic acid; A2: Reagents or kits for preparing L-malic acid; A3: Constructing a genetically engineered strain that produces high levels of L-malic acid; A4: Production of downstream products of malic acid, including at least succinic acid and fumaric acid.

3. The application as described in claim 2, characterized in that, In the application A3, the method for constructing a genetically engineered strain that produces high levels of L-malic acid includes mating the L-malic acid-producing Saccharomyces cerevisiae strain of claim 1 with the acid-resistant strain MA-α to obtain a diploid strain, and then knocking out alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase GPD1 / GPD2. The acid-resistant strain MA-α was obtained by laboratory evolution of *Saccharomyces cerevisiae* CEN.PK113-5D through a four-carbon dicarboxylic acid adaptation process. The strain had the pyruvate decarboxylase isoenzyme PDC1 knocked out, and its mating type was changed to... MATα ; The GenBank numbers of ADH1, GPD1 / GPD2, and PDC1 are KAF1902089.1, KAG2509011.1, KAG2511872.1, and CAA97575.1, respectively.

4. A genetically engineered strain that produces high levels of L-malic acid, obtained by application A3 according to claim 3.

5. A microbial inoculant, characterized in that, It is obtained by fermentation from the L-malic acid-producing Saccharomyces cerevisiae engineered strain as described in claim 1 or the high-L-malic acid-producing genetically engineered strain as described in claim 4.

6. The use of the genetically engineered strain with high L-malic acid production as described in claim 4 or the microbial agent as described in claim 5 in any one or more of the following: A11: Production of L-malic acid; A12: Reagents or kits for preparing L-malic acid; A13: Used to degrade glucose; A14: Reagents or kits for preparing glucose degradation products; A15: Production of downstream products of malic acid, including at least succinic acid and fumaric acid; A16: Kit for preparing downstream products of malic acid.

7. A method for producing L-malic acid, characterized in that, The L-malic acid-producing Saccharomyces cerevisiae engineered strain of claim 1 or the high L-malic acid-producing genetically engineered strain of claim 4 is obtained by fermentation in a culture medium. The culture medium is YPD medium; the YPD medium also contains 3-8 g / L CaCO3.

8. A method for constructing an engineered strain of *Saccharomyces cerevisiae* that produces L-malic acid, characterized in that, Starting with Saccharomyces cerevisiae BY-A0, malate dehydrogenase, malate transporter MAE1, pyruvate carboxylases PYC1 and PYC2, phosphoenolpyruvate carboxylase PPC, and glycerol-3-phosphate dehydrogenase GPD1 / 2 were sequentially overexpressed; then... ScMDH3ΔSKL Perform multi-copy integration Delta The region; while overexpressing phosphoenolpyruvate kinase PCK, the malate transport protein DIC1 was knocked out; glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA were overexpressed to obtain an engineered strain of Saccharomyces cerevisiae that produces L-malate. The malate dehydrogenase is a malate dehydrogenase ScMDH3ΔSKL, which is an endogenous peroxidase localizing peptide of Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.

1. The malate transporter MAE1 was derived from SpMAE1 of Schizosaccharomyces cerevisiae, with GenBank number CAC37422.

1. The pyruvate carboxylases include PYC1 and PYC2, with GenBank numbers CAA96765.1 and CAA85182.1, respectively. The phosphoenolpyruvate carboxylase PPC is a mutant PPC of phosphoenolpyruvate carboxylase derived from Escherichia coli. K620S In the amino acid sequence shown in GenBank: CAD6022922.1, K620 was mutated to S; The glycerol-3-phosphate dehydrogenases include GPD1 and GPD2, with GenBank numbers KAG2509011.1 and KAG2511872.1, respectively. The phosphoenolpyruvate kinase PCK has a GenBank number of AAC45394.1; The malate transport protein DIC1 has the GenBank number QHB10491.1; The glucose-6-phosphate dehydrogenase ZWF1 has the GenBank number CAA96146.1; The transhydrogenase EcSthA has the GenBank number CAD6022916.1; Multi-round integration of malate dehydrogenase will ScMDH3ΔSKL Gene expression cassettes are integrated into Delta The district has achieved this.

9. A method for constructing a genetically engineered strain that produces high levels of L-malic acid, characterized in that, Starting with Saccharomyces cerevisiae BY-A0, malate dehydrogenase, malate transporter MAE1, pyruvate carboxylases PYC1 and PYC2, phosphoenolpyruvate carboxylase PPC, and glycerol-3-phosphate dehydrogenase GPD1 / 2 were sequentially overexpressed; then... ScMDH3ΔSKL Perform multi-copy integration Delta The study involved: overexpressing phosphoenolpyruvate kinase (PCK) while simultaneously knocking out malate transport protein DIC1; overexpressing glucose-6-phosphate dehydrogenase (ZWF1) and transhydrogenase (EcSthA) to obtain an L-malic acid-producing Saccharomyces cerevisiae engineered strain; mating the L-malic acid-producing Saccharomyces cerevisiae engineered strain with the acid-tolerant strain MA-α to obtain a diploid strain, followed by knocking out alcohol dehydrogenase (ADH1) and glycerol-3-phosphate dehydrogenases (GPD1 / GPD2); The acid-resistant strain MA-α was obtained by laboratory evolution of *Saccharomyces cerevisiae* CEN.PK113-5D through a four-carbon dicarboxylic acid adaptation process. The strain had the pyruvate decarboxylase isoenzyme PDC1 knocked out, and its mating type was changed to... MATα ; The genetically engineered strain that produces high levels of L-malic acid is *Saccharomyces cerevisiae*. Saccharomyces cerevisiae MAT-2-2, which has been deposited at the China Center for Type Culture Collection on June 7, 2024, with accession number CCTCC NO:M 20241183; The malate dehydrogenase is a malate dehydrogenase ScMDH3ΔSKL, which is an endogenous peroxidase localizing peptide of Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.

1. The malate transporter MAE1 was derived from SpMAE1 of Schizosaccharomyces cerevisiae, with GenBank number CAC37422.

1. The pyruvate carboxylases include PYC1 and PYC2, with GenBank numbers CAA96765.1 and CAA85182.1, respectively. The phosphoenolpyruvate carboxylase PPC is a mutant PPC of phosphoenolpyruvate carboxylase derived from Escherichia coli. K620S In the amino acid sequence shown in GenBank: CAD6022922.1, K620 was mutated to S; The glycerol-3-phosphate dehydrogenases include GPD1 and GPD2, with GenBank numbers KAG2509011.1 and KAG2511872.1, respectively. The phosphoenolpyruvate kinase PCK has a GenBank number of AAC45394.1; The malate transport protein DIC1 has the GenBank number QHB10491.1; The glucose-6-phosphate dehydrogenase ZWF1 has the GenBank number CAA96146.1; The transhydrogenase EcSthA has the GenBank number CAD6022916.1; Multi-round integration of malate dehydrogenase will ScMDH3ΔSKL Gene expression cassettes are integrated into Delta The district has achieved this; The GenBank numbers of ADH1, GPD1 / GPD2, and PDC1 are KAF1902089.1, KAG2509011.1, KAG2511872.1, and CAA97575.1, respectively.

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