Acid-resistant kluyveromyces marxianus strain and application thereof in production of succinic acid

Abstract

The invention relates to the technical field of microorganisms, in particular to an acid-resistant kluyveromyces marxianus strain and application thereof in production of succinic acid. The Kluyveromyces marxianus DLY-KMPMFF-0329 provided by the invention is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the Kluyveromyces marxianus DLY-KMPMFF-0329 is CGMCC No. 27009. The strain can tolerate high-concentration succinic acid and low-pH conditions, can efficiently utilize glucose to synthesize succinic acid by reducing tricarboxylic acid circulation, can realize higher succinic acid yield and yield, has higher homologous recombination efficiency, and can efficiently perform gene editing. The strain not only can be used as a succinic acid production strain for fermentation production of succinic acid, but also can be used as an original strain for breeding or constructing a succinic acid high-yield strain, and has a relatively good application value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to an acid-resistant Kluyveromyces marxianus strain and application thereof in producing succinic acid. Background Art

[0002] Kluyveromyces marxianus is resistant to high temperatures, grows fast, and can naturally utilize multiple carbon sources such as xylose, lactose, and inulin. It is a good potential host for the production of bio-based chemicals (Qiu, et al., Kluyveromyces as promising yeast cell factories for industrial bioproduction: From bio-functional design to applications. Biotechnology Advances, 2023, 64: 108-125).

[0003] Succinic acid, also known as succinic acid (SA), is one of the important bio-based chemical raw materials and has important applications in agriculture, food, clothing, cosmetics and pharmaceutical industries. In addition to being used as a surfactant, ion chelator and food additive, succinic acid can also be used as a precursor for many high-value industrial chemicals, including adipic acid, 1,4-butanediol (1,4-BDO), tetrahydrofuran (THF), n-methylpyrrolidone (NMP), n-butyl lactone and other solvents and chemicals. In addition, succinic acid can also be used to produce biodegradable materials such as polyesters and polyamides. In 2004 and 2010, the U.S. Department of Energy (DOE) listed succinic acid as one of the five most promising bio-based platform chemicals (Ahn, et al., Production of succinic acid by metabolically engineered microorganisms. Current Opinion in Biotechnology, 2016, 42, 54-66). In recent years, the demand for succinic acid as a platform chemical has grown rapidly. Therefore, it is of great significance to improve the production efficiency of succinic acid and reduce the production cost.

[0004] Compared with the production process based on fossil raw materials, the production of succinic acid by microbial fermentation is more competitive. At present, microorganisms that can produce succinic acid include Yarrowia lipolytica, Saccharomyces cerevisiae, Escherichia coli, Actinobacillus succinogenes, Corynebacterium glutamicum, etc., but there are relatively few studies on the production of succinic acid by fermentation of Kluyveromyces marxianus, and Kluyveromyces marxianus is a yeast dominated by non-homologous recombination, which brings great difficulties to the efficient genetic manipulation of multiple gene sites in it.

[0005] Microorganisms can produce SA through three pathways: the oxidative tricarboxylic acid cycle pathway (oTCA), the glyoxylate pathway (GA), and the reductive tricarboxylic acid cycle pathway (rTCA). When produced simultaneously through the oTCA and GA pathways, pyruvate enters the mitochondria and generates acetyl-COA under the action of pyruvate dehydrogenase, and condenses with oxaloacetate to form citric acid, which is then oxidized and decarboxylated to form SA, with a theoretical yield of 1 mol SA / 1 mol glucose. In the rTCA pathway, pyruvate or phosphoenolpyruvate can fix 1 molecule of CO2 for each molecule of SA generated after carboxylation, so the theoretical yield is 1.71 mol SA / 1 mol glucose (Liu et al., Biosynthetic pathway and metabolic engineering of succinic acid. Frontiers in Bioengineering and Biotechnology, 2022, 10, 843887). Therefore, compared with oTCA, rTCA has a higher theoretical yield of SA production, and therefore has greater application potential.

[0006] At present, the main engineering host bacteria for producing SA using rTCA are Escherichia coli, actinomycetes, Corynebacterium glutamicum and other bacteria, but these bacteria are at risk of being infected by bacteriophages, and the acid resistance of bacteria is poor. Generally, the suitable pH range is 6.8-7.3. During the fermentation process, a large amount of neutralizer needs to be added to neutralize the acidic environment caused by the production of succinic acid, which increases the cost of fermentation and subsequent separation. Yeast cells do not have the risk of being infected by bacteriophages and are generally more acid-resistant than bacteria. Therefore, using yeast cells as chassis cells to produce SA using the rTCA pathway is a more promising production method. The reported production and yield of SA by Saccharomyces cerevisiae through the rTCA pathway are both low. Although Issatchenkia orientalis naturally has a strong tolerance to low pH, by overexpressing endogenous pyruvate carboxylase PYC1, malate dehydrogenase MDH3, fumarate hydratase FUM1, and heterologously expressing Aspergillus oryzae fumarate reductase FRDg, the final production of SA through the rTCA pathway was only 11.6 g / L, and the yield was only 0.12 g / g (Xiao et al., Exploiting Issatchenkia orientalis SD108 for succinic acid production. Microbial Cell Factories, 2014, 13, 121).

