Acid-resistant kluyveromyces marxianus strain and use thereof

Abstract

The present invention relates to the technical field of microorganisms, and in particular to an acid-resistant Kluyveromyces marxianus strain and the use thereof. The provided Kluyveromyces marxianus DLY-KMPMFF-0329 is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No.27009. The strain can tolerate a high concentration of succinic acid and low pH conditions, can efficiently use glucose to synthesize succinic acid by means of the reductive tricarboxylic acid cycle pathway, and can achieve a relatively high production and yield of succinic acid. In addition, the strain has a relatively high homologous recombination efficiency, so that efficient gene editing can be achieved. The strain can be used not only as a succinic acid-producing strain for the fermentation production of succinic acid, but also as a starting strain for breeding or constructing a high-yield succinic acid-producing strain, and has a good application value.

Description

Acid-resistant Kluyveromyces marxianus strain and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202311380695.6 filed on October 24, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of microorganisms, in particular to an acid-resistant Kluyveromyces marxianus strain and application thereof. Background Art

[0004] Kluyveromyces marxianus has the characteristics of high temperature resistance, fast growth rate, and natural utilization of 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).

[0005] Succinic acid, also known as succinic acid (SA), is an important biochemical raw material with significant applications in agriculture, food, clothing, cosmetics, and pharmaceutical industries. In addition to its use as a surfactant, ion chelator, and food additive, succinic acid is also 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. Furthermore, succinic acid can 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 biobased 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 production costs.

[0006] Compared to production processes based on fossil feedstocks, microbial fermentation is more competitive in producing succinic acid. Currently, microorganisms capable of producing succinic acid include Yarrowia lipolytica, Saccharomyces cerevisiae, Escherichia coli, Actinobacillus succinogenes, and Corynebacterium glutamicum. However, relatively little research has been conducted on succinic acid production by Kluyveromyces marxianus. Furthermore, Kluyveromyces marxianus is a yeast that primarily undergoes non-homologous recombination, making efficient genetic manipulation of multiple loci challenging.

[0007] 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 oxidatively decarboxylated to form SA, with a theoretical yield of 1 mol SA / 1 mol glucose. In the rTCA pathway, each molecule of SA generated after carboxylation of pyruvate or phosphoenolpyruvate can fix one molecule of CO2, 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.

[0008] Currently, the main engineered host bacteria for SA production using rTCA are Escherichia coli, Actinomycetes, and Corynebacterium glutamicum. However, these bacteria are at risk of phage infection and have poor acid resistance, generally with an optimal pH range of 6.8-7.3. During the fermentation process, a large amount of neutralizer must be added to neutralize the acidic environment caused by succinic acid production, increasing the costs of fermentation and subsequent separation. Yeast cells do not have the risk of phage infection and are generally more acid-resistant than bacteria. Therefore, using yeast cells as the base cell to produce SA using the rTCA pathway is a relatively promising production method. The reported SA production and yield of Saccharomyces cerevisiae via the rTCA pathway are both low. Although Issatchenkia orientalis naturally has strong low pH tolerance, by overexpressing endogenous pyruvate carboxylase PYC1, malate dehydrogenase MDH3, and fumarate hydratase FUM1, and heterologously expressing Aspergillus oryzae fumarate reductase FRDg, the final SA production via 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).

[0009] 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 et al., Highly efficient neutralizer-free L-malicacid 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.

[0010] 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.

[0011] Summary of the Invention

[0012] The invention provides an acid-resistant Kluyveromyces marxianus strain and application thereof.

[0013] During the research and development process, the applicant domesticated and screened Kluyveromyces marxianus (K. marxianus) NBRC1777 to improve its tolerance to low pH and succinic acid, obtaining a Kluyveromyces marxianus strain with strong low pH and succinic acid tolerance. Using this strain as the starting strain, the homologous recombination efficiency of the strain was further enhanced through genetic engineering, resulting in the acquisition of Kluyveromyces marxianus (K. marxianus) DLY-KMSP-0209. The strain was deposited on February 16, 2022 at the General Microbiology Center of the China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified as Kluyveromyces marxianus with a deposit number of CGMCC No. 24398. Kluyveromyces marxianus (K. marxianus) DLY-KMSP-0209 can tolerate low pH conditions and high succinate concentrations. Furthermore, the strain has a high homologous recombination efficiency, making it suitable as a base cell for breeding strains for the fermentation production of organic acids such as succinate. Furthermore, Kluyveromyces marxianus DLY-KMSP-0209 can grow using one or more carbon sources selected from glucose and lignocellulosic biomass (including xylose, lignocellulosic hydrolysate, etc.), with an optimal growth temperature of 30-42°C (preferably 37-42°C), enabling fermentation production at higher temperatures while tolerating low pH and high succinate concentrations.

