Recombinant corynebacterium glutamicum as well as construction method and application thereof

Abstract

The invention discloses recombinant corynebacterium glutamicum as well as a construction method and application thereof. According to the recombinant corynebacterium glutamicum, a lactic dehydrogenase gene ldhA is knocked out, and a xylose isomerase gene xylA derived from Neocallimatix calforniae is integrated at a protolactic dehydrogenase site of a genome. The recombinant corynebacterium glutamicum disclosed by the invention can be used for producing lysine through fermentation by directly utilizing lignocellulose hydrolysate which is not subjected to washing detoxification treatment, the yield can reach 38 g / L, and the recombinant corynebacterium glutamicum has a wide application prospect in biorefinery and conversion of lignocellulose raw materials.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering bacteria and relates to a recombinant Corynebacterium glutamicum and a construction method and application thereof. Background Art

[0002] Lysine is the second most demanded amino acid in the world after glutamate. It has multiple physiological functions, such as regulating metabolic balance in the body, improving the absorption and utilization of plant protein in animals, and promoting body development. It is known as a "special essential amino acid" and is widely used in the feed and food industries. As a compound precursor, it also has good application prospects in the field of value-added chemicals and bio-based materials.

[0003] The fermentation production of lysine usually uses corn starch saccharification liquid as raw material. However, with the rapid development of corn deep processing industry, the price of corn has also been rising, which has put a certain pressure on amino acid production companies. Different from the fermentation production of lysine from starch raw materials, the hydrolyzate of lignocellulose raw materials contains a large amount of xylose and a small amount of arabinose, galactose and mannose in addition to glucose. In addition, certain toxic by-products will be produced during the pretreatment of lignocellulose. These toxic by-products have a great inhibitory effect on enzymatic hydrolysis, fermentation and other processes, limiting its further application.

[0004] Based on the characteristics of lignocellulose raw materials, strains that produce lysine from hydrolysates need to have two basic characteristics in addition to having a high lysine synthesis capacity: (1) they can use carbon sources with high content in lignocellulose hydrolysates, especially glucose and xylose, for lysine biosynthesis; (2) the strains have a high tolerance to inhibitors contained in hydrolysates that have not been detoxified and can grow stably. Corynebacterium glutamicum is a commonly used lysine fermentation strain, but it cannot utilize xylose and has poor tolerance to toxic byproducts produced during lignocellulose pretreatment. Therefore, metabolic engineering to enable it to utilize xylose is a prerequisite for efficient fermentation of lignocellulose hydrolysates. Exploring a suitable fermentation method for lignocellulose hydrolysates is the key to ensuring the normal growth of the strain. Chinese patent application CN 115896152A discloses a Corynebacterium glutamicum and its application, wherein the xylose isomerase and xylulose kinase gene cluster xylAB derived from Escherichia coli are introduced into the genome of Corynebacterium glutamicum B253 to endow it with the ability to utilize xylose. However, it only utilizes 20.3 g / L xylose in 96 hours of fermentation, and the xylose utilization rate is low. Only 33.1 g / L lysine is obtained by fermentation in biologically detoxified wheat straw hydrolysate, and it is impossible to achieve efficient utilization of glucose and xylose in lignocellulose hydrolysate to efficiently produce lysine in one step fermentation. Summary of the invention

[0005] The object of the present invention is to provide a recombinant Corynebacterium glutamicum that can stably and efficiently utilize xylose and is tolerant to inhibitors in lignocellulose hydrolysate, and a construction method thereof, and an application thereof in producing lysine using lignocellulose hydrolysate. The recombinant Corynebacterium glutamicum can directly use lignocellulose hydrolysate as a carbon source without detoxification treatment, and reduces the inhibition of the growth of Corynebacterium glutamicum by inhibitors in lignocellulose hydrolysate in a high-density inoculation manner, thereby efficiently utilizing the carbon source in lignocellulose hydrolysate to achieve one-step production of lysine.

[0006] The technical solution for achieving the purpose of the present invention is as follows:

[0007] The recombinant Corynebacterium glutamicum is prepared by knocking out the lactate dehydrogenase gene ldhA and integrating the xylose isomerase gene xylA from Neocallimastix californiae shown in SEQ ID NO.5 into the original lactate dehydrogenase site of the genome.

[0008] Furthermore, the Corynebacterium glutamicum is CICC 23604.

