Saccharomyces cerevisiae engineering strain for producing L-malic acid as well as construction method and application thereof

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a saccharomyces cerevisiae engineering strain for producing L-malic acid as well as a construction method and application of the saccharomyces cerevisiae engineering strain. Specifically, the production strain of malic acid is constructed by introducing an rTCA approach, and the yield of malic acid is increased by 7.2 times compared with that of an initially constructed strain through metabolic engineering strategies of increasing precursor substances, multiple copies of key genes, regulating cofactor supply and the like. On the basis, the malic acid production strain and the acid-resistant strain are mated, so that the production efficiency of the strain is improved, the accumulation of byproducts is optimized, 12 g / L malic acid can be produced by fermenting for 24 hours in a shake flask, the conversion rate is 0.69 g / g, the productivity is 0.5 g / L / h, and 65.3 g / L malic acid can be obtained in a 1L fermentation tank. Meanwhile, a foundation is laid for low-pH fermentation production of malic acid.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to an L-malic acid-producing brewer's yeast engineering strain and a construction method and application thereof. Background Art

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

[0003] L-malic acid is an important platform compound with a wide range of applications in the food and pharmaceutical industries. Malic acid is mainly synthesized by chemical methods, enzymatic methods, and biological fermentation. Among them, the synthesis of malic acid using microbial cell factories has the advantages of being green and sustainable. The production strains of malic acid produced by fermentation mainly include filamentous fungi, Escherichia coli, yeast, etc. Filamentous fungi such as Aspergillus oryzae are natural malic acid production strains and have been used in industrial production, but the fermentation cycle 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 malic acid fermentation process requires the addition of alkali for neutralization. The use of a large amount of neutralizers brings inconvenience to the separation of the product and also increases the production cost.

[0004] Saccharomyces cerevisiae is a cell factory with great potential and good acid resistance. There are also some examples of modifying Saccharomyces cerevisiae to produce malic acid in the prior art. For example, Torulopsis glabrata CCTCC M202019 is a yeast used for industrial production of pyruvate. It produces malic acid through the following metabolic modification. The main operations are overexpression of endogenous pyruvate carboxylase and malate dehydrogenase to construct the rTCA pathway and overexpression of RoPYC, RoMDH and SpMAE1. In the final strain T. glabrata TG-PMS, 8.5 g / L of malic acid was produced, which is about 10 times higher than the initial strain. According to the investigation, there are still problems such as low malic acid production, limited growth of the strain itself or long fermentation time in the modified strain of Saccharomyces cerevisiae. Summary of the invention

[0005] To overcome the deficiencies of the above-mentioned prior art, the present invention provides an L-malic acid-producing Saccharomyces cerevisiae engineered strain and its construction method and application. This study constructed a malic acid production strain by introducing the rTCA pathway, and increased the production of malic acid by increasing precursor substances, multiple copies of key genes, regulating the supply of cofactors and other metabolic engineering strategies, which was 7.2 times higher than the initial constructed strain. On this basis, mating the malic acid production strain with the acid-resistant strain improved the production efficiency of the strain, optimized the accumulation of by-products, and produced 12g / L malic acid in a shake flask after 24h fermentation, with a conversion rate of 0.69g / g and a productivity of 0.5g / L / h, and 65.3g / L malic acid was obtained in a 1L fermenter. At the same time, it laid the foundation for the production of malic acid by low pH fermentation.

[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 producing L-malic acid, which is obtained by using Saccharomyces cerevisiae BY-A0 as a starting strain, constructing a malic acid production pathway, increasing the precursor substance oxaloacetate, integrating malate dehydrogenase for multiple rounds, overexpressing phosphoenolpyruvate kinase, knocking out malate internalization protein DIC1, and then overexpressing glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA;

[0008] The construction of the malic acid production pathway is achieved by overexpressing malate dehydrogenase and malate transporter MAE1;

[0009] The increase of the precursor substance 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 of a truncated peroxidase localization peptide endogenous to Saccharomyces cerevisiae, whose amino acid sequence is disclosed in the literature of 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 is from SpMAE1 of Schizosaccharomyces pombe, and its GenBank number is CAC37422.1;

[0012] The pyruvate carboxylase includes PYC1 and PYC2, and the GenBank numbers are CAA96765.1 and CAA85182.1 respectively;