[0014] In order to further improve the yield of succinic acid, the present invention uses Kluyveromyces marxianus DLY-KMSP-0209 as a starting strain, first acclimates Kluyveromyces marxianus DLY-KMSP-0209 to low pH and succinic acid tolerance, further genetically engineers the dominant strain obtained through acclimation screening to enable the strain to produce succinic acid using the reductive TCA pathway (rTCA), and finally subjects the genetically engineered strain to glucose substrate-directed screening to obtain a strain capable of efficiently utilizing glucose to synthesize succinic acid. Ultimately, a Kluyveromyces marxianus strain having good low pH and succinic acid tolerance, high ability to synthesize succinic acid using glucose, the ability to produce succinic acid using rTCA, and high succinic acid yield and yield is obtained, and is named DLY-KMPMFF-0329.

[0015] Specifically, the present invention provides the following technical solutions:

[0016] In the first aspect, the present invention provides Kluyveromyces marxianus DLY-KMPMFF-0329, which was deposited on April 3, 2023 in the General Microbiology Center of the China Culture Collection Administration (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 a deposit number of CGMCC No. 27009.

[0017] The present invention constructs rTCA in Kluyveromyces marxianus DLY-KMPMFF-0329 through genetic engineering modification. 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 encoding gene of malic enzyme MAE1, and inactivating the encoding gene of pyruvate decarboxylase PDC1.

[0018] 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.

[0019] 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 is expressed with an amino acid sequence as shown in SEQ ID NO.2, and / or the fumarate hydratase FUM1 is expressed with an amino acid sequence as shown in SEQ ID NO.3, and / or the gene encoding malic enzyme MAE1 is inactivated, and / or the gene encoding pyruvate decarboxylase PDC1 is inactivated.

[0020] Kluyveromyces marxianus DLY-KMPMFF-0329 can tolerate low pH conditions and high succinic acid concentrations, allowing the strain to adapt to more demanding production conditions and avoid product inhibition, thereby achieving higher yields and rates when using the strain for succinic acid fermentation production. Moreover, the strain has a high homologous recombination efficiency and is suitable as a chassis cell for breeding fermentation strains for organic acids such as succinic acid. In addition, Kluyveromyces marxianus DLY-KMPMFF-0329 can efficiently utilize glucose to synthesize succinic acid through rTCA, with higher succinic acid production and rate.

[0021] In a second aspect, the present invention provides Kluyveromyces marxianus comprising a gene expressing fumarate reductase FRDg derived from Trypanosoma brucei.

[0022] According to a preferred embodiment of the present invention, the amino acid sequence of the fumarate reductase FRDg is at least 70%, preferably at least 80%, and more preferably at least 90% identical to the amino acid sequence shown in SEQ ID NO. 4. Preferably, the activity of the fumarate reductase FRDg is at least 70%, more preferably at least 80%, and even more preferably at least 90% of the activity of the fumarate reductase FRDg having the amino acid sequence shown in SEQ ID NO. 4. Preferably, the fumarate reductase FRDg comprises 1000-1300 amino acids.

[0023] Preferably, the amino acid sequence of the fumarate reductase FRDg is 95-100% identical to the amino acid sequence shown in SEQ ID NO. 4. For example, the identity may be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the above values, or any intermediate value within the range.

[0024] According to a preferred embodiment of the present invention, the genome of Kluyveromyces marxianus contains a gene encoding a fumarate reductase FRDg having a nucleotide sequence identity of at least 60% with the nucleotide sequence of SEQ ID NO. 5. Preferably, the gene encoding the fumarate reductase FRDg has an identity of at least 70% with the nucleotide sequence of SEQ ID NO. 5, more preferably at least 80%, and even more preferably at least 90%.

[0025] Preferably, the gene encoding the fumarate reductase FRDg has an identity of 95-100% with the nucleotide sequence shown in SEQ ID NO. 5. For example, the identity may be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the above values, or any intermediate value within the range.

[0026] According to a preferred embodiment of the present invention, compared with the wild-type Kluyveromyces marxianus, the expression of pyruvate carboxylase PYC2 in the Kluyveromyces marxianus is enhanced, and / or, malate dehydrogenase MDH3 is expressed, and / or, fumarate hydratase FUM1 is expressed, and / or, the gene encoding malic enzyme MAE1 is inactivated, and / or, the gene encoding pyruvate decarboxylase PDC1 is inactivated.

[0027] Preferably, the amino acid sequence of the malate dehydrogenase MDH3 is not less than 70% identical to the amino acid sequence shown in SEQ ID NO. 2, preferably not less than 80%, more preferably not less than 90%.

[0028] More preferably, the amino acid sequence of the malate dehydrogenase MDH3 has an identity of 95-100% with the amino acid sequence of SEQ ID NO. 2, for example, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the foregoing values, or any intermediate value within the range.

[0029] Preferably, the amino acid sequence of the fumarate hydratase FUM1 is not less than 70% identical to the amino acid sequence shown in SEQ ID NO. 3, preferably not less than 80%, more preferably not less than 90%.

[0030] More preferably, the amino acid sequence of the fumarate hydratase FUM1 is 95-100% identical to the amino acid sequence of SEQ ID NO. 3. For example, the identity may be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the foregoing values, or any intermediate value within the range.