[0009] The above-mentioned method for constructing the recombinant Corynebacterium glutamicum first constructs the recombinant plasmid pJYS3-ΔldhA, then constructs the recombinant plasmid pJYS3-neoxylA, and finally transforms the recombinant plasmid pJYS3-neoxylA into Corynebacterium glutamicum by electroporation. The specific steps are as follows:

[0010] (1) PCR amplified the backbone fragment of the plasmid pJYS3-ΔcrtYf, fused the upstream and downstream homologous arms ldhA-left and ldhA-right of the Corynebacterium glutamicum gene ldhA and the crRNA sequence of the gene ldhA by overlap extension PCR, and then assembled the fused fragments into the backbone fragment of the plasmid pJYS3-ΔcrtYf to obtain the recombinant plasmid pJYS3-ΔldhA, wherein the nucleotide sequences of the ldhA-left, ldhA-right and crRNA sequences are shown in SEQ ID NOs. 1 to 3, respectively;

[0011] (2) The recombinant plasmid pJYS3-ΔldhA was digested with restriction endonucleases to obtain a vector backbone, and the RBS sequence shown in SEQ ID NO.4 and the xylose isomerase gene xylA from Neocallimastix californiae shown in SEQ ID NO.5 were fused by overlap extension PCR, and then the fusion fragments were assembled into the vector backbone to obtain the recombinant plasmid pJYS3-neoxylA;

[0012] (3) The recombinant plasmid pJYS3-neoxylA was first transferred into the competent Escherichia coli JM109 by heat shock method, the plasmid was extracted by amplification culture, and then the extracted plasmid was transferred into Corynebacterium glutamicum by electroporation to obtain a recombinant Corynebacterium glutamicum in which the lactate dehydrogenase gene was knocked out and the xylose isomerase gene xylA from Neocallimastix californiae was integrated into the original lactate dehydrogenase site of the genome.

[0013] The present invention also provides the use of the above-mentioned recombinant Corynebacterium glutamicum in the production of lysine. The specific application method is: inoculating the recombinant Corynebacterium glutamicum seed liquid into a fermentation medium containing lignocellulose hydrolyzate to ferment and produce lysine.

[0014] Further, the initial inoculation density of the recombinant Corynebacterium glutamicum seed solution was OD 600 =5~15, preferably OD 600 =10.

[0015] The lignocellulose raw material described in the present invention is a lignocellulose-containing biomass raw material conventionally used in the field of biorefining, and is selected from one or more of wheat straw, corn straw, rice straw, barley straw, sorghum straw, soybean straw, recycled wood pulp fiber, sawdust, softwood, hardwood, water grass, aquatic plants, algae and animal feces.

[0016] The lignocellulose hydrolysate of the present invention is a hydrolysate obtained by hydrolyzing lignocellulose raw materials with acid, alkali, etc., which is common in the field of biorefining. In a specific embodiment of the present invention, a hydrolysate obtained by pretreatment with sulfuric acid and enzymatic hydrolysis with cellulase is taken as an example.

[0017] Furthermore, the formula of the culture medium of the recombinant Corynebacterium glutamicum seed solution is: 25 g / L glucose, 2.5 g / L urea, 25 g / L corn steep liquor, 0.6 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 2.0 mg / L ferrous sulfate, 2.0 mg / L manganese sulfate, and sodium hydroxide is used to adjust the pH to 7.0-7.3.

[0018] Furthermore, the formula of the fermentation medium containing lignocellulose hydrolysate is: lignocellulose hydrolysate, corn steep liquor 20.0 g / L, ammonium sulfate 20.0 g / L, threonine 0.5 g / L, methionine 0.5 g / L, dipotassium hydrogen phosphate 1.0 g / L, anhydrous magnesium sulfate 0.6 g / L, and sodium hydroxide is used to adjust the pH to 7.0-7.3.

[0019] Furthermore, the fermentation temperature is 30°C and the fermentation time is 96 to 120 hours.

[0020] Furthermore, the fermentation method is dynamic fermentation, and the shaking speed is 200 rpm.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention integrates the xylose isomerase gene xylA from Neocallimastix californiae into Corynebacterium glutamicum, which has good biological activity and higher xylose utilization ability than other xylose isomerase genes, and can promote the efficient utilization of xylose by Corynebacterium glutamicum;

[0023] (2) The present invention uses recombinant Corynebacterium glutamicum as a fermentation strain, which has a good tolerance to inhibitors in lignocellulose hydrolysate, can directly use lignocellulose hydrolysate without water washing and detoxification as a carbon source, and has a high biomass. It can efficiently utilize glucose and xylose to produce lysine, and the yield can reach 38g / L lysine. It has good application prospects in the biorefining and conversion of lignocellulose raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The utilization of xylose by the genetically engineered bacteria in a composite culture medium containing glucose and xylose was shown after xylose isomerases from different sources were expressed in Corynebacterium glutamicum.