[0007] At present, the production and yield of SA produced by yeast using glucose as a carbon source through the oTCA or rTCA pathway are still low. One of the reasons is that the pH gradually decreases during the accumulation of SA, which inhibits the acid production of the strain. According to literature reports, in experiments using yeast to produce malic acid, the production of malic acid was significantly improved by enhancing the tolerance of the production strain to low pH (Sun L eal., Highly efficient neutralizer-free L-malic acid production using engineered Saccharomyces cerevisiae. Bioresource Technology. 2023, 370: 128580), which shows that the high tolerance of chassis cells to low pH is beneficial to the production of organic acids.

[0008] In summary, there is still a need to develop production strains with higher acid resistance and succinic acid production capacity in order to further improve the yield and production efficiency of succinic acid. Summary of the invention

[0009] The invention provides an acid-resistant Kluyveromyces marxianus strain and application of the strain in producing succinic acid.

[0010] During the early research and development process, the applicant domesticated and screened Kluyveromyces marxianus NBRC1777 to improve its tolerance to low pH and succinic acid, and obtained a Kluyveromyces marxianus strain with strong tolerance to low pH and succinic acid; and used this strain as the starting strain to further enhance the homologous recombination efficiency of the strain through genetic engineering, and obtained Kluyveromyces marxianus DLY-KMSP-0209, which was deposited on February 16, 2022 in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC for short, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), classified and named Kluyveromyces marxianus, and the preservation number is CGMCC No. 24398. K. marxianus DLY-KMSP-0209 can tolerate low pH conditions and high succinic acid concentrations. Moreover, the strain has a high homologous recombination efficiency and is suitable as a chassis cell for breeding fermentation production strains of organic acids such as succinic acid. In addition, K. marxianus DLY-KMSP-0209 can be selected from one or more of glucose and lignocellulosic biomass (including xylose, lignocellulose hydrolysate, etc.) as carbon sources for growth, and the suitable growth temperature is 30-42°C (preferably 37-42°C), which can achieve tolerance to low pH and high succinic acid concentrations at higher temperatures for fermentation production.

[0011] In order to further improve the yield of succinic acid, the present invention uses Kluyveromyces marxianus DLY-KMSP-0209 as a starting strain, firstly performs low pH and succinic acid tolerance acclimation on Kluyveromyces marxianus DLY-KMSP-0209, further performs genetic engineering modification on the dominant strain obtained by acclimation screening so that the strain can produce succinic acid by using the reduced TCA pathway (rTCA), and finally performs glucose substrate-oriented screening on the genetically engineered strain to obtain a strain that can efficiently utilize glucose to synthesize succinic acid, and finally obtains a Kluyveromyces marxianus strain with good low pH and succinic acid tolerance, high ability to synthesize succinic acid by using glucose, and capable of producing succinic acid by using rTCA, and with high succinic acid yield and yield, which is named DLY-KMPMFF-0329.

[0012] Specifically, the present invention provides the following technical solutions:

[0013] The present invention provides Kluyveromyces marxianus DLY-KMPMFF-0329, which was deposited on April 3, 2023 in the General Microbiology Center of China Culture Collection Administration (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Kluyveromyces marxianus, with a deposit number of CGMCC No. 27009.

[0014] The invention constructs rTCA in Kluyveromyces marxianus DLY-KMPMFF-0329 through genetic engineering modification, and the specific modification includes: expressing fumarate reductase FRDg with an amino acid sequence as shown in SEQ ID NO.4, enhancing the expression of pyruvate carboxylase PYC2, expressing malate dehydrogenase MDH3 with an amino acid sequence as shown in SEQ ID NO.2, expressing fumarate hydratase FUM1 with an amino acid sequence as shown in SEQ ID NO.3, inactivating the coding gene of malic enzyme MAE1, and inactivating the coding gene of pyruvate decarboxylase PDC1.

[0015] Therefore, the Kluyveromyces marxianus DLY-KMPMFF-0329 has the following genotype: it expresses fumarate reductase FRDg with an amino acid sequence as shown in SEQ ID NO.4 and / or, its genome contains a DNA fragment with a nucleotide sequence as shown in SEQ ID NO.5.

[0016] In addition, compared with the wild-type Kluyveromyces marxianus NBRC1777, the expression of pyruvate carboxylase PYC2 in the Kluyveromyces marxianus DLY-KMPMFF-0329 is enhanced, and / or, the malate dehydrogenase MDH3 with an amino acid sequence as shown in SEQ ID NO.2 is expressed, and / or, the fumarate hydratase FUM1 with an amino acid sequence as shown in SEQ ID NO.3 is expressed, and / or, the coding gene of malic enzyme MAE1 is inactivated, and / or, the coding gene of pyruvate decarboxylase PDC1 is inactivated.