[0031] According to a preferred embodiment of the present invention, the wild-type Kluyveromyces marxianus is NBRC1777.

[0032] According to some preferred embodiments of the present invention, the gene encoding malic enzyme MAE1 in the Kluyveromyces marxianus is inactivated compared to Kluyveromyces marxianus CGMCC No. 24398.

[0033] According to some preferred embodiments of the present invention, the gene encoding pyruvate decarboxylase PDC1 in the Kluyveromyces marxianus is inactivated compared to Kluyveromyces marxianus CGMCC No. 24398.

[0034] Preferably, the Kluyveromyces marxianus is DLY-KMPMFF-0329, and its deposit number is CGMCC No. 27009.

[0035] Furthermore, the present invention provides a method for producing an engineered Kluyveromyces marxianus strain that produces high succinate production, comprising introducing a gene expressing a fumarate reductase (FRDg) from Trypanosoma brucei into the genome of wild-type Kluyveromyces marxianus. Preferably, the amino acid sequence of the fumarate reductase (FRDg) is at least 70% identical to the amino acid sequence of SEQ ID NO. 4, preferably at least 80%, and more preferably at least 90%.

[0036] Preferably, the amino acid sequence of the fumarate reductase FRDg is 95-100% identical to the amino acid sequence shown in SEQ ID NO. 4. For example, the identity may be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the above values, or any intermediate value within the range.

[0037] According to a preferred embodiment of the present invention, the gene encoding fumarate reductase FRDg has an identity of not less than 60% with the nucleotide sequence shown in SEQ ID NO. 5. Preferably, the gene encoding fumarate reductase FRDg has an identity of not less than 70% with the nucleotide sequence shown in SEQ ID NO. 5, more preferably not less than 80%, and even more preferably not less than 90%.

[0038] Preferably, the gene encoding the fumarate reductase FRDg has an identity of 95-100% with the nucleotide sequence shown in SEQ ID NO. 5. For example, the identity may be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the above values, or any intermediate value within the range.

[0039] According to a preferred embodiment of the present invention, the method further includes enhancing the expression of pyruvate carboxylase PYC2 in the engineered Kluyveromyces marxianus compared to the wild-type Kluyveromyces marxianus, and / or expressing malate dehydrogenase MDH3 in the engineered Kluyveromyces marxianus, and / or expressing fumarate hydratase FUM1 in the engineered Kluyveromyces marxianus, and / or inactivating the gene encoding malic enzyme MAE1 in the engineered Kluyveromyces marxianus, and / or inactivating the gene encoding pyruvate decarboxylase PDC1 in the engineered Kluyveromyces marxianus.

[0040] Preferably, the amino acid sequence of the malate dehydrogenase MDH3 is not less than 70% identical to the amino acid sequence shown in SEQ ID NO. 2, preferably not less than 80%, more preferably not less than 90%.

[0041] More preferably, the amino acid sequence of the malate dehydrogenase MDH3 has an identity of 95-100% with the amino acid sequence of SEQ ID NO. 2, for example, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the foregoing values, or any intermediate value within the range.

[0042] Preferably, the amino acid sequence of the fumarate hydratase FUM1 is not less than 70% identical to the amino acid sequence shown in SEQ ID NO. 3, preferably not less than 80%, more preferably not less than 90%.

[0043] More preferably, the amino acid sequence of the fumarate hydratase FUM1 is 95-100% identical to the amino acid sequence of SEQ ID NO. 3. For example, the identity may be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, or a range consisting of any two of the foregoing values, or any intermediate value within the range.

[0044] In the present invention, gene inactivation can be achieved using methods commonly used in the art. Gene inactivation means that the expression level of the gene does not exceed 5% of the original expression level, and preferably, the gene is not expressed at all. Specific methods of inactivation include (complete) gene knockout, replacement / knockout of the gene's expression regulatory region, or introduction of other gene sequences that can inhibit gene expression.

[0045] According to a particularly preferred embodiment of the present invention, the method comprises:

[0046] (1) Domesticating, screening, and genetically modifying wild-type Kluyveromyces marxianus to obtain a base strain with improved homologous recombination efficiency;

[0047] (2) domesticating, screening, and genetically modifying the base bacteria obtained in step (1) to obtain the engineered Kluyveromyces marxianus bacteria.

[0048] Preferably, in step (1), the homologous recombination efficiency of the chassis bacteria is increased by at least 0.5%, preferably by 0.5-90%, compared to that of wild-type Kluyveromyces marxianus. For example, the homologous recombination efficiency of the chassis bacteria can be increased by 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or can be a range consisting of any two of the above values, or any intermediate value within the range.

[0049] Preferably, the wild-type Kluyveromyces marxianus is NBRC1777.

[0050] More preferably, the base plate bacteria is Kluyveromyces marxianus DLY-KMSP-0209, with a deposit number of CGMCC No. 24398.

[0051] The present invention further provides an engineered Kluyveromyces marxianus strain obtained according to the above method.

