A malate dehydrogenase, an engineered pichia pastoris containing the same and application thereof

Abstract

The present application relates to the technical field of enzyme engineering, and discloses a malate dehydrogenase, an engineered Pichia kudriavzevii containing the same, and application of the engineered Pichia kudriavzevii. In the present application, the Pichia kudriavzevii engineering strain with NADH selection pressure after double knockout of ethanol / glycerol synthesis pathway is used as a chassis strain, in vivo directed evolution screening is carried out, and various malate dehydrogenase mutants are obtained. The malate dehydrogenase mutants have high activity and can be used as a novel and efficient tool enzyme for preparing malic acid.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a malate dehydrogenase, an engineered Pichia kudriaz yeast containing the same, and their applications. Background Technology

[0002] In the field of enzyme engineering, particularly in the modification of malate dehydrogenase (MDH), traditional in vitro directed evolution methods have been widely used to screen for mutants with improved catalytic performance. However, these methods still have several significant limitations in practical applications.

[0003] First, the in vitro screening process based on existing methods lacks physiological relevance. Current techniques typically involve screening in purified enzyme systems or cell lysates, where conditions such as pH, ionic strength, cofactor, and substrate concentration are artificially set, making it difficult to accurately simulate the complex metabolic microenvironment within cells. Therefore, these methods are prone to missing functional MDH mutants that exhibit low in vitro activity but can well adapt to the host's metabolic network in real cells.

[0004] Secondly, traditional in vitro methods struggle to effectively couple cofactor regeneration with intracellular metabolic flux. The MDH-catalyzed reduction of oxaloacetate to malate is strictly dependent on NADH and accompanied by NAD. + Regeneration. However, in vitro experiments typically drive the reaction by adding an excess of NADH. This high cofactor concentration under non-physiological conditions masks the enzyme's true affinity for NADH (Km) and makes it impossible to accurately assess its catalytic efficiency (e.g., kcat / Km) under cofactor-limited conditions. This leads to a discrepancy between the screening results and the enzyme's functional requirements in actual industrial fermentation scenarios.

[0005] Therefore, developing an in vivo screening strategy that can directly evaluate and screen MDH mutants under physiologically relevant conditions is of great significance for obtaining enzyme mutants with excellent performance in actual metabolic environments, and is also more in line with the application needs of industrial biocatalysis and metabolic engineering. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a malate dehydrogenase, an engineered *Pichia gondii* containing the same, and its applications. Addressing the shortcomings of existing in vitro directed evolution methods for malate dehydrogenase, this invention uses engineered *Pichia gondii* strains with NADH selection pressure after double knockout of the ethanol / glycerol synthesis pathway as the substrate strain for in vivo directed evolution screening, obtaining various malate dehydrogenase mutants. These malate dehydrogenase mutants exhibit high activity and can serve as novel and efficient tool enzymes for the preparation of malate.

[0007] A first aspect of the present invention provides a malate dehydrogenase or a variant thereof, said malate dehydrogenase comprising:

[0008] (1) A malate dehydrogenase obtained by at least one amino acid substitution based on the amino acid sequence shown in SEQ ID NO: 2; or

[0009] (2) Based on the malate dehydrogenase described in (1), a malate dehydrogenase variant obtained by at least one amino acid substitution, addition or deletion, wherein the malate dehydrogenase variant has a malate yield and / or substrate consumption that is comparable to or higher than the amino acid sequence shown in SEQ ID NO: 2.

[0010] In this invention, "comprising," "containing," "having," or "including" means that at least the specified substance, component, element, or method step is present in the product, article, or method, but does not exclude the presence of other substances, components, elements, or method steps, even if other such substances, components, elements, or method steps have the same function as the specified ones.

[0011] In some embodiments of the present invention, the term and / or refers to: malic acid production and / or substrate consumption per unit time.

[0012] In some embodiments of the present invention, the malic acid yield and substrate consumption are expressed by means including but not limited to: concentration, mass, volume, amount of substance (moles), equivalent, etc., or a ratio formed by at least one of these.