[0017] Kluyveromyces marxianus DLY-KMPMFF-0329 can tolerate low pH conditions and high succinic acid concentrations. Moreover, the strain has a high homologous recombination efficiency and is suitable as a chassis cell for breeding fermentation production strains of organic acids such as succinic acid. In addition, Kluyveromyces marxianus DLY-KMPMFF-0329 can efficiently utilize glucose to synthesize succinic acid through rTCA, and has a higher succinic acid production and yield.

[0018] The present invention also provides a microbial preparation, which comprises the above-mentioned Kluyveromyces marxianus DLY-KMPMFF-0329.

[0019] The microbial preparation described above can be a solid preparation or a liquid preparation. In the microbial preparation, Kluyveromyces marxianus DLY-KMPMFF-0329 exists in the form of live bacteria. In addition to containing Kluyveromyces marxianus DLY-KMPMFF-0329 or a culture of Kluyveromyces marxianus DLY-KMPMFF-0329, the microbial preparation can also contain carriers or other auxiliary materials permitted in the field of microbial preparations.

[0020] The present invention provides a method for preparing the above-mentioned microbial preparation, which comprises the step of culturing the Kluyveromyces marxianus DLY-KMPMFF-0329.

[0021] In some embodiments of the present invention, Kluyveromyces marxianus DLY-KMPMFF-0329 is cultured to obtain a culture solution, and the culture solution is directly prepared as a liquid microbial preparation or mixed with an auxiliary material to prepare a liquid microbial preparation, or the culture solution is directly mixed with an auxiliary material and freeze-dried to obtain a solid microbial preparation, or the culture solution is subjected to solid-liquid separation to obtain bacterial cells, and the bacterial cells are mixed with an auxiliary material and freeze-dried to obtain a solid microbial preparation.

[0022] The present invention provides any of the following uses of the above-mentioned Kluyveromyces marxianus DLY-KMPMFF-0329 or the microbial preparation:

[0023] (1) Application in the fermentation production of organic acids or their derivatives;

[0024] (2) Application in breeding or constructing strains producing organic acids or their derivatives.

[0025] In the above (2), Kluyveromyces marxianus DLY-KMPMFF-0329 is used as the starting strain to select or construct a strain for producing organic acids or their derivatives. Among them, selection refers to obtaining a new strain by non-rational transformation methods such as mutagenesis or domestication; construction refers to obtaining a new strain by rational transformation methods such as genetic engineering.

[0026] In the present invention, the starting strain refers to the initial strain used for strain selection or construction, and the starting strain is subjected to genetic engineering, mutagenesis or domestication to obtain the target strain.

[0027] Kluyveromyces marxianus DLY-KMPMFF-0329 has a high tolerance to low pH and succinic acid, and at the same time, has a high homologous recombination efficiency, and can efficiently utilize glucose to produce succinic acid through the rTCA pathway with a higher theoretical yield. Therefore, this strain is suitable as a starting strain for breeding or constructing a production strain of organic acids (especially succinic acid) or their derivatives.

[0028] Preferably, the organic acid is succinic acid.

[0029] The invention provides a method for constructing a succinic acid-producing Kluyveromyces marxianus engineered bacterium. The method comprises: taking Kluyveromyces marxianus DLY-KMPMFF-0329 as a starting strain and genetically modifying the starting strain.

[0030] Preferably, the genetic modification comprises introducing exogenous nucleic acid into the starting strain using homologous recombination technology, and / or editing endogenous genes of the starting strain using homologous recombination technology.

[0031] The homologous recombination technology includes but is not limited to CRISPR / Cas9, Cre-Loxp, TALEN, etc.

[0032] The present invention provides a method for producing succinic acid by fermentation, which comprises the step of culturing the above-mentioned Kluyveromyces marxianus DLY-KMPMFF-0329.

[0033] Preferably, the culture temperature is 37-42°C.

[0034] Preferably, the culturing includes a fermentation culturing stage, and the fermentation culturing stage includes the step of culturing the Kluyveromyces marxianus DLY-KMPMFF-0329 under oxygen-limited conditions.

[0035] The above oxygen-limited condition is to control the dissolved oxygen DO to be below 2%, preferably to control the dissolved oxygen DO to be below 1%.

[0036] The present invention finds that when producing succinic acid using the rTCA pathway, the dissolved oxygen level has a significant impact on the fermentation process, succinic acid conversion, and by-product generation. Controlling the dissolved oxygen level under the above-mentioned oxygen-limited conditions is conducive to promoting the synthesis of succinic acid and reducing the generation of by-products.

[0037] Preferably, in the fermentation culture stage, carbonate is added at the beginning of fermentation and / or during fermentation; the amount of carbonate added is 10-50 g / L.