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

[0014] The glycerol-3-phosphate dehydrogenase includes GPD1 and GPD2, and their GenBank numbers are KAG2509011.1 and KAG2511872.1 respectively;

[0015] The GenBank number of the phosphoenolpyruvate kinase PCK is AAC45394.1; the GenBank number of the malate internalization protein DIC1 is QHB10491.1;

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

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

[0018] In a specific embodiment of the present invention, the ScMDH3ΔSKL gene is regulated by the promoter UAS TEF-CIT-CLB -P GPD and terminator T CYC1 wherein 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 disclosed plasmid PIYC04 (addgene number: 64741).

[0019] In a specific embodiment of the present invention, the expression of the transporter is driven by the promoter P TEF1 and terminator T PGK1 Control, all amplified from the public plasmid pJFE3 (PMID: 36636342).

[0020] In a specific embodiment of the present invention, the multiple rounds of integration of malate dehydrogenase are achieved by integrating the expression cassette of the ScMDH3ΔSKL gene into the Delta region.

[0021] In a second aspect, the present invention provides an application of an engineered strain of Saccharomyces cerevisiae producing L-malic acid in any one or more of the following:

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

[0023] A2: Prepare a reagent or kit for producing L-malic acid;

[0024] A3: Construction of genetically engineered strains with high production of L-malic acid;

[0025] A4: Produce downstream products of malic acid; such as succinic acid, fumaric acid, etc.

[0026] In a specific embodiment of the present invention, in the application A3, the method for constructing a genetically engineered strain with high L-malic acid production comprises 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-resistant strain MA-α is specifically obtained by knocking out the isozyme PDC1 of pyruvate decarboxylase in Saccharomyces cerevisiae CEN.PK113-5D, and changing its mating type to MATα; preferably, in order to further improve its acid (malic acid) resistance, before knocking out the isozyme PDC1 of pyruvate decarboxylase, Saccharomyces cerevisiae CEN.PK113-5D can be firstly screened based on the Adaptive Laboratory Evolution strategy to obtain Saccharomyces cerevisiae with higher tolerance to malic acid; specifically, the specific method of the adaptive laboratory evolution strategy is: the Saccharomyces cerevisiae CEN.PK113-5D strain is shake-cultured in a culture medium containing 20 g / L malic acid, and grown to an OD of 0.04 within 48 hours. 600 When the concentration of the culture medium is 15-20, the culture medium is transferred to a medium with a malic acid concentration of 25 g / L for further evolution; otherwise, the culture medium is transferred to a medium with a malic acid concentration of 20 g / L for further culture, and the above operation is repeated.

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

[0029] In a third aspect, the present invention provides a genetically engineered strain for obtaining a high yield of L-malic acid prepared according to the second aspect.

[0030] In a fourth aspect, the present invention provides a fermentation product / microbial agent obtained by fermenting the above-mentioned L-malic acid-producing Saccharomyces cerevisiae engineered strain or high-yield L-malic acid genetically engineered strain.

[0031] In a fifth aspect, the present invention provides the use of the above-mentioned genetically engineered strain or fermentation product / microbial agent with high L-malic acid production in any one or more of the following:

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

[0033] A12: preparing a reagent or a kit for producing L-malic acid;

[0034] A13: used to degrade glucose;

[0035] A14: preparing a reagent or a kit for degrading glucose;

[0036] A15: Produce downstream products of malic acid, such as succinic acid, fumaric acid, etc.;

[0037] A16: Preparation of a kit for producing malic acid downstream products.

[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-yield L-malic acid-producing genetically engineered strain described in the third aspect is placed in a culture medium and fermented;

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

[0040] In a seventh aspect, the present invention provides a method for constructing an L-malic acid-producing Saccharomyces cerevisiae engineered strain or a high-yield L-malic acid-producing genetically engineered strain, comprising:

[0041] Using Saccharomyces cerevisiae BY-A0 as the starting strain, malate dehydrogenase, malate transporter MAE1, pyruvate carboxylase PYC1 and PYC2, phosphoenolpyruvate carboxylase PPC were overexpressed in sequence, and oleate-3-phosphate dehydrogenase GPD1 / 2 were knocked out; then multiple copies of ScMDH3ΔSKL were integrated into the Delta region; phosphoenolpyruvate kinase PCK was overexpressed and malate internalization protein DIC1 was knocked out; glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA were overexpressed to obtain an L-malic acid-producing Saccharomyces cerevisiae engineered strain;