[0025] Figure 2 To investigate the growth of genetically engineered bacteria for the production of lysine using lignocellulose hydrolysate at different inoculation densities ( Figure 2 a) and lysine production ( Figure 2 b).

[0026] Figure 3 These are the fermentation curves of growth, sugar consumption and lysine production of engineered bacteria using lignocellulose hydrolysate at different inoculation densities. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0028] Unless otherwise specified in the following examples, the enzymes and plasmids used were purchased, and the molecular cloning experiments involved, including bacterial genome extraction, DNA fragment amplification, digestion, recovery, ligation, transformation, colony PCR, plasmid extraction, etc., refer to the operating instructions of the corresponding reagents for the operating methods.

[0029] Example 1

[0030] This example describes the construction of recombinant Corynebacterium glutamicum, the steps are as follows:

[0031] (1) Construction of recombinant plasmid pJYS3-ΔldhA: The upstream and downstream homologous arms ldhA-left, ldhA-right and crRNA sequences of the gene ldhA were obtained from the genome of Corynebacterium glutamicum, and their gene sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; the vector backbone was obtained from the original plasmid pJYS3-ΔcrtYf by conventional PCR amplification; overlap extension PCR was performed using PrimeSTAR Max DNA Polymer (Bao Ri Yi Biotechnology Co., Ltd.) to fuse the ldhA-right, ldhA-left and crRNA sequence fragments, and the ldhA-right, ldhA-left and crRNA sequence fragments were fused using The fused fragments were assembled into the vector backbone using IIOne Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.) to obtain the plasmid pJYS3-ΔldhA.

[0032] (2) Construction of recombinant plasmid pJYS3-neoxylA: The xylose isomerase gene obtained from Neocallimastix californiae was codon-optimized by a commercial company to obtain xylA, whose nucleotide sequence is shown in SEQ ID NO.5. PrimeSTAR Max DNA Polymer was used to extend the RBS sequence shown in SEQ ID NO.4 and xylA to form a fragment. The plasmid pJYS3-ΔldhA was digested with SmaⅠ restriction endonuclease (Thermo Fisher Scientific) to obtain the vector backbone. The IIOne Step Cloning Kit was used to assemble the fused fragments onto the vector backbone to obtain the plasmid pJYS3-neoxylA; the initially constructed plasmid pJYS3-neoxylA was transferred into Escherichia coli JM109 competent cells by heat stimulation to modify the plasmid, and the plasmid was extracted after expanded culture to obtain the plasmid pJYS3-neoxylA that can be further transformed.

[0033] (3) Construction of recombinant Corynebacterium glutamicum: The Corynebacterium glutamicum used in this example can be purchased from the China Industrial Microbiological Culture Collection Center, numbered CICC 23604. First, Corynebacterium glutamicum 23604 competent cells were prepared, and the plasmid pJYS3-neoxylA was transferred into Corynebacterium glutamicum by electroporation to obtain recombinant Corynebacterium glutamicum NeoX.

[0034] The competent culture of Corynebacterium glutamicum was prepared as follows: streaking on a bacterial tube plate and culturing at 30°C for 2 days; picking a single colony and transferring it to a test tube containing 5 mL of LB medium and culturing it at 30°C overnight; adding the cultured bacteria in the test tube to a 250 mL shake flask containing 30 mL of Epo competent culture medium at a 1% addition rate, and culturing in a 30°C shaker until the OD 600 At about 0.6, (about 4h); put the cultured bacteria into a 50mL centrifuge tube and place it in ice water to cool for 20min; after cooling, centrifuge the centrifuge tube containing the bacteria at 4°C, 5000rpm for 10min, discard the supernatant, add 30mL of pre-cooled 10% (v / v) glycerol to the centrifuge tube, blow the bacteria to make them evenly suspended in glycerol; repeat the first washing and centrifugation twice; after discarding the supernatant for the last time, add 1mL of pre-cooled 10% glycerol, and mix the bacteria thoroughly, divide the mixed bacteria into 1.5mL centrifuge tubes according to the volume of 100μL per tube, and place them in ice water for later use.