[0013] In some embodiments of the present invention, when only one of the above representation methods is selected, at least one additional value is required to form the ratio. In the present invention, the selection of the additional value is not limited; it can be any meaningful or meaningless value in the art.

[0014] In some embodiments of the present invention, when two or more of the above representations are selected, the ratio may be formed without additional values.

[0015] In some embodiments of the present invention, the malic acid yield and substrate consumption are the same as the malic acid yield and substrate consumption in the same system.

[0016] In some embodiments of the present invention, the term "equivalent" means that the difference (absolute value) between the malate yield and substrate consumption of the modified malate dehydrogenase variant and the amino acid sequence shown in SEQ ID NO: 2 is less than or equal to 5%, including ±5%, ±4%, ±3%, ±2%, ±1%, or equal.

[0017] In some embodiments of the invention, the malate dehydrogenase variant has a malate yield and substrate consumption that are at least 5% higher than the amino acid sequence shown in SEQ ID NO: 2.

[0018] In some embodiments of the present invention, the high percentage is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0019] In some embodiments of the present invention, the same system is understood to be the same reaction vessel or fermentation vessel.

[0020] In some embodiments of the present invention, the number of amino acid substitutions, additions, or deletions is 1-5.

[0021] In some embodiments of the present invention, the number of amino acid substitutions, additions, or deletions is 1, 2, 3, 4, or 5.

[0022] In some embodiments of the present invention, the amino acid substitutions described in (1) occur at positions including, with reference to SEQ ID NO: 2, glutamic acid (E) at position 103, leucine (L) at position 133, and alanine (A) at position 254.

[0023] In some embodiments of the present invention, with reference to SEQ ID NO: 2, the amino acid substitution includes or only includes at least one of the following amino acid substitutions: E103K, L133F and A254T.

[0024] In some embodiments of the present invention, with reference to SEQ ID NO: 2, the amino acid substitution includes or only includes at least two of the following amino acid substitutions: E103K, L133F and A254T; for example, E103K and L133F, L133F and A254T, or E103K and A254T.

[0025] In some embodiments of the present invention, with reference to SEQ ID NO: 2, the amino acid substitution includes or only includes all three of the following amino acid substitutions: E103K, L133F and A254T.

[0026] In this invention, the maintenance or enhancement of such production and / or consumption (function) can be based on conservative amino acid substitution (such as amino acid substitution achieved by conventional substitution methods, such as alanine scanning).

[0027] In some embodiments of the present invention, the malate dehydrogenase variant described in (2) has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0028] In this invention, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and (if necessary) vacancies are introduced to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed by various methods known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art can determine appropriate parameters for determining the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0029] In some embodiments of the present invention, the malate dehydrogenase variant described in (2) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0030] In some embodiments of the present invention, the malate dehydrogenase variant described in (2) contains the amino acid sequence shown in SEQ ID NO: 2 (i.e., 100% sequence identity).

[0031] In some embodiments of the present invention, the malate dehydrogenase or its variants are provided with functional elements or are chemically modified.

[0032] In some embodiments of the present invention, the functional element includes: a decorative element and a reinforcing element.

[0033] In some embodiments of the present invention, the modifying element includes: a tag sequence, a targeting peptide, a dye, biotin, and an affinity ligand.

[0034] In some embodiments of the present invention, the modifying elements include, but are not limited to: His-tag, GST-tag, maltose-binding protein tag, Strep-tag, c-Myc tag, HA tag, FLAG tag, V5 tag, AviTag, SNAP-tag, and SUMO tag.

[0035] In this invention, "enhancing element" refers to improving at least one function of malate dehydrogenase or its variants, or conferring or enhancing the purity, enzyme activity, catalytic efficiency, substrate affinity, substrate selectivity, condition adaptability (such as temperature, pH, etc.), and stability (such as heat resistance, low temperature resistance, acid and alkali resistance, and resistance to inhibitors, etc.) of malate dehydrogenase or its variants.

[0036] In some embodiments of the present invention, the reinforcing element can improve at least one functionality of malate dehydrogenase or its variants, such as purity, enzyme activity, catalytic efficiency, substrate affinity, substrate selectivity, adaptability to conditions (such as temperature, pH, etc.), and stability (such as heat resistance, low temperature resistance, acid and alkali resistance, and resistance to inhibitors, etc.).