[0052] In a third aspect, the present invention further provides a microbial preparation comprising the above-mentioned Kluyveromyces marxianus.

[0053] According to a preferred embodiment of the present invention, the Kluyveromyces marxianus is DLY-KMPMFF-0329.

[0054] The microbial preparation described above can be a solid preparation or a liquid preparation. The Kluyveromyces marxianus in the microbial preparation is present in the form of live bacteria. In addition to containing Kluyveromyces marxianus or a culture of Kluyveromyces marxianus, the microbial preparation can also contain carriers or other excipients permitted in the field of microbial preparations.

[0055] Preferably, the excipients include at least one of a protective agent (such as a lyophilization protective agent, etc.), a buffer and a stabilizer.

[0056] The present invention also provides a method for preparing the above-mentioned microbial preparation, which comprises the step of culturing the Kluyveromyces marxianus.

[0057] In some embodiments of the present invention, Kluyveromyces marxianus is cultured to obtain a culture solution, and the culture solution is directly prepared into 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.

[0058] In a fourth aspect, the present invention provides any of the following uses of the above-mentioned Kluyveromyces marxianus or the microbial preparation:

[0059] (1) Application in the fermentation production of organic acids or their derivatives;

[0060] (2) Application in the breeding and / or construction of strains producing organic acids or their derivatives.

[0061] In the above application (2), the Kluyveromyces marxianus provided by the present invention (e.g., the Kluyveromyces marxianus described in the first or second aspect, or the Kluyveromyces marxianus constructed according to the above method) can be used as a starting strain to carry out the selection or construction of a strain producing an organic acid or its derivatives. Wherein, 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. The derivative of the organic acid can be a derivative compound generated with the organic acid as the main substrate, such as a salt (such as an ammonium salt, a metal salt, etc.), an ester, etc.

[0062] In the present invention, the starting strain refers to the initial strain used for strain selection or construction, and the starting strain is genetically engineered, mutagenized or domesticated to obtain the target strain.

[0063] The Kluyveromyces marxianus provided in the first and second aspects of the present invention, especially the 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 also 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.

[0064] Preferably, the organic acid is succinic acid.

[0065] The present invention further provides a method for constructing an engineered Kluyveromyces marxianus strain that produces succinic acid, the method comprising: using the Kluyveromyces marxianus provided by the present invention (eg, Kluyveromyces marxianus DLY-KMPMFF-0329) as a starting strain, and genetically modifying the starting strain.

[0066] Preferably, the genetic modification comprises introducing exogenous nucleic acid into the starting strain using homologous recombination technology, and / or performing gene editing on endogenous genes of the starting strain using homologous recombination technology.

[0067] The homologous recombination technology includes but is not limited to CRISPR / Cas9, Cre-Loxp, TALEN, etc.

[0068] A fifth aspect of the present invention provides a method for producing succinic acid by fermentation, the method comprising the step of culturing Kluyveromyces marxianus as described in the first or second aspect, or prepared by the method described in the third aspect.

[0069] Preferably, the culture temperature is 37-42°C.

[0070] According to a preferred embodiment of the present invention, the Kluyveromyces marxianus is Kluyveromyces marxianus DLY-KMPMFF-0329, and its deposit number is CGMCC No.27009.

[0071] 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.

[0072] The oxygen-limiting condition is to control the dissolved oxygen (DO) to below 2%. Preferably, the dissolved oxygen (DO) is controlled to below 1%. For example, the dissolved oxygen (DO) can be controlled at 2%, 1.8%, 1.5%, 1.2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%, or can be controlled within a range consisting of any two of the above values, or any intermediate value within the range.

[0073] The present invention discovered that when producing succinate using the rTCA pathway, dissolved oxygen levels significantly affect the fermentation process, succinate conversion, and byproduct formation. Controlling dissolved oxygen levels within the aforementioned oxygen-limited conditions can promote succinate synthesis and reduce byproduct formation.

[0074] 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.

[0075] The present invention discovered that carbon dioxide can be fixed by adding carbonates when producing succinate using the rTCA pathway. Carbon dioxide can be utilized by Kluyveromyces marxianus DLY-KMPMFF-0329 in the form of carbonates (e.g., calcium carbonate), significantly increasing succinate production and providing new insights and solutions for addressing carbon emission reduction.

[0076] The carbonate salt mentioned above preferably includes at least one of calcium carbonate, sodium carbonate, potassium carbonate and ammonium carbonate, and is most preferably calcium carbonate.

[0077] Preferably, the carbonate can be added directly to the fermentation medium, and / or the carbonate can 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.

[0078] 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.

[0079] Preferably, the mass ratio of the carbon source to the nitrogen source is (3-6):1 (calculated as C and N, this ratio may also be referred to as the "carbon-nitrogen ratio"). The present invention has found that appropriately increasing the carbon-nitrogen ratio in the fermentation medium is beneficial for increasing the yield of succinic acid produced by Kluyveromyces marxianus DLY-KMPMFF-0329 fermentation, and preferably controlling the carbon-nitrogen ratio within the range of (3-6):1.