[0035] The electroporation method for competent cells of Corynebacterium glutamicum is carried out as follows: 10 μL of plasmid pJYS3-neoxylA is added to 100 μL of competent cells of Corynebacterium glutamicum prepared in the previous step, mixed gently, and placed in ice water for 30 min; the competent cells mixed with the plasmid are added to a dry and pre-cooled electrode cup, the water droplets on the wall of the electroporation cup are wiped off, and the cells are placed in an electroporation instrument and electroporated twice at a voltage of 3000 V; the competent cells after electroporation are taken out from the electroporation cup, placed in a 1.5 mL centrifuge tube, and 800 μL of LBHIS culture medium are added, and inverted evenly. Then place the centrifuge tube containing the competent state in a 46°C water bath for heat shock for 6 minutes; place the centrifuge tube containing the competent cells in a 30°C shaker for incubation for 2 hours. After the incubation is completed, centrifuge the centrifuge tube at 5000 rpm for 2 minutes, remove most of the supernatant, and evenly spread the remaining bacteria on the kana resistance screening plate, and place the plate in a 30°C incubator for 2-3 days.

[0036] The high-fidelity enzyme KOD Plus Neo (Shanghai Toyobo Biotechnology Co., Ltd.) was used for verification PCR to screen positive transformants, and the genetically engineered bacteria with correct genome sequencing were extracted, namely the recombinant Corynebacterium glutamicum NeoX.

[0037] The formula of Epo medium is as follows: 10 g / L yeast powder, 5 g / L peptone, 5 g / L sodium chloride, 3% glycine, 1% Tween 80 and 0.4% isoniazid in a mass ratio.

[0038] The formula of LBHIS medium is as follows: 10 g / L yeast powder, 5 g / L peptone, 5 g / L sodium chloride, 91 g / L sorbitol and 34 g / L brain heart extract, wherein sorbitol is filtered and sterilized separately.

[0039] Example 2

[0040] This example compares the xylose utilization effects of recombinant Corynebacterium glutamicum expressing xylose isomerases from different sources.

[0041] 1. Construction of recombinant Corynebacterium glutamicum expressing xylose isomerase from different sources: The xylose isomerase gene (SEQ ID NO.8) from Escherichia coli BL21 (DE3), the xylose isomerase gene (SEQ ID NO.9) from Lactobacillus pentosus, the codon-optimized xylose isomerase gene (SEQ ID NO.6) from Bacterium J10 (2018), and the codon-optimized xylose isomerase gene (SEQ ID NO.7) from Hallella seregens were constructed by the method described in Example 1 and named EcoX, LacX, BajX, and HasX, respectively.

[0042] 2. Utilization of xylose by recombinant Corynebacterium glutamicum expressing xylose isomerase from different sources

[0043] (1) Single colonies of recombinant Corynebacterium glutamicum EcoX, LacX, BajX, HasX, and NeoX were picked up and cultured on primary seeds, respectively, using a 150 mL shake flask with a liquid volume of 15 mL, sealed with eight layers of gauze, and the shaking conditions were 30°C, 200 rpm, and the culture time was 18 h; the formula of the primary seed culture medium is as follows: 25.0 g / L glucose, 2.5 g / L urea, 25.0 g / L corn steep liquor, 0.6 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 2.0 mg / L ferrous sulfate, 2.0 mg / L manganese sulfate, and the pH was adjusted to 7.0-7.3 with sodium hydroxide.

[0044] (2) The activated primary seed liquid was transferred to the secondary culture medium for cultivation, using a 150 mL shaking flask with a liquid volume of 15 mL, sealed with eight layers of gauze, and cultured in a shaking incubator at 30°C and 200 rpm for 12 h; the formula of the secondary seed culture medium is as follows: 25.0 g / L glucose, 2.5 g / L urea, 5.0 g / L corn steep liquor, 0.6 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, 2.0 mg / L ferrous sulfate, 2.0 mg / L manganese sulfate, and the pH was adjusted to 7.0-7.3 with sodium hydroxide.

[0045] (3) Inoculate the secondary seed solution into the fermentation medium, and the inoculation amount is controlled to be the OD of the medium after inoculation. 6002, using 250mL shaking flask, the liquid volume is 20mL, sealed with eight layers of gauze, shaking conditions are 30℃, 200rpm, and the culture time is 72h. The fermentation medium is a composite medium with glucose and xylose as carbon sources, and its formula is as follows: glucose 100.0g / L, xylose 50.0g / L, urea 7.0g / L, corn steep liquor 5.0g / L, magnesium sulfate 0.6g / L, potassium dihydrogen phosphate 1.0g / L, ferrous sulfate 5.0mg / L, manganese sulfate 5.0mg / L, sodium hydroxide is used to adjust the pH to 7.0-7.3.