[0037] In some embodiments of the present invention, the reinforcing element includes, but is not limited to: cyclic peptide structures, fatty acid groups, cell-penetrating peptides, PEG, albumin, etc.

[0038] In some embodiments of the present invention, the chemical modification includes: phosphorylation, acetylation, methylation, ubiquitination or ubiquitination-like processes, glycosylation, esterification, cyclization, enzymatic cleavage, formation of disulfide bonds, and hydroxylation.

[0039] In some embodiments of the present invention, the method of obtaining the malate dehydrogenase or its variants is not limited, and it can be obtained by any conventional method in the art, including but not limited to: solid-phase synthesis, bioextraction, biosynthesis, etc.

[0040] In some embodiments of the present invention, the biosynthesis includes high-density fermentation production achieved through a heterologous expression system.

[0041] In some embodiments of the present invention, the malate dehydrogenase or a variant thereof is synthesized using a solid-phase synthesis method.

[0042] Conservative substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of other amino acid sites of the above-mentioned malate dehydrogenase or its variants are also included within the scope of this invention.

[0043] In some embodiments of the present invention, the number of amino-terminal truncations and carboxyl-terminal truncations is 1-10 amino acid residues, specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid residues.

[0044] A second aspect of the present invention provides a biomaterial comprising any one of the following (1)-(4):

[0045] (1) A nucleic acid molecule encoding the malate dehydrogenase or a variant thereof described above;

[0046] (2) An expression vector containing the nucleic acid molecule described in (1);

[0047] (3) Transformants containing the nucleic acid molecules described in (1);

[0048] (4) A transformant containing the expression vector described in (2).

[0049] In some embodiments of the present invention, the expression vector includes a plasmid.

[0050] In some embodiments of the present invention, the transformants include bacteria, fungi, viruses, plant cells, or animal cells.

[0051] In some embodiments of the present invention, the transformant does not involve plant or animal reproductive materials.

[0052] In some embodiments of the present invention, the expression vector and transformant can be constructed based on any conventional techniques in the art.

[0053] In some embodiments of the present invention, the expression vector further includes a promoter, a terminator, and an integration site homologous arm.

[0054] In some embodiments of the present invention, the promoter includes FBA1, the terminator includes TEF1, and the integration site includes Ⅲ-14.

[0055] In some embodiments of the present invention, the expression vector further includes a screening tag.

[0056] In some embodiments of the present invention, the screening label includes an resistance label, such as an antibiotic resistance label.

[0057] In some embodiments of the present invention, the transformant includes Pichia kudriaz.

[0058] In some embodiments of the present invention, the Pichia kudriaz yeast lacks glycerol metabolism genes and / or ethanol metabolism genes.

[0059] In some embodiments of the present invention, the *Pichia kudriaz* is an engineered strain of *Pichia kudriaz* S9-103 with double knockout of the ethanol / glycerol synthesis pathway, as disclosed in the applicant's prior Chinese patent CN 119614400A.

[0060] In some embodiments of the present invention, the engineered strain S9-103 of Pichia kudriaz, which has the dual knockout of the ethanol / glycerol synthesis pathway, is constructed based on the original strain S9 (Pichia kudriaz) and the construction method and the original strain S9 are disclosed in the applicant's prior Chinese patent CN 119614400 A.

[0061] A third aspect of the invention provides a composition comprising at least one of the malate dehydrogenase described in the foregoing aspects or a variant thereof and a biological material.

[0062] In some embodiments of the present invention, the composition further includes excipients.

[0063] In some embodiments of the present invention, the excipients are rationally selected based on factors such as the product form of the composition, its intended use, and the fermentation method, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, defoamers, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0064] A fourth aspect of the invention provides the use of at least one of the malate dehydrogenases described above, or variants thereof, biological materials, or compositions, in biosynthesis.

[0065] In some embodiments of the present invention, the biosynthesis includes at least one of biocatalytic reaction, biofermentation and bioenzymatic hydrolysis.