[0080] Preferably, the fermentation medium comprises: 20-100 g / L glucose, 0-20 g / L peptone, and 5-15 g / L yeast powder.

[0081] 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 carried out under aerobic conditions, and the second fermentation and culture stage is carried out under oxygen-limited conditions.

[0082] The present invention finds that a two-stage fermentation process for the fermentation of 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.

[0083] The culturing may further include a seed culturing stage.

[0084] The beneficial effects of the present invention include at least the ability of the Kluyveromyces marxianus DLY-KMPMFF-0329 strain to tolerate high concentrations of succinic acid and low pH conditions, the ability to efficiently utilize glucose to synthesize succinic acid via the rTCA pathway, and the increased production and yield of succinic acid. Furthermore, the strain exhibits high homologous recombination efficiency and is capable of efficient gene editing. Based on these advantages, the Kluyveromyces marxianus DLY-KMPMFF-0329 strain can be used as a succinic acid-producing strain for fermentation production, or as a starting strain for breeding or constructing high-yielding strains of succinic acid, thus possessing considerable application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 is a schematic diagram of the expression cassette for constructing the reductive TCA pathway in Example 2 of the present invention, wherein IMTCP2p-kmPYC2-CP1t indicates that the endogenous pyruvate carboxylase gene kmPYC2 of Kluyveromyces marxianus is overexpressed using the endogenous promoter IMTCP2p of Kluyveromyces marxianus and the terminator IMTCP1 (represented by CP1t in the figure); IMTCP1p-MDH3-R-CP2t indicates that the endogenous malate dehydrogenase gene MDH3-R of Kluyveromyces marxianus is overexpressed 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 indicate that the gene PDC1 encodes The homology arms of 650 bp upstream and 657 bp downstream of the frame (used for the target fragment to target PDC1); the fragment TEF1p-FRDg-INUt indicates heterologous overexpression of fumarate reductase FRDg using the endogenous promoter TEF1p of Kluyveromyces marxianus and the INU1 gene terminator INU1t; TEF3p-FUM1-14n-PDC1t indicates expression of endogenous fumarate hydratase using the endogenous promoter TEF3p of Kluyveromyces marxianus and the PDC1 gene terminator PDC1t (14n indicates the mitochondrial localization sequence with 14 amino acids removed); 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.

[0086] FIG2 shows the metabolite detection results of the glucose substrate-directed screening experiment performed on four transformants with correct genotypes in Example 3 of the present invention, wherein #28, #25, #21, and #17 are the four transformants with correct genotypes.

[0087] FIG3 shows the detection results of succinic acid production and by-product 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.

[0088] FIG4 shows the effect of different rotation speeds on succinic acid production under oxygen-limited conditions in Example 5 of the present invention, wherein a) is succinic acid production, b) is glucose consumption, c) is lactic acid accumulation, and d) is glycerol accumulation.

[0089] Figure 5 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 labeled curve represents glucose consumption, and the hollow labeled curve represents succinic acid production; glucose 50, 60, 80, and 100 represent added glucose concentrations of 50, 60, 80, and 100 g / L, respectively, and peptone 0, 5, and 10 g / L represent the addition of no peptone and the addition of 5 and 10 g / L peptone, respectively.

[0090] Figure 6 is a metabolite analysis of succinate produced in a 5 L fermentation tank in Example 7 of the present invention, wherein a) shows the changes in glucose and SA; b) shows the accumulation of byproducts citric acid, acetic acid, and malic acid; and c) shows the accumulation of byproducts glycerol and lactic acid.

[0091] In Figures 1 to 6, SA stands for succinic acid. DETAILED DESCRIPTION

[0092] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0093] Example 1 Acclimation of Kluyveromyces marxianus to Low pH and Succinic Acid Tolerance

[0094] Using 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 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 medium and 3 mL of 60 g / L SA mother solution was added (SA final concentration of 45 g / L). The culture was continued under the same conditions for 90 days. Finally, the strain was inoculated in 0.5 mL of YPD liquid 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). After 20 days of culture under the same conditions, the bacterial solution was taken to spread on plates, and single clones that tolerated low pH and high concentration succinic acid were selected to obtain strain DLY-KMSP-0209M. The strain can grow under the conditions of pH 2.3 and SA concentration of 52.5g / L. The tolerance test results showed that the strain DLY-KMSP-0209M had 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%.

[0095] Example 2 Construction of a succinate-producing strain using the TCA reduction pathway

[0096] The production of succinate by the reductive TCA pathway requires four key genes, which encode pyruvate carboxylase PYC2, which converts pyruvate to HCO3 - The enzyme catalyzes the production of oxaloacetate; malate dehydrogenase (MDH3) reduces oxaloacetate to malate while consuming one NADH molecule; fumarate hydratase (FUM1) converts malate to fumarate; and fumarate reductase (FRDg) reduces fumarate to succinate while consuming one NADH molecule. The first three genes, PYC2, MDH3, and FUM1, can be endogenously amplified from the Kluyveromyces marxianus strain NBRC1777. However, one of the key genes, FRDg, originates from Trypanosoma brucei and requires heterologous synthesis and codon optimization. This enzyme's coenzyme is NADH, which facilitates the unification of the coenzyme and the establishment of redox balance.