[0046] (4) The method for determining the content of the fermentation broth is as follows: After the fermentation broth is centrifuged, the supernatant is taken, diluted 10 times and then filtered, and the solution content is determined by high performance liquid chromatography. The consumption of xylose by recombinant Corynebacterium glutamicum expressing xylose isomerase from five different sources is shown in Table 1. Figure 1 The control strain expressing the empty plasmid pJYS3-ΔcrtYf was almost unable to utilize xylose. The recombinant Corynebacterium glutamicum BajX expressing the codon-optimized xylose isomerase gene from Bacterium J10 (2018) utilized 28.8 g / L xylose, the recombinant Corynebacterium glutamicum EcoX expressing the xylose isomerase gene from Escherichia coli BL21 (DE3) utilized 23.9 g / L xylose, the recombinant Corynebacterium glutamicum HasX expressing the codon-optimized xylose isomerase gene from Hallella seregens utilized 20.0 g / L xylose, the recombinant Corynebacterium glutamicum expressing the xylose isomerase gene from Lactobacillus pentosus utilized 4.8 g / L xylose, and the recombinant Corynebacterium glutamicum expressing the xylose isomerase gene from Neocallimastix californiae and codon-optimized recombinant Corynebacterium glutamicum NeoX utilized 39.7 g / L xylose, showing the best xylose utilization effect.

[0047] Example 3

[0048] This example describes the growth and lysine yield of recombinant Corynebacterium glutamicum NeoX when it is fermented in corn straw hydrolysate at different inoculation densities to produce lysine.

[0049] 1. Preparation of corn straw hydrolyzate: corn straw was thoroughly mixed with sulfuric acid at a rate of 0.075 g sulfuric acid / g corn straw, and then pelletized using a pelletizer. The resulting DLC(sa) pellets were immediately sterilized at 121°C for 30 minutes to promote their decomposition. Subsequently, enzymatic hydrolysis was performed directly in a 2L shake flask at a solid loading of 25% without undergoing a detoxification step, and 20 mg protein / g glucan was added. CTec3 (cellulase, purchased from Nanjing Novozyme Biotechnology Co., Ltd.), enzymatic hydrolysis at 50°C and 250 rpm for 72 hours, and then centrifuged at 8000 rpm for 10 minutes to obtain a clarified corn straw hydrolysate. The glucose concentration in the obtained corn straw hydrolysate was 87.9 g / L and the xylose concentration was 47.6 g / L.

[0050] 2. Preparation of fermentation medium: Add 20.0 g / L corn steep liquor, 20.0 g / L ammonium sulfate, 0.5 g / L threonine, 0.5 g / L methionine, 1.0 g / L dipotassium hydrogen phosphate, and 0.6 g / L anhydrous magnesium sulfate to the corn straw hydrolyzate, and adjust the pH to 7.0-7.3 with sodium hydroxide.

[0051] 3. Fermentation production of lysine: Recombinant Corynebacterium glutamicum NeoX was inoculated in a fermentation medium at different inoculation densities, and the shaking culture conditions were 30°C, 200 rpm, and the culture time was 72 h.

[0052] The fermentation sample was diluted with pure water and the absorbance OD of the sample at 600 nm was measured by UV spectrophotometer. 600 After centrifugation, the supernatant was diluted and passed through a membrane. The glucose and xylose contents in the solution were determined by high performance liquid chromatography, and the lysine content was determined by biosensor S-10 (Shenzhen Hillman Technology Co., Ltd.). The inoculation OD at the beginning of the fermentation was controlled 600 The growth rate (OD 600 The production of lysine and Figure 2 Figure a and Figure b in . Figure 2 It can be seen that increasing the inoculation density to OD 600 When the inoculation density was 10, the growth of recombinant Corynebacterium glutamicum NeoX in corn straw hydrolysate was significantly increased, and the effect of inhibitors on growth was weakened; the yield of lysine also improved with the inoculation density, and the initial OD 600 When the temperature reaches 10, the lysine production after 72 hours of fermentation reaches 37.5g / L.

[0053] Example 4

[0054] This example describes the production of lysine by fermentation of recombinant Corynebacterium glutamicum NeoX in corn straw hydrolysate. The preparation method of the fermentation medium containing corn straw hydrolysate and the sample measurement method used in this example are the same as those in Example 3.