[0042] Alternatively, an L-malic acid-producing Saccharomyces cerevisiae engineered strain was mated with the acid-tolerant strain MA-α to obtain a diploid strain, and then the alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase GPD1 / GPD2 were knocked out;

[0043] The acid-resistant strain MA-α is specifically obtained by knocking out the isozyme PDC1 of pyruvate decarboxylase in Saccharomyces cerevisiae CEN.PK113-5D and changing its mating type to MATα; preferably, in order to further improve its acid (malic acid) resistance, before knocking out the isozyme PDC1 of pyruvate decarboxylase, Saccharomyces cerevisiae CEN.PK113-5D can be firstly screened based on the Adaptive Laboratory Evolution strategy to obtain Saccharomyces cerevisiae CEN.PK113-5D with higher tolerance to malic acid; specifically, the specific method of the adaptive laboratory evolution strategy is: the Saccharomyces cerevisiae CEN.PK113-5D strain is cultured in a shake flask in a culture medium containing 20 g / L malic acid, and the OD 600 If it reaches 10 or above, transfer to a culture medium with a malic acid concentration of 25 g / L to continue evolution. Otherwise, continue to transfer to a culture medium with a malic acid concentration of 20 g / L to continue cultivation. Repeat the above steps to gradually increase the malic acid concentration and eventually increase the malic acid concentration to 40 g / L.

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

[0045] One or more of the above technical solutions have the following beneficial effects:

[0046] The present invention provides an engineered strain of Saccharomyces cerevisiae that produces L-malic acid, which constructs a malic acid biosynthetic pathway, increases the precursor substance oxaloacetate, and integrates multiple copies of ScMDH3ΔSKL to increase the expression level. Subsequently, the transport protein DIC1 that transports malic acid to mitochondria is knocked out to weaken the malic acid consumption pathway, and finally, transhydrogenase is expressed to increase intracellular NADH synthesis, further promoting malic acid synthesis, and the yield reaches 3.7 g / L, which is 7.2 times higher than that of the initial modified strain.

[0047] The present invention also uses an L-malic acid-producing yeast engineered strain as a starting strain to construct a genetically engineered strain with high L-malic acid production. The genetically engineered strain with high L-malic acid production provided by the present invention has a significantly higher malic acid production than the L-malic acid-producing yeast engineered strain, and 65.3 g / L malic acid is obtained by batch feeding fermentation in a 1L fermenter. At the same time, the strain is more acid-resistant, has fewer by-products, has more stable bacterial activity, and has a short fermentation time.

[0048] This invention broadens the thinking on metabolic engineering strategies for malic acid production by brewer's yeast, 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, and provides a new strain for high-yield L-malic acid, which has practical production significance.

[0049] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0051] Figure 1 The fermentation conditions of different malic acid producing strains in Example 1 of the present invention are shown in FIG.

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

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

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

[0055] Figure 5 This is the fermentation condition of strain MAT-2-2 in Example 2 of the present invention in a 1 L fermenter. DETAILED DESCRIPTION

[0056] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0057] It should be noted that the details of the present invention are known to those skilled in the art. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The experimental steps that are not recorded in detail refer to "Molecular Cloning Laboratory Manual" (Edited by (US) MR Green (Michael R. Green), (US) J. Sambrook (Joseph. Sambrook), fourth edition), pathophysiological experiments, online databases, etc.

[0058] In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified.

[0059] In the present invention, the starting strain BY-A0 used is obtained by the inventor's research group in the early stage. It is a strain deficient in pyruvate decarboxylase (PDC), genotype: MATa; PDC1Δ::LEU2, PDC5Δ::URA3, PDC6Δ::TRP1,LEU2-3,112,URA3-52,TRP1-92,GAL, adaptive evolution in glucose; BY-A0 specific information is disclosed in the article [1] Wang Zhikun. Directed evolution and coenzyme engineering modification of pyruvate production by brewer's yeast BY5419 [D]. Shandong University, 2010. DOI:10.7666 / d.y1790924.