[0038] The present invention finds that when producing succinic acid using the rTCA pathway, carbon dioxide can be fixed by adding carbonate. Carbon dioxide can be used by Kluyveromyces marxianus DLY-KMPMFF-0329 in the form of carbonate (such as calcium carbonate), which can significantly increase the yield of succinic acid and provide a new idea and solution for solving the problem of carbon emission reduction.

[0039] The carbonates mentioned above include calcium carbonate.

[0040] The carbonate may be added directly to the fermentation medium, and / or the carbonate may be added once or in multiple additions during the fermentation process. When added in multiple additions, the total amount of carbonate added is 10-50 g / L.

[0041] The culture medium used in the above-mentioned fermentation culture includes a carbon source and a nitrogen source; the carbon source includes glucose, and the nitrogen source includes peptone and / or yeast powder.

[0042] Preferably, the mass ratio of the carbon source to the nitrogen source is (3-6): 1. The present invention finds that appropriately increasing the carbon-nitrogen ratio in the fermentation medium is beneficial to increasing the yield of succinic acid produced by fermentation of Kluyveromyces marxianus DLY-KMPMFF-0329, and preferably the carbon-nitrogen ratio is controlled within the range of (3-6): 1.

[0043] Preferably, the fermentation medium comprises: 20-100 g / L glucose, 0-20 g / L peptone, and 5-15 g / L yeast powder.

[0044] Preferably, the fermentation and culture stage includes a first fermentation and culture stage and a second fermentation and culture stage; the first fermentation and culture stage is cultured under aerobic conditions, and the second fermentation and culture stage is cultured under oxygen-limited conditions.

[0045] The present invention finds that the two-stage fermentation process for fermenting Kluyveromyces marxianus DLY-KMPMFF-0329 (the first stage is mainly used for bacterial growth, and the second stage is mainly used for succinic acid production) is more conducive to promoting the production of succinic acid, further significantly improving the fermentation level, and shortening the fermentation cycle.

[0046] The culturing may further include a seed culturing stage.

[0047] The beneficial effects of the present invention include at least: the Kluyveromyces marxianus DLY-KMPMFF-0329 provided by the present invention can tolerate high concentrations of succinic acid and low pH conditions, can efficiently utilize glucose to synthesize succinic acid through the rTCA pathway, can achieve a high succinic acid production and yield, and has a high homologous recombination efficiency, and can efficiently perform gene editing. The Kluyveromyces marxianus DLY-KMPMFF-0329 can be used as a succinic acid production strain for succinic acid fermentation production, and can also be used as a starting strain for breeding or construction of a high-yield strain of succinic acid, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 Schematic diagram of the expression frame for constructing the reductive TCA pathway in Example 2 of the present invention, wherein IMTCP2p-kmPYC2-CP1t represents the overexpression of the endogenous pyruvate carboxylase gene kmPYC2 of Kluyveromyces marxianus using the endogenous promoter IMTCP2p of Kluyveromyces marxianus and the terminator IMTCP1 (represented by CP1t in the figure); IMTCP1p-MDH3-R-CP2t represents the overexpression of the endogenous malate dehydrogenase gene MDH3-R of Kluyveromyces marxianus (removing the three amino acids "TKL" at the C-terminus) using the endogenous promoter IMTCP1p of Kluyveromyces marxianus and the terminator IMTCP2 (represented by CP2t in the figure); gene fragments PDC1_U650 and PDC1_D657 represent the coding frame of gene PDC1 The homology arms of 650 bp upstream and 657 bp downstream (used for the target fragment to target PDC1); the fragment TEF1p-FRDg-INUt represents the heterologous overexpression of fumarate reductase FRDg using the endogenous promoter TEF1p of Kluyveromyces marxianus and the INU1 gene terminator INU1t, TEF3p-FUM1-14n-PDC1t represents the expression of endogenous fumarate hydratase using the endogenous promoter TEF3p of Kluyveromyces marxianus and the PDC1 gene terminator PDC1t (14n represents the mitochondrial localization sequence with 14 amino acids removed), and the gene fragments MAE1_U1000 and MAE1_D1000 represent the homology arms of 1000 bp upstream and downstream of the malate dehydrogenase gene (MAE1), which are used for large fragment targeting at the gene MAE1 position.

[0050] Figure 2The metabolite detection results of the glucose substrate directed screening experiment were performed on the four transformants with correct genotypes in Example 3 of the present invention, among which #28, #25, #21, and #17 were the four transformants with correct genotypes.

[0051] Figure 3 These are the test results of succinic acid production and byproduct accumulation in the microaerobic fermentation of Kluyveromyces marxianus DLY-KMPMFF-0329 in Example 4 of the present invention, wherein the peak time of succinic acid is 11.903 minutes.

[0052] Figure 4 The figure shows the effect of different rotation speeds on the succinic acid production under oxygen-limited conditions in Example 5 of the present invention, wherein a) is the succinic acid production, b) is the consumption of glucose, c) is the accumulation of lactic acid, and d) is the accumulation of glycerol.