[0055] The recombinant Corynebacterium glutamicum NeoX was grown at the initial OD 600 The high density of 10 was used for inoculation and aerobic fermentation was carried out in a 250 mL shake flask. Figure 3As shown in Figure 2, after 96 hours of fermentation in corn stover hydrolysate, the recombinant Corynebacterium glutamicum NeoX grew vigorously, with an OD 600 It reached 134, and after consuming all the glucose in 48 hours, it began to utilize xylose, and finally utilized 87.9g / L of glucose and 38.8g / L of xylose, and fermented to produce 38.0g / L of lysine.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that To knock out the lactate dehydrogenase gene ldhA , the original lactate dehydrogenase site of the genome was integrated with the gene from SEQ ID NO.5 Neocallimastix californiae Xylose isomerase gene xylA of Corynebacterium glutamicum.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The Corynebacterium glutamicum was CICC23604.

3. The method for constructing a recombinant Corynebacterium glutamicum according to claim 1, wherein The specific steps are as follows: (1) PCR amplified the backbone fragment of plasmid pJYS3-ΔcrtYf, and the C. glutamicum gene was inserted into the C. glutamicum gene by overlap extension PCR. ldhA Upstream and downstream homology arms ldhA-left, ldhA-right and gene ldhA The crRNA sequence was fused, and the fused fragment was assembled into the backbone fragment of the plasmid pJYS3-ΔcrtYf to obtain the recombinant plasmid pJYS3-ΔldhA, wherein the nucleotide sequences of the ldhA-left, ldhA-right and crRNA sequences are shown in SEQ ID NOs. 1 to 3, respectively; (2) The recombinant plasmid pJYS3-ΔldhA was digested with restriction endonucleases to obtain the vector backbone. The RBS sequence shown in SEQ ID NO.4 and the sequence from SEQ ID NO.5 were overlapped by PCR. Neocallimastix californiae Xylose isomerase gene xylA The fusion fragments were then assembled into the vector backbone to obtain the recombinant plasmid pJYS3-neoxylA; (3) The recombinant plasmid pJYS3-neoxylA was first transformed into the competent Escherichia coli JM109 by heat shock method, the plasmid was extracted by amplification culture, and then the extracted plasmid was transformed into Corynebacterium glutamicum by electroporation to obtain the knockout of lactate dehydrogenase gene and the integration of the original lactate dehydrogenase site from Neocallimastix californiae Xylose isomerase gene xylA of recombinant Corynebacterium glutamicum.

4. The use of the recombinant Corynebacterium glutamicum in the production of lysine according to claim 1, characterized in that The specific application method is: recombinant Corynebacterium glutamicum seed liquid is inoculated into a fermentation medium containing lignocellulose hydrolyzate to ferment and produce lysine.

5. The use according to claim 4, characterized in that The initial inoculation density of the recombinant Corynebacterium glutamicum seed solution was OD 600 =5~15.

6. The use according to claim 4, characterized in that The initial inoculation density of the recombinant Corynebacterium glutamicum seed solution was OD 600 =10.

7. The use according to claim 4, characterized in that The lignocellulose raw material is one or more selected from wheat straw, corn straw, rice straw, barley straw, sorghum straw, soybean straw, recycled wood pulp fiber, sawdust, softwood, hardwood, water grass, aquatic plants, algae and animal feces.

8. The use according to claim 4, characterized in that The lignocellulose hydrolyzate is a hydrolyzate obtained by pre-treating the lignocellulose raw material with sulfuric acid and then enzymatically hydrolyzing it with cellulase.

9. The use according to claim 4, characterized in that The culture medium for the recombinant Corynebacterium glutamicum seed solution was formulated as follows: 25 g / L glucose, 2.5 g / L urea, 25 g / L corn steep liquor, 0.6 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 2.0 mg / L ferrous sulfate, and 2.0 mg / L manganese sulfate, with the pH adjusted to 7.0-7.3 using sodium hydroxide. The fermentation medium containing lignocellulose hydrolysate was formulated as follows: lignocellulose hydrolysate, 20.0 g / L corn steep liquor, 20.0 g / L ammonium sulfate, 0.5 g / L threonine, 0.5 g / L methionine, 1.0 g / L dipotassium hydrogen phosphate, and 0.6 g / L anhydrous magnesium sulfate, with the pH adjusted to 7.0-7.3 using sodium hydroxide.

10. The use according to claim 4, characterized in that The fermentation temperature is 30°C, the fermentation time is 96~120 h; the fermentation method is dynamic fermentation, and the shaking speed is 200 rpm.

Images