[0060] Strain deposit information

[0061] Abbreviation of depository institution: CCTCC

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

[0063] Address of depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code: 430072 Deposit date: June 7, 2024

[0064] CCTCC No.: M 20241183

[0065] Classification: Saccharomyces cerevisiae

[0066] Strain ID: MAT-2-2

[0067] Materials used in the present 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 the volume to 2 mL, dilute step by step into 5 concentration gradients, and 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 of glucose monohydrate, 25.3 μL of anhydrous ethanol, 15.8 μL of glycerol, and 0.02 g of succinic acid standard, dilute to 2 mL, and prepare a standard curve by the same operation as above.

[0073] 2. Experimental methods

[0074] Saccharomyces cerevisiae shake flask fermentation:

[0075] Pick three different transformants on the plate and activate them twice, then transfer them to a 100 mL Erlenmeyer flask containing 50 mL YPD medium and adjust the initial OD 600 The culture temperature was 30℃, the rotation speed was 200r / min, and samples were taken every 24h to measure OD 600 , and stored at −20°C after centrifugation for subsequent metabolite analysis.

[0076] Pre-treatment of fermentation products:

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

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

[0079] HPLC:

[0080] A Shimadzu high performance liquid chromatograph was used in all experiments.

[0081] Pyruvic acid liquid phase conditions:

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

[0083] Liquid phase conditions of glucose and organic acid:

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

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

[0086] Strain mating:

[0087] Two strains with different nutritional deficiency types and different sexes were inoculated in YPD for activation for 24 hours. In this experiment, the nutritional deficiency selection marker of the malic acid production strain was URA3, and the mating type was MATa; the nutritional deficiency selection marker of the strain with improved acid resistance was HIS3, and the mating type was MATα. Transfer the above two strains and activate them in YPD for 12 hours. Take 50μL of each of the two strains and inoculate it into 2mL YPD for shaking culture for 3 hours. Take a small amount of bacterial liquid and streak it on the SC-URA-HIS plate for separation. Culture in a 30℃ incubator for 2-3 days until a single colony grows. Pick different transformants for fermentation experiments.

[0088] Yeast fed-batch fermentation:

[0089] The first-level seeds were cultured in 30 mL culture flasks with 5 mL YPD medium at 30 ° C, 220 r / min for 20 h, and then transferred to 300 mL conical flasks with 1% inoculum for secondary activation and cultured at 30 ° C, 220 r / min for 20 h. OD 600 About 20, with a 60mL syringe at a 5% inoculation amount, added into the fermenter, and samples were retained every 24 hours for fermentation component determination.

[0090] The conditions of batch fed fermentation were set at a speed of 500 r / min and a ventilation volume of 1 vvm. YPD was used as the fermentation medium, with an initial sugar concentration of 60 g / L glucose, which was supplemented every day according to the consumption of the previous day. 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 in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0092] Embodiment 1

[0093] This example discloses an L-malic acid-producing Saccharomyces cerevisiae engineered strain BY-MAD2-2-3 and a construction method and application thereof.

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

[0095] The malate dehydrogenase ScMDH3ΔSKL from Saccharomyces cerevisiae and the SpMAE1 from Schizosaccharomyces pombe were overexpressed in the starting strain BY-A0 and expressed using UAS TEF-CIT-CLB -P GPDThe promoter and TCYC1 terminator or the PTEF1 promoter and TPGK1 terminator were site-specifically integrated into chromosomes II and VIII using CRISPR-Cas9 technology, and the sgRNA sequences were shown in targeting sequences 1 and 2. Subsequently, PYC1 / 2 was inserted into the position of the glycerol-3-phosphate dehydrogenase gene GPD1 / 2 to increase the accumulation of precursor substances, and the sgRNA sequences were shown in targeting sequences 3 and 4. The expression of the PYC1 and PYC2 genes was controlled by the PTEF1 promoter.