[0097] Because yeast cell mitochondria have a double-membrane structure, the products of the TCA pathway (malic acid, fumaric acid, and succinic acid) need to use transport proteins to cross the double membrane into the cytoplasm and then be secreted outside the cell, which undoubtedly hinders the production and excretion of succinic acid. However, the mechanism of transport of these organic dicarboxylic acids from mitochondria to the cytoplasm is not yet fully understood. To minimize this effect, the enzymes related to the reduction pathway located in the mitochondria were directly transferred to the cytoplasm. PYC2p (amino acid sequence shown in SEQ ID NO.1, encoded by gene KLMA_10253) is itself a cytoplasmic protein. It was ligated with the endogenous Kluyveromyces marxianus promoter IMTCP2p and terminator IMTCP1 to construct the IMTCP2p-kmPYC2-CP1t expression cassette and inserted into the genomic location of the PDC1 gene. MDH3p is localized in peroxisomes. The three "TKL" amino acids at the C-terminus of MDH3p (amino acid sequence shown in SEQ ID NO. 2) were removed and placed in the IMTCP1p-MDH3-R-CP2t expression cassette for cytoplasmic expression. FUM1p (encoded by gene KLMA_80028), a protein that is bilocalized in mitochondria and the cytoplasm, was localized in the cytoplasm by removing the 14 amino acids at the N-terminus (including a 14-amino acid mitochondrial localization sequence and a start amino acid, amino acid sequence shown in SEQ ID NO. 3). A new translation start codon was then introduced to localize it in the cytoplasm. The protein was then expressed in the TEF3p-FUM1-14n-PDC1t expression cassette. 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 encoding gene sequence is shown in SEQ ID NO.5), and the protein was ligated with the endogenous promoter TEF1p of Kluyveromyces marxianus and the INU1 gene terminator INU1t to obtain a TEF1p-FRDg-INUt expression cassette for cytoplasmic expression.

[0098] In addition, the malic enzyme encoded by the gene MAE1 (NCBI-GeneID: 34717552) can decarboxylate oxaloacetate to pyruvate, resulting in a futile cycle, so MAE1 was further inactivated. Considering that K. marxianus may produce large amounts of ethanol during cultivation at high glucose concentrations, the gene PDC1 (NCBI-GeneID: 34717303), encoding pyruvate decarboxylase, was further inactivated.

[0099] In summary, the low pH-tolerant and succinate-tolerant strain DLY-KMSP-0209M obtained by domestication in Example 1 was subjected to the following genetic manipulations to construct a cytoplasmic reduction TCA pathway: overexpression of four key genes of the reduction TCA pathway (encoding amino acid sequences such as pyruvate carboxylase PYC2p, malate dehydrogenase MDH3-R, fumarate hydratase FUM1, and fumarate reductase FRDg, respectively), while disrupting genes MAE1 and PDC1. An expression cassette plasmid was constructed for the above-mentioned establishment of a cytoplasmic reduction TCA succinate production pathway. The plasmid schematic is shown in Figure 1. The above genetic manipulations were completed using CRISPR / cas9 genome editing technology to obtain four transformants with correct genotypes, named #17, #21, #25, and #28, respectively.

[0100] Example 3 Glucose substrate-directed screening of succinate-producing strains using the TCA reduction pathway

[0101] Initially, transformants #17, #21, #25, and #28 obtained in the above screening showed little growth 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.

[0102] First, transformants #17, #21, #25, and #28 were cultured in 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 bacteria. The bacteria 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 after 48 hours, 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. As shown in Figure 2, the succinic acid yield of transformant #28 can reach 0.77 g / L, the accumulation of by-product glycerol is 1.5 g / L, lactic acid is 2.5 g / L, and the accumulation of by-products acetic acid and citric acid is less. Transformant #28 produced 7.7 times more succinate than the starting strain DLY-KMSP-0209M under the same fermentation conditions. Therefore, transformant #28 was selected for further study and designated DLY-KMPMFF-0329.

[0103] Kluyveromyces marxianus DLY-KMPMFF-0329 was deposited on April 3, 2023 at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified as Kluyveromyces marxianus with the deposit number CGMCC No. 27009.

[0104] The above experimental results demonstrate the feasibility of synthesizing succinic acid using the reductive TCA pathway in Kluyveromyces marxianus, and also show that its byproduct is 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 microaerobic culture methods to inhibit lactic acid production and allow more pyruvate to flow to the reductive pathway of succinic acid. Therefore, subsequent studies were carried out on the effect of the reductive TCA pathway on succinic acid production under different oxygen levels, in order to conduct microaerobic fermentation within a range that allows the reductive TCA pathway to perform its catalytic function. First, the dissolved oxygen level was changed by changing the shaker speed during the shake flask exploration stage, and then the dissolved oxygen control study was carried out at the fermentation tank scale-up level.