[0066] A fifth aspect of the invention provides the use of at least one of the malate dehydrogenases described above, or variants thereof, biological materials, or compositions, in the preparation of malate or derivatives thereof.

[0067] In some embodiments of the present invention, the derivative comprises a salt of malic acid.

[0068] A sixth aspect of the present invention provides a method for preparing malic acid, comprising the following steps:

[0069] At least one of the malate dehydrogenases described above, or variants thereof, biological materials, or compositions thereof, is used to catalyze the substrate.

[0070] In some embodiments of the present invention, the substrate is a carbohydrate.

[0071] In some embodiments of the present invention, the substrate is glucose.

[0072] A seventh aspect of the present invention provides a method for directed in vivo evolution of malate dehydrogenase, comprising the following steps:

[0073] Transformers lacking glycerol metabolism genes and / or ethanol metabolism genes were used as substrates to transfer malate dehydrogenase expression vectors to be evolved. The vectors were cultured on substrate-containing medium, and positive clones containing evolved malate dehydrogenase were screened out. The evolved malate dehydrogenase was then extracted.

[0074] In some embodiments of the present invention, the transformant includes Pichia kudriaz.

[0075] In some embodiments of the present invention, the *Pichia kudriaz* is an engineered strain of *Pichia kudriaz* S9-103 with double knockout of the ethanol / glycerol synthesis pathway, as disclosed in the applicant's prior Chinese patent CN 119614400A.

[0076] In some embodiments of the present invention, the malate dehydrogenase expression vector includes the expression vector described above.

[0077] In some embodiments of the present invention, the substrate is a carbohydrate.

[0078] In some embodiments of the present invention, the substrate is glucose.

[0079] In some embodiments of the present invention, the screening includes screening based on the quality of malic acid production and / or substrate consumption.

[0080] In some embodiments of the present invention, the screening further includes screening for the growth rate of transformants.

[0081] The beneficial effects of this invention are:

[0082] This invention develops an in vivo directed evolution method for malate dehydrogenase. Using an engineered strain of *Pichia gondii* with NADH selection pressure as the chassis cell, the method performs in vivo directed evolution of malate dehydrogenase, screening for mutants with increased yield and significantly enhanced growth rate and sugar consumption rate of the host strain. This method effectively overcomes the limitations of conventional in vitro screening methods in the prior art, enabling direct evaluation and screening of MDH mutants under physiologically relevant conditions. It is simple, rapid, and suitable for large-scale screening, allowing for the acquisition of a large number of mutants in a single operation.

[0083] This invention introduces specific mutations into wild-type malate dehydrogenase through in vivo directed evolution, resulting in a variety of malate dehydrogenase mutants. These mutants can significantly increase malate production, up to 254%, ultimately increasing biomass by 39% and sugar consumption rate by 40%. Attached Figure Description

[0084] Figure 1 For the upstream and downstream homologous arms of the genomic integration site (III-14) in the embodiments of the present invention, ZrMDH A schematic diagram showing the connection relationship between the expression element (promoter, terminator) segment and the expression element (promoter, terminator).

[0085] Figure 2 A comparison of glucose residues in different malate dehydrogenase mutants obtained through screening. Detailed Implementation

[0086] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0087] In the following embodiments, the engineered strain S9-103 of Pichia kudriaz, which has the ethanol / glycerol synthesis pathway double knockout, is disclosed in the applicant’s prior Chinese patent CN 119614400 A, and the original strain S9 used to construct this strain is also disclosed therein, which is incorporated herein by reference.

[0088] Example 1

[0089] In this embodiment, the effects of NADH toxicity on existing chassis cells were examined.

[0090] The specific verification method is as follows:

[0091] (1) Preparation of seed solution:

[0092] The original strain S9 and the engineered strain S9-103 of *Pichia gondii* were inoculated into test tubes containing seed culture medium and cultured at 30 °C and 200 rpm for 24 h. The seed culture medium consisted of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, based on the final concentration.