[0053] Figure 5 The figure shows the effect of optimization of shake flask culture conditions on succinic acid production in Example 6 of the present invention, wherein a) is the effect of different glucose concentrations on succinic acid accumulation, and b) is the effect of different peptone concentrations on succinic acid accumulation. The solid marked curve represents glucose consumption, and the hollow marked curve represents succinic acid production; glucose 50, 60, 80, 100 represents the addition of glucose concentrations of 50, 60, 80, 100 g / L, respectively, and peptone -0, 5, 10 g / L represents the addition of no peptone and the addition of 5 and 10 g / L peptone, respectively.

[0054] Figure 6 This is an analysis of the metabolites of succinic acid produced in a 5L fermentation tank in Example 7 of the present invention, wherein a) is the change in glucose and SA; b) is the accumulation of by-products citric acid, acetic acid and malic acid; and c) is the accumulation of by-products glycerol and lactic acid.

[0055] Figure 1-Figure 6 In the formula, SA stands for succinic acid. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Example 1 Acclimation of Kluyveromyces marxianus to Succinic Acid Tolerance

[0058] Taking Kluyveromyces marxianus DLY-KMSP-0209 as the starting strain, it was acclimated to low pH and high concentration succinic acid tolerance. The specific method is as follows: the strain was inoculated in 2 mL of YPD liquid culture medium and cultured at 37°C and 200 rpm until the bacterial solution began to become turbid. The strain was then inoculated in 1 mL of YPD liquid culture medium and 3 mL of 60 g / L SA mother solution (SA final concentration of 45 g / L) was added. The culture was continued under the same conditions for 90 days. Finally, the strain was inoculated in 0.5 mL of YPD liquid culture medium with a pH of 2.3 and 3.5 mL of 60 g / L SA mother solution (SA final concentration of 52.5 g / L) was added. After 20 days of culture under the same conditions, the bacterial solution was taken to plate, and a single clone that tolerated low pH and high concentration succinic acid was selected to obtain the strain DLY-KMSP-0209M. The strain can grow under the conditions of pH 2.3 and SA concentration of 52.5 g / L. The tolerance test results show that the strain DLY-KMSP-0209M has a lower OD value in the logarithmic growth phase than the original strain DLY-KMSP-0209 under the same stress conditions. 600 The value increased by more than 20%.

[0059] Example 2 Construction of a succinate-producing strain using the TCA reduction pathway

[0060] The production of succinate by the reductive TCA pathway requires four key genes, which encode pyruvate carboxylase PYC2, which converts pyruvate into HCO3 - Catalyzes the production of oxaloacetate; malate dehydrogenase MDH3 reduces oxaloacetate to malate under the condition of consuming one NADH molecule; fumarate hydratase FUM1 converts malate to fumarate; fumarate reductase FRDg reduces fumarate to succinate under the condition of consuming one NADH molecule. The first three genes can be endogenously amplified from the Kluyveromyces marxianus NBRC1777 strain, while one of the key genes FRDg is derived from Trypanosoma brucei and needs to be heterologously synthesized and codon optimized. The coenzyme of this enzyme is NADH, which is conducive to unifying the coenzyme to establish a redox balance.

[0061] Since the mitochondria of yeast cells are double-layer membrane structures, the products (malic acid, fumaric acid, succinic acid) in the reduction TCA pathway need to use transporters to cross the double membrane into the cytoplasm and then secrete to the outside of the cell, which will undoubtedly hinder the production and exocytosis of succinic acid. However, the transport principle of the above-mentioned organic dicarboxylic acids from mitochondria to the cytoplasm is not completely clear at present. In order to eliminate this effect as much as possible, the reduction pathway-related enzymes located in the mitochondria are directly transferred to the cytoplasm. PYC2p (amino acid sequence as shown in SEQ ID NO.1, gene KLMA_10253 encoding) is itself a cytoplasmic protein, which is connected to the endogenous promoter IMTCP2p and terminator IMTCP1 of Kluyveromyces marxianus, and the IMTCP2p-kmPYC2-CP1t expression cassette is constructed and inserted into the genomic position where the PDC1 gene is located. MDH3p is localized in peroxisomes. The three amino acids "TKL" at the C-terminus of MDH3p (the amino acid sequence is shown in SEQ ID NO.2) are removed and placed in the IMTCP1p-MDH3-R-CP2t expression frame for cytoplasmic expression. The protein FUM1p (encoded by gene KLMA_80028) that is dual-localized in mitochondria and cytoplasm is removed by removing the 14 amino acids at the N-terminus (including the 14 amino acid mitochondrial localization sequence and 1 start amino acid, the amino acid sequence is shown in SEQ ID NO.3) and then a new translation start codon is introduced to localize it in the cytoplasm, and it is expressed in the TEF3p-FUM1-14n-PDC1t expression frame. The three "SKI" amino acids at the C-terminus of FRDgp were removed (the amino acid sequence is shown in SEQ ID NO.4, and the coding gene sequence is shown in SEQ ID NO.5), and connected with the endogenous promoter TEF1p of Kluyveromyces marxianus and the INU1 gene terminator INU1t to obtain the TEF1p-FRDg-INUt expression frame to enable its cytoplasmic expression.