[0105] Example 4 Microaerobic fermentation of succinate-producing strains using the reduced TCA pathway

[0106] 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 medium (marking the liquid level) was loaded into a 250 mL Erlenmeyer flask, about 1.2 g of CaCO3 was added (final concentration was about 24 g / L) and an oxygen-limiting 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 microaerobic conditions had a significant effect on the yield of succinic acid and the accumulation of by-products, so the effect of different oxygen levels on succinic acid production was investigated.

[0107] Example 5 Effect of Different Oxygen Supply on Succinic Acid Production by Strain DLY-KMPMFF-0329

[0108] In order to explore the effect of oxygen supply rate (under oxygen-limited conditions) on succinic acid production, fermentation experiments with different rotation speeds were designed, and the metabolites of the strains were detected. In YPD (50 g / L glucose) and with the addition of CaCO3 at a final concentration of 30 g / L, using oxygen-limited rubber stoppers, at 37 ° C, the rotation speeds were set to 200, 150, 100, and 75 rpm for testing, and 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 in XY-75r (oxygen-limited, 75 rpm). With the increase in rotation speed, the production rate of succinic acid was the fastest under XY-200r (oxygen-limited, 200 rpm), but the yield was not as high as that of XY-75r, indicating that the oxygen supply 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).

[0109] It can be seen that in the above-mentioned microaerobic fermentation process, the consumption rate of glucose is relatively slow as a whole. Even under aerobic conditions, it takes 96h to consume 50g / L of glucose. This is probably because after adding CaCO3, the metabolic flow is biased towards producing succinic acid, causing the environmental pH to become low. In addition, as the speed gradually decreases, the glucose consumption characteristic approaches the straight line from the curve, and the consumption of glucose every 24h is almost the same. This is probably caused by the speed of pyruvic acid produced by the metabolism of glucose under a specific oxygen supply and the flow to the succinic acid synthesis rate. As can be seen from Figure 4, there is a certain margin between the sugar consumption curves at a speed of 150rpm and 100rpm. Therefore, it is inferred that 125rpm may be more conducive to balancing the sugar consumption rate and the succinic acid yield, reducing the generation of by-products. For the generation of by-products (such as lactic acid, 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). As shown in Figure 4c), under oxygen-limited conditions, lactate production gradually decreases with increasing rotational speed, reaching a maximum of approximately 3 g / L before being consumed as a carbon source in the later stages. With more oxygen, pyruvate metabolism favors the more productive respiratory chain. These results further demonstrate the importance of controlling oxygen levels for succinate production. As shown in Figure 4d), glycerol production decreases with increasing rotational speed under oxygen-limited conditions, reaching a maximum of no more than 2 g / L. Glycerol production consumes one molecule of NADH, competing with succinate for both NADH and carbon sources. Theoretically, for every additional gram of glycerol produced as a byproduct, 1.46 g of malic acid is converted to 1.28 g of succinate, thereby reducing malic acid byproducts and increasing succinate production and yield. Based on the results in Figure 4, succinate production is higher at 200 rpm under oxygen-limited conditions, while byproduct accumulation is lower. Furthermore, optimization of the CaCO3 addition level revealed that adding 15 g / L CaCO3 significantly increased succinate production.

[0110] Example 6 Production and Detection of Succinic Acid by Strain DLY-KMPMFF-0329 in Shake Flask Fermentation

[0111] In order to shorten the culture period, the culture medium was YPD. 50 The strain DLY-KMPMFF-0329 was fermented in a shake flask with 50 mL of (glucose 50 g / L) + 15 g / L CaCO3 in a 250 mL triangular bottle at 200 rpm and 37°C to produce succinic acid. The results showed that the SA production reached 9.73 g / L at 96 h, with a yield of 0.24 g / g. 80Under the conditions of (glucose 80g / L) + 15g / L CaCO3, after 72h of feeding 15g / L CaCO3, glucose was completely consumed at 168h, and SA production reached 16.5g / L, with a yield of 0.23g / g. However, when the initial sugar concentration was 100g / L, both the production and yield were not the highest, as shown in Figure 5a). Surprisingly, when the medium was YD 50 Under the conditions of (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 after 120h, with a yield of 0.26g / g (see Figure 5b for details). This shows that increasing the carbon-nitrogen ratio is beneficial to the production of SA and also provides a good reference for reducing the culture medium cost for SA production in the future.

[0112] Example 7 Production and Detection of Succinic Acid by Strain DLY-KMPMFF-0329 in Fermentor Fermentation

[0113] Through the early shake flask experiments, 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 and production of SA by strain DLY-KMPMFF-0329 in a fermenter, the fermentation and 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 in the middle as a basis for adjusting the speed and ventilation size. By 250 h of cultivation, a total of 175 g / L of glucose was consumed. Four feedings were performed during this process, as shown in Figure 6a). 37.5 g / L CaCO3 was added three times, one at 15 g / L in the culture medium before fermentation, the second time at 15 g / L at 96 h of cultivation, and the third time at 7.5 g / L at 144 h (the need for additional CaCO3 can be determined by microscopic examination and changes in the pH of the fermentation liquid). The production of succinic acid was 25.2 g / L, and the yield was 0.144 g / g of glucose.