[0093] (2) Fermentation:

[0094] With initial OD 600The original strain S9 and the engineered strain S9-103 of *Pichia gondii* were transferred to Erlenmeyer flasks containing fermentation medium at a concentration of 0.1% and cultured at 30 °C and 200 rpm for 48 h. The fermentation medium consisted of 20 g / L peptone, 10 g / L yeast extract, and 50 g / L glucose, based on the final concentration.

[0095] Fermentation broth was collected at 0 h and 48 h after transfer, and OD was measured using a UV spectrophotometer. 600 .

[0096] The results showed that the original strain S9 had an OD of 48 h. 600 The OD value was 25.6, while that of the engineered strain S9-103 of Pichia pastoris at 48 h was... 600 The value was 7.8, significantly lower than that of S9. This indicates that, phenotypically, the knockout of the ethanol / glycerol synthesis pathway significantly inhibited the growth of the strain, resulting in a markedly slower growth rate.

[0097] Further malic acid detection was performed. Specifically, fermentation broth was collected 48 hours after transfer, centrifuged to remove bacterial cells, filtered through a 0.22 μm aqueous filter membrane, and the filtrate was used to determine the malic acid concentration using a Waters 2695 high-performance liquid chromatograph (HPLC). The mobile phase was 2.5 mM sulfuric acid, and the detector was a Milford RI. 2414 differential refractive index detector, Aminex HPX column. 87H (Bio The parameters were: Rad (300×7.8 mm), injection volume 10 μL, mobile phase flow rate maintained at 0.6 mL / min, column oven temperature 60℃, analysis time 25 min, detector operating temperature 40 ℃, and sensitivity set to 32.

[0098] The results showed that the malic acid content in the fermentation broth of the original strain S9 after 48 h of fermentation was 1.5 g / L, while that in the fermentation broth of the engineered strain S9-103 of Pichia kudriaz was 1.7 g / L, indicating that the malic acid yields of the two strains were basically the same.

[0099] Further glucose consumption levels were determined. The detection method was the same as for malic acid, using the initial glucose concentration of 50.0 g / L in the fermentation medium as the initial glucose concentration, and calculating the consumption level based on the measured residual glucose.

[0100] The results showed that the residual glucose in the fermentation broth of the original strain S9 after 48 h of fermentation was 0.0 g / L, while that in the fermentation broth of the engineered strain S9-103 of Pichia kudriaz was 26.4 g / L. This indicates that the glucose consumption level of the strain was significantly inhibited after the ethanol / glycerol synthesis pathway was knocked out.

[0101] Based on the growth data, it can be determined that due to the toxic effects of NADH, the growth and glucose metabolism rate of the strain are significantly inhibited, which may even lead to cell death.

[0102] Example 2

[0103] In this embodiment, in vivo directed evolution was performed on existing chassis cells, and mutants were obtained through screening. In this embodiment, the upstream and downstream homologous arms, promoter, terminator, and mutant library of the integration site were integrated into Pichia pastoris via homologous recombination. After screening, target transformants were obtained, and malate dehydrogenase mutants were isolated from them.

[0104] The specific method is as follows:

[0105] (1) ZrMDH DNA sequence synthesis:

[0106] In this embodiment, ZrMDH The DNA sequence was synthesized by Qingke Biotechnology. The specific construction method involved double digestion with SacⅠ and XmaⅠ to obtain the optimized DNA sequence. ZrMDH The gene was inserted into a commercially available pUC19 plasmid. Positive clones were obtained by screening with ampicillin resistance plates (containing 100 µg / mL ampicillin). The plasmid was extracted from the positive clones to obtain... ZrMDH Recombinant plasmid.

[0107] Among them, the optimized ZrMDH

[0108] The corresponding amino acid sequence is:

[0109] MPHSINGDVKIAVLGAAGGIGQSLSLLLKTQLTRELPNHRHAQLALYDVNADAVRGVAADLSHIDTGVTVTGYEGDRIGEALEGTDIVLIPAGVPRKPGMTREDLLVVNAKIVKSIGSSIAQHCDLNKVFILLISNPINSLVPVLVKELESKSQGTQVERRVLGLTKLDSVRASAFLHEV TIKHGLKPKSNTLDDVPVVGGHSGETIVPLFSQAPNGNRLSQDALEALVQRVQFGGDEVVRAKNGAGSATLCMAHAAYTVAASFIPLITGQKRSISGTFYVALKDAQGQPINSSAKRLLGSINDLPYFAVPLEITSQGVDELDTSVLERMTKYERERLLAPCLGKLEGGIRNGLSL (SEQ ID NO: 2).