[0096] Using the strong promoter PENO2 to control the mutant PPC of phosphoenolpyruvate carboxylase from Escherichia coli K620S , site-specifically integrated into chromosome VII of the genome, sgRNA sequence sees targeting sequence 5. To increase malate dehydrogenase activity, the expression cassette of the ScMDH3ΔSKL gene was integrated into the Delta region twice, sgRNA sequence sees targeting sequence 6, there are at least 100 Delta repeat sequences in the chromosomal DNA of Saccharomyces cerevisiae, which can achieve multi-copy integration. On the basis of multiple rounds of integration, in order to balance the rTCA pathway, phosphoenolpyruvate kinase PCK, which can synthesize ATP, was introduced. pckA from Anaerobiospirillum succiniciproducens was overexpressed, sgRNA sequence sees targeting sequence 7, to reduce malate consumption, DIC1, a protein transporting malate to mitochondria, was knocked out, sgRNA was designed to locate inside the DIC1 gene, the specific location sees targeting sequence 8 and the homologous arm of DIC1, to guide the nuclease part of Cas9 to cut, at the resulting gap, homologous arms of about 160bp were used for homologous recombination, and stop codons were introduced into the homologous arms to terminate translation early and knock out the gene. Then, the soluble pyridine nucleotide transhydrogenase SthA from Escherichia coli was overexpressed and integrated into chromosome XI through upstream and downstream homology arms. The specific location is shown in targeting sequence 9. Before expressing EcsthA, the strong promoter UAS was replaced. TEF-CIT-CLB -P GPD The gene ZWF1 encoding the key enzyme glucose-6-phosphate-1-dehydrogenase that produces NADPH in the PPP pathway was overexpressed. The specific replacement position is shown in the targeting sequence 10. The sgRNA sequences used to construct the strains are shown in Table 1-2, the homology 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] The co-expression of ZWF1 and Ecsth in the strain resulted in a significant increase in malic acid content to 3.7 g / L, an increase of 131%. The malic acid production of the comprehensive strain is shown in Figure 1 .

[0098] The primers, plasmids and strain information used in this example are as follows:

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

[0100]

[0101]

[0102] Table 1-2 Example 1 sgRNA targeting sequence

[0103]

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

[0105]

[0106]

[0107]

[0108]

[0109] Embodiment 2

[0110] This embodiment provides a genetically engineered strain with high L-malic acid production, a construction method thereof, and an application of the strain in fermentation to produce malic acid.

[0111] In order to improve the metabolic activity of the strain in Example 1 and the strain's tolerance to malic acid, the strain was mated with a strain with increased malic acid tolerance obtained through adaptive evolution.

[0112] 1. Mating of malic acid producing strains with malic acid tolerant strains

[0113] The cerevisiae CEN.PK113-5D strain was subjected to laboratory evolution of tetracarbonyl dicarboxylic acid adaptation (the cerevisiae CEN.PK113-5D strain was cultured in a shake flask in a medium containing 20 g / L malic acid, and the OD 600 If the concentration of malic acid reaches 10 or above, it is transferred to a medium with a malic acid concentration of 25 g / L to continue evolution; otherwise, it is transferred to a medium with a malic acid concentration of 20 g / L to continue culturing, and the above operation is repeated to gradually increase the malic acid concentration, and finally the malic acid concentration is increased to 40 g / L). The strain obtained after knocking out the isozyme PDC1 of pyruvate decarboxylase and changing its mating type to MATα is a yeast strain (i.e., the acid-resistant strain MA-α) and mating with the malic acid producing strain BY-MAD2-2-3 constructed in Example 1 to improve the metabolic efficiency and acid resistance of the malic acid producing strain, and further increase the malic acid synthesis efficiency.

[0114] The strains obtained by mating were tested for malic acid tolerance and growth in a medium containing 20 g / L malic acid, as compared to the strains before mating. Figure 2 As shown, the strain MAT after mating has good growth in the medium with 20 g / L malic acid, while the strain before mating can hardly grow. The acid resistance of the strain is significantly improved.

[0115] Next, it was fermented in shake flasks. Figure 3 The MAT strain obtained the highest malic acid titer of 4.4 g / L at 24 h of fermentation, and the metabolic rate of the diploid strain was significantly improved.