[0114] Furthermore, the fermentation cycle was relatively long, and SA accumulation only accelerated in the latter half of the fermentation. This is presumably due to an increase in bacterial cell count and an increase in dissolved oxygen (DO). The rate gradually increased from 100 mL / min (DO value approximately 0%) to 250 mL / min and then to 1 vvm (DO approximately 1%). The slow SA accumulation rate in the early stages was likely due to insufficient oxygen, resulting in slow biomass growth. After the dissolved oxygen level increased, the sugar consumption rate significantly accelerated, and the SA accumulation rate increased significantly. Simultaneously, lactic acid accumulation began to decrease and eventually disappeared (see Figure 6(c)). These results suggest that the growth phase can be completed quickly before entering the SA production phase.

[0115] It can be seen from the results of the above examples that the Kluyveromyces marxianus DLY-KMPMFF-0329 provided by the present invention not only has good low pH and high concentration succinic acid tolerance, but also can produce high succinic acid by reducing the TCA pathway. The strain produces succinic acid by fermentation through cytoplasmic expression, which greatly improves the yield and yield of succinic acid. In addition, by coordinating the preferred fermentation methods and conditions of the present invention (such as controlling the dissolved oxygen during the fermentation process, i.e., fermenting under oxygen-limited conditions), the growth rate of DLY-KMPMFF-0329 is increased, and it can quickly enter the SA production stage, shortening the SA fermentation production cycle and improving production efficiency.

[0116] 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. Kluyveromyces marxianus, characterized in that The Kluyveromyces marxianus contains a gene for expressing fumarate reductase FRDg derived from Trypanosoma brucei.

3. The Kluyveromyces marxianus according to claim 2, wherein The amino acid sequence of the fumarate reductase FRDg has an identity of not less than 70%, preferably not less than 80%, and more preferably not less than 90% with the amino acid sequence shown in SEQ ID NO.4; Preferably, the amino acid sequence of the fumarate reductase FRDg is 95-100% identical to the amino acid sequence shown in SEQ ID NO.

4.

4. The Kluyveromyces marxianus according to claim 2 or 3, wherein The genome of Kluyveromyces marxianus contains a gene encoding fumarate reductase FRDg, the nucleotide sequence of which is more than 60% identical to SEQ ID NO.

5.

5. The Kluyveromyces marxianus according to claim 4, wherein The identity of the gene encoding fumarate reductase FRDg to the nucleotide sequence shown in SEQ ID NO.5 is not less than 70%, preferably not less than 80%, more preferably not less than 90%; Preferably, the encoding gene of the fumarate reductase FRDg has an identity of 95-100% with the nucleotide sequence shown in SEQ ID NO.

5.

6. The Kluyveromyces marxianus according to any one of claims 2 to 5, wherein Compared with Kluyveromyces marxianus CGMCC No. 24398, the encoding gene of malic enzyme MAE1 is inactivated; And / or, compared with Kluyveromyces marxianus CGMCC No. 24398, the coding gene of pyruvate decarboxylase PDC1 is inactivated in the Kluyveromyces marxianus; Preferably, the Kluyveromyces marxianus is DLY-KMPMFF-0329, and its deposit number is CGMCC No.27009.

7. A microbial preparation, characterized in that: The invention comprises the Kluyveromyces marxianus according to any one of claims 1 to 6 and auxiliary materials.

8. The microbial preparation according to claim 7, wherein The auxiliary material includes at least one of a protective agent, a buffer and a stabilizer.

9. Any of the following uses of the Kluyveromyces marxianus according to any one of claims 1 to 6 or the microbial preparation according to claim 7 or 8: (1) Application in the fermentation production of organic acids or their derivatives; (2) Application in breeding and / or constructing strains producing organic acids or their derivatives.

10. The use according to claim 11, wherein: The organic acid is succinic acid.

11. A method for producing succinic acid by fermentation, characterized in that: The method comprises the step of culturing the Kluyveromyces marxianus according to any one of claims 1 to 6.

12. The method according to claim 11, wherein: The culturing includes a fermentation culturing stage, and the fermentation culturing stage includes the step of culturing the Kluyveromyces marxianus under oxygen-limited conditions; Preferably, the oxygen-limited condition is to control the dissolved oxygen DO to be below 2%.

13. The method according to claim 11 or 12, wherein: In the fermentation culture stage, carbonate is added at the beginning of fermentation and / or during fermentation; Preferably, the addition amount of the carbonate is 10-50 g / L, and preferably the carbonate includes at least one of calcium carbonate, sodium carbonate, potassium carbonate and ammonium carbonate.

14. The method according to any one of claims 11 to 13, wherein: The Kluyveromyces marxianus is Kluyveromyces marxianus DLY-KMPMFF-0329, and its deposit number is CGMCC No.27009.

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