[0110] (2) ZrMDH Construction of mutant libraries:

[0111] In this embodiment, the amplification was performed using an error-prone PCR amplification method (based on the QuickMutation™ random gene mutation kit, purchased from Shanghai Beyotime Biotechnology Co., Ltd.), following the steps described above. ZrMDH A ZrMDH mutant library was constructed using recombinant plasmids as templates. The PCR amplification primers used were:

[0112] ZrMDH -F: 5'-CCTTCCAATAAATTTGTTCAATCAGTACACAGCTAATACTATTATTACAGCTACTACTAATACTACTACTACTATTACTACCACCCCCAACACAAACACAATGCCTCACTCTATTAACGG-3' (SEQ ID NO: 3);

[0113] ZrMDH -R: 5'-TTGTTGTTGTTTTTGCTTCTTGTGAGATTACAGACATGCTGTAACAATCAAAGAGATGGAAATACAAAGTTTATTAAACACAGCCTAAAATAGAACATGTTTATAGGGACAAGCCATTAC-3' (SEQ ID NO: 4).

[0114] Refer to the instruction manual for the amplification reaction system and reaction procedure.

[0115] After amplification, the amplification product (i.e., the ZrMDH mutant library) was recovered, purified using a HiPure PCR Pure Mini Kit (purchased from Guangzhou Meiji Biotechnology Co., Ltd.), and then used for subsequent transformation.

[0116] (3) Obtaining upstream and downstream homologous arms and expression element (promoter, terminator) fragments of the integration site (III-14):

[0117] In this embodiment, the genome integration site is site III-14 of the *Pichia gondii* genome, with its homologous arms consisting of 1000 bp genomic DNA fragments on each side, and the promoter is... FBA1 (S9 endogenous gene), terminator is TEF1 (S9 endogenous gene). The connection methods of each segment are as follows: Figure 1 As shown.

[0118] Specifically, the upstream homologous arm fragment of site III-14 and the promoter fragment were seamlessly fused using overlap extension PCR technology to obtain the "III-14 upstream homologous arm- FBA1 "Promoter" fusion fragment.

[0119] The specific steps are as follows:

[0120] Design and synthesize primers specific to site III-14 and the promoter:

[0121] III-14-up-F: 5'-ATTTGTTCTTTGCTGCCTAGAGGG-3' (SEQ ID NO: 5);

[0122] III-14-up-R: 5'-TTATAGTATAAAATAGTATACAGATAAACACTTTTTGTCTCTC-3' (SEQ ID NO: 6).

[0123] FBA1 promoter-F: 5'-TATACTATTTTATACTATAATAACC-3' (SEQ ID NO: 7);

[0124] FBA1 promoter-R: 5'-TGTGTTTGTGTTGGGGGTGGTAG-3' (SEQ ID NO: 8).

[0125] In terms of primer design, the 5' end of III-14-up-R contains FBA1 Within the overlapping region of the 20 complementary base sequences at the 5' end of the promoter, it is possible to connect at the 3' end of the upstream homologous arm of III-14 and... FBA1 Sequence overlap occurs between the 5' ends of the promoter.

[0126] Simultaneously, using genomic DNA extracted from *Pichia gondii* (either S9 or S9-103, as these variants do not alter the sequence of the relevant sites to be amplified) as a template, PCR amplification was performed using the aforementioned specific primers. The amplification products were recovered to obtain the upstream homologous arm fragment of III-14 and... FBA1 Promoter segment.