[0116] 2. Weakened byproduct synthesis of diploid strains

[0117] In order to retain part of the ethanol synthesis ability of the bacteria and reduce the competition of ethanol for carbon sources as much as possible, the expression frame of ScMDH3ΔSKL was integrated into the inside of ADH1 respectively. When designing the homology arm, it was ensured that ScMDH3ΔSKL was inserted and the frameshift mutation was caused inside the ADH1 gene to ensure that it could not function normally. After 24 hours of shake flask fermentation, the ethanol accumulation was reduced to 1.7g / L. However, the reduction of ethanol also led to a decrease in biomass and a decrease in malic acid accumulation, with malic acid accumulation decreasing by 11%, and a significant increase in glycerol accumulation, reaching 4.1g / L. Although the glycerol synthesis pathway has been knocked out in the strain before mating, the other parent involved in mating still retains the glycerol synthesis ability. Thus, the sgRNA of knocking out GPD1 / 2 in Example 1 was used to insert ScMDH3ΔSKL into the GPD2 gene, thereby strengthening malic acid synthesis and weakening glycerol synthesis. On this basis, PYC2 was inserted into the GPD1 gene to strengthen the carboxylation reaction while weakening the glycerol pathway. After GPD1 and GPD2 were knocked out simultaneously, glycerol accumulation was significantly reduced and malic acid production was significantly increased. Figure 4 As shown, it reached 6g / L. The sgRNA sequence used to construct this strain is shown in Table 2-2, the primers used to amplify the homology arms are shown in Table 2-3, and the description of the intermediate strains and their genotypes is shown in Table 2-1.

[0118] Fed-batch fermentation of strain MAT-2-2

[0119] The fermentation was carried out in batches with an initial glucose concentration of 60 g / L. The glucose concentration was measured every 24 h, and the feed was made according to the concentration of sugar consumed the previous day. 20 g / L CaCO3 was added at the beginning of the fermentation to slowly release CO3 2- , thereby replenishing the substrate HCO3 in the carboxylation reaction - , add 10g / L CaCO3 in the middle of fermentation to prevent CaCO3 from being consumed during fermentation. Figure 5The entire fermentation process consumed 146.2 g / L of glucose, and the final malic acid production reached 65.3 g / L, the productivity reached 0.54 g / L / h, and the conversion rate reached 0.45 g / g glucose. This shows that strain MAT-2-2 can obtain a higher malic acid yield after fermentation in a fermenter.

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

[0121] The primers, plasmids and strain information used in this example are as follows:

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

[0123]

[0124]

[0125] Table 2-2 Example 2 sgRNA targeting sequences

[0126]

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

[0128]

[0129]

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered strain of Saccharomyces cerevisiae producing L-malic acid, characterized in that: Using Saccharomyces cerevisiae BY-A0 as the starting strain, the malate production pathway was constructed, the precursor oxaloacetate was added, malate dehydrogenase was integrated in multiple rounds, phosphoenolpyruvate kinase PCK was overexpressed, malate internalization protein DIC1 was knocked out, and glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA were overexpressed; The construction of the malic acid production pathway is achieved by overexpressing malate dehydrogenase and malate transporter MAE1; The increase of the precursor substance oxaloacetate is achieved by overexpressing pyruvate carboxylase and phosphoenolpyruvate carboxylase PPC, and knocking out glycerol-3-phosphate dehydrogenase.

2. The L-malic acid producing Saccharomyces cerevisiae engineered strain according to claim 1, characterized in that: The malate dehydrogenase is the malate dehydrogenase ScMDH3ΔSKL of the truncated peroxidase localization peptide endogenous to Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.1; The malate transporter MAE1 is derived from SpMAE1 of Schizosaccharomyces pombe, and its GenBank number is CAC37422.1 The pyruvate carboxylase includes PYC1 and PYC2, and the GenBank numbers are CAA96765.1, CAA85182.1; The phosphoenolpyruvate carboxylase PPC is a mutant PPC of phosphoenolpyruvate carboxylase derived from Escherichia coli. K620S , K620 was mutated to S in the amino acid sequence shown in GenBank: CAD6022922.1; The glycerol-3-phosphate dehydrogenase includes GPD1 and GPD2, and their GenBank numbers are KAG2509011.1 and KAG2511872.1 respectively; The phosphoenolpyruvate kinase PCK has a GenBank number of AAC45394.1; The malate internalization protein DIC1 has a GenBank number of QHB10491.1; The glucose-6-phosphate dehydrogenase ZWF1 has a GenBank number of CAA96146.1; The transhydrogenase EcSthA has a GenBank number of CAD6022916.