[0062] In addition, the malic enzyme encoded by the gene MAE1 (NCBI-Gene ID: 34717552) can decarboxylate oxaloacetate to pyruvate, resulting in an ineffective cycle, so MAE1 is further inactivated. Considering that K. marxianus may produce a large amount of ethanol during cultivation at high glucose concentrations, the gene PDC1 (NCBI-Gene ID: 34717303), encoding pyruvate decarboxylase, is further inactivated.

[0063] In summary, the superior succinate-resistant strain DLY-KMSP-0209M obtained by domestication in Example 1 was subjected to the following genetic manipulations to construct a cytoplasmic reduction TCA pathway: four key genes of the reduction TCA pathway (encoding amino acid sequences such as pyruvate carboxylase PYC2, malate dehydrogenase MDH3-R, fumarate hydratase FUM1, and fumarate reductase FRDg, respectively) were overexpressed, and genes MAE1 and PDC1 were destroyed at the same time. An expression cassette plasmid was constructed for the above-mentioned establishment of a cytoplasmic reduction TCA succinate production pathway, and the plasmid schematic diagram is shown in FIG. Figure 1 As shown, the above genetic manipulation was completed using CRISPR / cas9 genome editing technology, and 4 transformants with correct genotypes were obtained, which were named #17, #21, #25, and #28.

[0064] Example 3 Glucose substrate-directed screening of succinate-producing strains using the TCA reduction pathway

[0065] Initially, the transformants #17, #21, #25, and #28 obtained from the above screening hardly grew under anaerobic conditions due to the destruction of PDC1. However, after two generations of transfer under aerobic conditions, they resumed growth under aerobic conditions, but the lag phase and growth cycle were slightly extended.

[0066] First, the #17, #21, #25, and #28 transformants were placed in an aerobic YPD medium (10 g / L yeast powder, 20 g / L peptone, and 20 g / L glucose) at 37°C and 200 rpm to quickly obtain a large number of cells. The cells were then collected by centrifugation, transferred to fresh YPD medium, and 10 g / L CaCO3 was added for anaerobic fermentation (80 mL round-bottom centrifuge tubes, sealed, and statically cultured). It was found that at 48 h, the transformants could consume 20 g / L glucose and accumulate succinic acid. It was also found that compared with the other transformants, the succinic acid yield of transformant #28 was significantly higher and the accumulation of by-products was significantly less, such as Figure 2 As shown, the succinic acid yield of transformant #28 can reach 0.77 g / L, the accumulation of byproduct glycerol is 1.5 g / L, lactic acid is 2.5 g / L, and the accumulation of byproduct acetic acid and citric acid is less. The succinic acid yield of transformant #28 is 7.7 times that of the starting strain DLY-KMSP-0209M under the same fermentation conditions. Therefore, transformant #28 was selected for further study and named DLY-KMPMFF-0329.

[0067] Kluyveromyces marxianus DLY-KMPMFF-0329 was deposited on April 3, 2023 in the General Microbiology Center of China Culture Collection (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Kluyveromyces marxianus, with the deposit number CGMCC No.27009.

[0068] The above experimental results prove the feasibility of synthesizing succinic acid using the reductive TCA pathway in Kluyveromyces marxianus, and also show that its byproducts are mainly lactic acid, followed by glycerol, and then acetic acid. Considering that the dissolved oxygen level during the fermentation process may affect the accumulation of lactic acid, subsequent attempts were made to use micro-aerobic culture methods to inhibit lactic acid production and allow more pyruvate to flow to the reductive pathway of succinic acid. Therefore, subsequent studies were conducted on the effects of the reductive TCA pathway on succinic acid production under different oxygen levels, in order to conduct micro-aerobic fermentation within a range that enables the reductive TCA pathway to perform its catalytic function. First, the dissolved oxygen level was changed by changing the speed of the shaker during the flask exploration stage, and then the dissolved oxygen control study was conducted at the fermenter scale-up level.

[0069] Example 4 Microaerobic fermentation of succinate-producing strains by reducing TCA pathway

[0070] Since the strain DLY-KMPMFF-0329 does not grow under completely anaerobic conditions and produces lactic acid, the feasibility of producing succinic acid under microaerobic conditions was tested. 50 mL of YPD liquid culture medium (marking the liquid level) was loaded into a 250 mL conical flask, about 1.2 g of CaCO3 (final concentration of about 24 g / L) was added and an oxygen-limited rubber stopper was used. After continuous cultivation at 37°C and 75 rpm for 7 days (the liquid level dropped slightly due to evaporation, and water was added to correct the volume), HPLC detection unexpectedly found that the only metabolite left was succinic acid, with almost no accumulation of by-products ( Figure 3 ). The yield of the target product succinic acid was 3.89 g / L, and the yield was 0.195 g / g. The experimental results showed that the micro-oxygen conditions had a significant effect on the yield of succinic acid and the accumulation of by-products, so the effect of different oxygen amounts on the production of succinic acid was studied.