[0127] The equimolar amounts of the III-14 upstream homologous arm fragment obtained in the above steps and FBA1 The promoter fragment mixture was used as a template for the first round of PCR amplification (including the complete denaturation-annealing-extension process) without exogenous primers. During the annealing stage, the upstream homologous arm fragment of III-14 and... FBA1 The promoter fragments anneal to each other through their terminal overlap regions, extend under the action of DNA polymerase, and form a partial fusion product. Then, III-14-up-F and... FBA1 promoter-R was used for a second round of PCR amplification to obtain the complete "III-14 upstream homologous arm- FBA1 "Promoter" fusion fragment.

[0128] The obtained "III-14 upstream homologous arm- FBA1 The promoter fusion fragment was analyzed by agarose gel electrophoresis, and the target band was purified using a gel recovery kit to obtain the correctly sequenced "III-14 upstream homologous arm-". FBA1 "Promoter" fusion fragment.

[0129] for" TEF1 The terminator-III-14 downstream homologous arm adopts the same design as the aforementioned "III-14 upstream homologous arm-" FBA1 The promoter fusion fragment was prepared using the same method.

[0130] The primers used are:

[0131] TEF1 terminator -F: 5'-ACATGTTCTATTTTAGGCTGTG-3' (SEQ ID NO: 9);

[0132] TEF1terminator -R:5’-GATGAGATCAATGAGTTGCA-3’(SEQ ID NO:10)。

[0133] III-14-down-F:5’-TGCAACTCATTGATCTCATCAGGGAGACACCTCTTCAAAGC-3’(SEQ IDNO:11);

[0134] III-14-down-R:5’-TGTTTTCGGTGGATTGTTCAG-3’(SEQ ID NO:12);

[0135]

[0136]

[0137] (4) Construction of yeast strains overexpressing ZrMDH mutant:

[0138] The obtained ZrMDH mutant library, gRNA targeting the III-14 site of Pichia kudriaz, and the upstream homologous arm of "III-14" were used to... FBA1 promoter fusion fragment, " TEF1 The terminator-III-14 downstream homologous arm fusion fragment was co-transformed into the competent cells of *Pichia gondii* strain S9-103. Clones were screened using Nourseothricin-resistant plates (containing 20 μg / L Nourseothricin) to obtain positive clones (i.e., strains overexpressing the ZrMDH mutant library), which were named S9. 103-M.

[0139] At the same time, wild type ZrMDH The gene (NCBI ID: XM_002495726.1) and the sgRNA targeting the III-14 site of *Pichia kudriazica* were transformed into competent cells of *Pichia kudriazica* engineered strain S9-103. Clones were screened using Norilskia succinate resistance plates, yielding positive clones (i.e., ZrMDH wild-type overexpressing strains), which were named S9. 103-M0.

[0140] Among them, the gene editing plasmid (III-14-gRNA) at the III-14 site was constructed with the assistance of the CRISPR-Cas9 system, as referenced in CN118910116A. The target sequence at the III-14 site is: 5'-CGTGTTTGTGGGAGTCATCC-3' (SEQ ID NO: 15).

[0141] The method for preparing competent cells of Pichia kudriaz, and the cellular chemical transformation process are well known in the art, and the specific steps are as described in the applicant's prior Chinese patent CN 119614400 A, which is incorporated herein by reference.

[0142] Among them, wild type ZrMDH

[0143] (5) Screening for ZrMDH mutants:

[0144] From S9 360 positive transformants were randomly selected from a plate containing 103-M (all transformants overexpressed in the ZrMDH mutant library), and further transformed from S9... Three transformants were picked from the 103-M0 (ZrMDH wild-type overexpression transformant) plate as a control group. These transformants were then inoculated into 96-well deep-well plates containing seed culture medium and cultured at 30 ℃ and 800 rpm for 24 h. After culture, 20 μL of the culture medium was transferred to 96-well deep-well plates containing fermentation medium and cultured at 30 ℃ and 800 rpm for 48 h. After culture, the plates were centrifuged at 5000 rpm for 3 min, and the supernatant was collected. S9 was detected using DNS reagent (purchased from Beijing Solarbio Science & Technology Co., Ltd.). 103-M and S9 The residual glucose content 48 h after fermentation of 103-M0. Refer to the instruction manual for the procedure of detecting glucose using the DNS reagent.

[0145] In this embodiment, a total of up to 360 mutants were obtained, and the residual glucose content of each mutant was measured.