1.

3. The L-malic acid producing Saccharomyces cerevisiae engineered strain according to claim 1, characterized in that: The multiple rounds of integration of malate dehydrogenase are achieved by integrating the expression cassette of the ScMDH3ΔSKL gene into the Delta region.

4. Use of the engineered strain of Saccharomyces cerevisiae producing L-malic acid according to any one of claims 1 to 3 in any one or more of the following: A1: Production of L-malic acid; A2: Prepare a reagent or kit for producing L-malic acid; A3: Construction of genetically engineered strains with high production of L-malic acid; A4: Produce downstream products of malic acid, including at least succinic acid and fumaric acid.

5. The use according to claim 4, characterized in that In the application A3, the method for constructing a genetically engineered strain with high L-malic acid production comprises mating the L-malic acid-producing Saccharomyces cerevisiae engineered strain according to 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 is a strain obtained by laboratory evolution of Saccharomyces cerevisiae CEN.PK113-5D to adapt to tetracarbonyl dicarboxylic acids, in which the isozyme PDC1 of pyruvate decarboxylase is knocked out and its mating type is changed to MATα; The GenBank numbers of ADH1, GPD1 / GPD2, and PDC1 are: KAF1902089.1, KAG2509011.1, KAG2511872.1, and CAA97575.1, respectively.

6. The genetically engineered strain for high L-malic acid production according to claim 5.

7. A fermentation product / microbial agent, characterized in that: The product is obtained by fermentation of the L-malic acid-producing Saccharomyces cerevisiae engineered strain according to any one of claims 1 to 3 or the high-yield L-malic acid-producing genetically engineered strain according to claim 6.

8. Use of the genetically engineered strain with high L-malic acid production according to claim 6 or the fermentation product / microbial agent according to claim 7 in any one or more of the following: A11: Production of L-malic acid; A12: preparing a reagent or a kit for producing L-malic acid; A13: used to degrade glucose; A14: preparing a reagent or a kit for degrading glucose; A15: Produce downstream products of malic acid, including at least succinic acid and fumaric acid; A16: Preparation of a kit for producing malic acid downstream products.

9. A method for producing L-malic acid, characterized in that: The L-malic acid-producing Saccharomyces cerevisiae engineered strain according to claim 1 or the high-yield L-malic acid-producing genetically engineered strain according to claim 6 is placed in a culture medium and fermented to obtain; The culture medium is YPD culture medium; the YPD culture medium also includes 3-8 g / L CaCO3.

10. A method for constructing an L-malic acid-producing Saccharomyces cerevisiae engineered strain or a high-yield L-malic acid-producing genetically engineered strain, characterized in that: Using Saccharomyces cerevisiae BY-A0 as the starting strain, malate dehydrogenase, malate transporter MAE1, pyruvate carboxylase PYC1 and PYC2, phosphoenolpyruvate carboxylase PPC, and oleate-3-phosphate dehydrogenase GPD1 / 2 were overexpressed in sequence; then multiple copies of ScMDH3ΔSKL were integrated into the Delta region; phosphoenolpyruvate kinase PCK was overexpressed and malate internal transporter DIC1 was knocked out; glucose-6-phosphate dehydrogenase ZWF1 and transhydrogenase EcSthA were overexpressed to obtain an L-malic acid-producing Saccharomyces cerevisiae engineered strain; or, the L-malic acid-producing Saccharomyces cerevisiae engineered strain was mated with the acid-resistant strain MA-α to obtain a diploid strain, and then alcohol dehydrogenase ADH1 and glycerol-3-phosphate dehydrogenase GPD1 / GPD2 were knocked out; The acid-resistant strain MA-α is a strain obtained by laboratory evolution of Saccharomyces cerevisiae CEN.PK113-5D to adapt to tetracarbonyl dicarboxylic acids, in which the isozyme PDC1 of pyruvate decarboxylase is knocked out and its mating type is changed to MATα; Furthermore, the genetically engineered strain that produces high L-malic acid is Saccharomyces cerevisiae MAT-2-2, which has been deposited in the China Center for Type Culture Collection on June 7, 2024, with a deposit number of CCTCC NO: M 20241183.

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