[0071] Example 5 Effect of different oxygen supply on succinic acid production by strain DLY-KMPMFF-0329

[0072] In order to explore the effect of oxygen supply rate (under oxygen-limited conditions) on succinic acid production, fermentation experiments with different speeds were designed and the metabolites of the strains were detected. In YPD (50 g / L glucose) and CaCO3 with a final concentration of 30 g / L, oxygen-limited rubber stoppers were used, and the speeds were set to 200, 150, 100, and 75 rpm for testing at 37°C. The metabolites of each group of strains were detected by HPLC. The results are shown in Figure 4 The yield of succinic acid reached 7.5 g / L at XY-75r (oxygen-limited, 75 rpm). With the increase of the rotation speed, the production rate of succinic acid was the fastest at XY-200r (oxygen-limited, 200 rpm), but the yield was not as high as that of XY-75r, indicating that the oxygen flow rate has a direct effect on the production of succinic acid, including the effect on the enzyme activity in the reduction TCA pathway, such as the oxygen-sensitive catalytic pyruvate and HCO3 - The enzyme activity of PYC2p, the key enzyme for producing oxaloacetate, and the effect of oxygen on the rate of glycolysis (high oxygen flow will also affect the growth of the strain).

[0073] It can be seen that in the above-mentioned microaerobic fermentation process, the overall consumption rate of glucose is relatively slow. Even under aerobic conditions, it takes 96 hours to consume 50 g / L of glucose. This may be because after the addition of CaCO3, the metabolic flow tends to produce succinic acid, causing the environmental pH to become lower. In addition, as the rotation speed gradually decreases, the glucose consumption characteristic approaches a straight line from a curve, and the consumption of glucose every 24 hours is almost the same. This may be caused by the fact that the rate of pyruvate produced by the metabolism of glucose under a specific oxygen supply is equivalent to the rate of flow to succinic acid synthesis. Figure 4 It can be seen that there is a certain margin between the sugar consumption curves at 150 rpm and 100 rpm, so it is inferred that 125 rpm may be more conducive to balancing the sugar consumption rate and the yield of succinic acid and reducing the generation of by-products. Regarding the generation of by-products (such as lactic acid and glycerol), it is speculated that it is mainly due to the overflow of pyruvic acid (the production rate of pyruvic acid is greater than the rate for succinic acid production). Figure 4 From the c, we can see that under oxygen-limited conditions, as the rotation speed increases, the production of lactic acid gradually decreases, and the overall maximum is about 3g / L, and it will be consumed as a carbon source in the later stage. The more oxygen there is, the more pyruvate metabolism tends to be more productive respiratory chain. The above results once again prove the importance of oxygen control for succinic acid production. Figure 4It can be seen from the d that the production of glycerol decreases with the increase of rotation speed under oxygen-limited conditions, and the maximum does not exceed 2g / L. The production of glycerol requires the consumption of one molecule of NADH, competing with succinic acid for NADH and carbon sources. Theoretically, the NADH consumed by each additional 1g of glycerol by-product can convert 1.46g of malic acid into 1.28g of succinic acid, which not only reduces the by-product of malic acid but also increases the production and yield of succinic acid. Figure 4 The results showed that the succinic acid yield was higher at 200rpm under limited conditions, and the by-product accumulation was lower. In addition, the results of the optimization of CaCO3 addition showed that the addition of 15g / L CaCO3 had a better effect on improving the succinic acid yield.

[0074] Example 6 Production and detection of succinic acid by strain DLY-KMPMFF-0329 in shake flask fermentation

[0075] In order to shorten the culture period, the culture medium was YPD. 50 The strain DLY-KMPMFF-0329 was fermented in a shake flask to produce succinic acid at 37°C with a volume of 50 mL (glucose 50 g / L) + 15 g / L CaCO3 and a rotation speed of 200 rpm. The results showed that the production of SA reached 9.73 g / L at 96 h, with a yield of 0.24 g / g. 80 Under the condition of (glucose 80g / L) + 15g / L CaCO3, after 72h of feeding 15g / L CaCO3, glucose was completely consumed at 168h, SA production reached 16.5g / L, and the yield was 0.23g / g. However, when the initial sugar concentration was 100g / L, the production and yield were not the highest ( Figure 5 a). Surprisingly, in the medium of YD 50 (peptone 0g / L+10g / L yeast powder+glucose 50g / L)+15g / L CaCO3, rotation speed 200rpm and 37℃, the SA production reached 13.2g / L in 120h, and the yield was 0.26g / g( Figure 5 b), indicating that increasing the carbon-nitrogen ratio is beneficial to the production of SA, and also provides a good reference for reducing the cost of culture medium for producing SA in the future.