[0146] The results are as follows Figure 2 As shown.

[0147] It was found that the mutant strain S9-103-M25 had the lowest residual glucose level at 9.4 g / L, while the residual glucose levels of other mutant strains ranged from 22.0 to 12.0 g / L. S9-103-M10 was randomly selected for retesting, and its residual glucose level was 15.3 g / L. Meanwhile, the expression of wild-type... ZrMDH S9 of the gene The glucose residue of 103-M0 at 48 h was 20.5 g / L. This indicates that the glucose consumption capacity of the mutant strain S9-103-M25 is relatively higher than that of the wild type. ZrMDH Gene strain (S9) 103-M0) showed a significant improvement, and its influence from NADH was low.

[0148] Example 3

[0149] In this embodiment, the effects of the selected mutants are verified.

[0150] Malic acid production and glucose consumption levels were measured in S9-103-M0 and mutant strains S9-103-M10 and S9-103-M25, respectively. The malic acid production and glucose consumption levels were the same as in Example 1.

[0151] The results showed that after 48 h of fermentation, the S9-103-MO OD in the fermentation broth was... 600 The OD value was 9.7, the malic acid content was 5.2 g / L, the residual glucose was 20.1 g / L, and the corresponding glucose consumption rate was 0.62 g / L / h. The OD value of the fermentation broth of mutant S9-103-M10 was... 600 The OD value of the mutant strain S9-103-M25 was 11.2, the malic acid content was 9.4 g / L, the residual glucose was 16.0 g / L, and the corresponding glucose consumption rate was 0.71 g / L / h. 600 The concentration was 13.5, the malic acid content was 18.4 g / L, the glucose residue was 8.1 g / L, and the corresponding glucose consumption rate was 0.87 g / L / h.

[0152] The malate dehydrogenase mutant (named ZrMDH-25) was isolated after lysis of the mutant strain S9-103-M25 and sequenced.

[0153] Sequencing results showed that, with SEQ ID NO:2 as a reference, it had E103K, L133F and A254T mutations.

[0154] In summary, it can be found that the optimized strain obtained based on the *Pichia gondii* engineered strain S9-103, which has a dual knockout of the ethanol / glycerol synthesis pathway, is the best candidate for this method. ZrMDH Directed evolution in vivo was achieved under NADH selection pressure, and the optimal malate dehydrogenase mutant ZrMDH-25 obtained exhibited good function in recombinant cells compared to the expression of the original mutant. ZrMDH The recombinant strain S9-103-M0 showed a 254% increase in malic acid production, a 39% increase in the final biomass of its recombinant cells, and a 40% increase in sugar consumption rate, demonstrating significant technological advantages.

[0155] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A malate dehydrogenase, characterized in that, The malate dehydrogenase is: The malate dehydrogenase was obtained by substituting three amino acids, E103K, L133F, and A254T, based on the amino acid sequence shown in SEQ ID NO:

2.

2. A biomaterial, characterized in that, The biomaterial includes any one of the following (1)-(4): (1) A nucleic acid molecule encoding the malate dehydrogenase of claim 1; (2) An expression vector containing the nucleic acid molecule described in (1); (3) Transformants containing the nucleic acid molecules described in (1); (4) A transformant containing the expression vector described in (2).

3. The biomaterial according to claim 2, characterized in that, The expression vector includes plasmids, and the transformants include bacteria, fungi, viruses, plant cells, or animal cells.

4. The biomaterial according to claim 3, characterized in that, The transformants include Pichia kudriaz.

5. The biomaterial according to claim 4, characterized in that, The *Pichia kudriaz* yeast lacks glycerol metabolism genes and / or ethanol metabolism genes.

6. A composition, characterized in that, The composition contains at least one of the malate dehydrogenase of claim 1 and the biomaterial of any one of claims 2-5; The composition also includes excipients.

7. Use of the malate dehydrogenase of claim 1 in the preparation of malate.

8. A method for preparing malic acid, comprising the following steps: Catalysis of the substrate using the malate dehydrogenase described in claim 1; in, The substrate is glucose.

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