[0076] Example 7 Production and detection of succinic acid by strain DLY-KMPMFF-0329 in fermenter fermentation

[0077] Through the early shake flask experiment, it was found that the DO level has a very important influence on the production and yield of SA of strain DLY-KMPMFF-0329. In order to explore the conditions for the fermentation of SA by strain DLY-KMPMFF-0329 in a fermenter, the fermentation production of SA was carried out in a 5L fermenter ( Figure 6 ). The fermentation medium formula is as follows: peptone 20g / L, yeast powder 10g / L, glucose 50g / L, initial calcium carbonate 15g / L, the feeding strategy is to add mother liquor glucose (500g / L) once each time the sugar is exhausted, the temperature is 37°C, and the DO level is controlled by manually adjusting the speed and ventilation volume (generally not more than 1%). Continuous sampling and HPLC are used to detect changes in metabolites as a basis for adjusting the speed and ventilation size. As of 250h of cultivation, a total of 175g / L of glucose was consumed (four feedings see Figure 6 a), 37.5 g / L CaCO3 was added three times, the first time was 15 g / L in the culture medium before fermentation, the second time was 15 g / L at 96 h of culture, and the third time was 7.5 g / L at 144 h (the need for additional CaCO3 can be determined by microscopic examination and pH changes in the fermentation liquid). The yield of succinic acid was 25.2 g / L.

[0078] In addition, the fermentation cycle is relatively long and the accumulation of SA begins to accelerate in the second half of the fermentation. It is speculated that this is mainly due to the increase in the number of bacteria and the increase in dissolved oxygen, which gradually increases from 100 mL / min (the dissolved oxygen DO value is about 0%) to 250 mL / min and then to 1 vvm (DO is about 1%). The slow SA accumulation rate in the early stage may be due to the slow growth of biomass caused by insufficient oxygen. After the dissolved oxygen is increased, the sugar consumption rate is significantly accelerated, the SA accumulation rate is significantly increased, and the accumulation of lactic acid begins to decrease to no ( Figure 6 c). The above results suggest that the growth phase can be completed quickly before entering the SA production phase.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Kluyveromyces marxianus DLY-KMPMFF-0329, characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.27009.

2. The Kluyveromyces marxianus DLY-KMPMFF-0329 according to claim 1, characterized in that The Kluyveromyces marxianus DLY-KMPMFF-0329 expresses a fumarate reductase FRDg whose amino acid sequence is shown in SEQ ID NO.4; And / or, the genome of the Kluyveromyces marxianus DLY-KMPMFF-0329 contains a DNA fragment with a nucleotide sequence as shown in SEQ ID NO.

5.

3. Kluyveromyces marxianus DLY-KMPMFF-0329 according to claim 1 or 2, characterized in that Compared with the wild-type Kluyveromyces marxianus NBRC1777, the expression of pyruvate carboxylase PYC2 in the Kluyveromyces marxianus DLY-KMPMFF-0329 is enhanced, and / or the malate dehydrogenase MDH3 with an amino acid sequence as shown in SEQ ID NO.2 is expressed, and / or the fumarate hydratase FUM1 with an amino acid sequence as shown in SEQ ID NO.3 is expressed, and / or the coding gene of malic enzyme MAE1 is inactivated, and / or the coding gene of pyruvate decarboxylase PDC1 is inactivated.

4. A microbial preparation, characterized in that: The yeast comprises the Kluyveromyces marxianus DLY-KMPMFF-0329 according to any one of claims 1 to 3.

5. The method for preparing the microbial preparation according to claim 4, characterized in that: The method comprises the step of culturing the Kluyveromyces marxianus DLY-KMPMFF-0329.

6. Any of the following uses of Kluyveromyces marxianus DLY-KMPMFF-0329 according to any one of claims 1 to 3 or the microbial preparation according to claim 4: (1) Application in the fermentation production of organic acids or their derivatives; (2) Application in breeding or constructing strains producing organic acids or their derivatives; Preferably, the organic acid is succinic acid.

7. A method for producing succinic acid by fermentation, characterized in that: The method comprises the step of culturing the Kluyveromyces marxianus DLY-KMPMFF-0329 according to any one of claims 1 to 3.

8. The method according to claim 7, characterized in that The culture comprises a fermentation culture stage, and the fermentation culture stage comprises the step of culturing the Kluyveromyces marxianus DLY-KMPMFF-0329 under oxygen-limited conditions.

9. The method according to claim 8, characterized in that The oxygen-limited condition is to control the dissolved oxygen DO to be below 2%.

10. The method according to claim 8 or 9, characterized in that: In the fermentation and culturing stage, carbonate is added at the beginning of fermentation and / or during fermentation; The added amount of the carbonate is 10-50 g / L.

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