Expression cassette for preparing l-lysine, genetically engineered bacteria and application thereof

Abstract

The application discloses an expression cassette for preparing L-lysine, genetically engineered bacteria and application thereof. The expression cassette comprises a promoter, a transaldolase gene and / or a transketolase gene. After the expression cassette is introduced into a starting bacterium, the expression enhances the mode of transketolase and transaldolase, enhances the pentose phosphate pathway, effectively improves the electron supply in the fermentation process, thereby improves the glucose utilization efficiency, and further improves the yield of L-lysine. When the straw hydrolysate is used, the yield of lysine of the genetically engineered bacteria of the application is obviously improved compared with the starting bacterium. The genetically engineered bacteria provided by the application can effectively utilize agricultural waste such as straw for fermentation, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an expression cassette for preparing L-lysine, a genetically engineered bacterium, and their applications. Background Technology

[0002] Lysine, also known as 2,6-diaminohexanoic acid, is a basic amino acid. Lysine plays important nutritional and physiological roles and is widely used in the pharmaceutical, food, and feed industries. It can also be used as a precursor in the synthesis of nylon polymer materials. The main production methods for lysine include protein hydrolysis, chemical synthesis, and fermentation. Among these, microbial fermentation is the most widely used method for industrial production of lysine due to its low production cost, high production intensity, high specificity, and minimal environmental pollution.

[0003] Prokaryotic microorganisms used for lysine production mainly include Corynebacterium, Brevibacterium, Nocardia, Pseudomonas, Escherichia, and Bacillus. Corynebacterium glutamicum is the most important and safest strain for amino acid fermentation, and improving its ability to oversynthesize various amino acids through metabolic modification has been a research hotspot. For example, overexpressing genes related to the lysine synthesis pathway and feedback inhibition desensitization, strengthening the energy supply pathway starting from glucose metabolism, and optimizing lysine transport proteins on the cell membrane have all effectively improved lysine productivity.

[0004] Besides continuously enhancing the metabolic pathway for lysine production, fermentation methods that open up alternative pathways to non-lysine metabolism are also worth exploring in depth. Transketolases and transaldolases, which have the function of transferring aldehyde or ketone groups, play an important role in the pentose phosphate metabolic pathway. Currently, there are no reports on the application of transketolases and transaldolases in lysine production by Corynebacterium glutamicum. There is a lack of genetically engineered bacteria for high-yield lysine production in existing technologies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an expression cassette for preparing L-lysine, a genetically engineered bacterium, and their applications. Specifically, this invention provides an expression cassette that increases lysine production in *Corynebacterium glutamicum* and a genetically engineered bacterium that efficiently produces lysine using straw hydrolysate. The genetically engineered bacterium can effectively utilize glucose in straw hydrolysate. This invention improves the fermentation efficiency of *Corynebacterium glutamicum* under glucose and hydrolysate conditions by enhancing the functions of transketolase and transaldolase in *Corynebacterium glutamicum*, significantly increasing L-lysine production. After the expression cassette provided by this invention is introduced into the starting bacterium, it enhances the expression of transketolase and transaldolase, strengthens the pentose phosphate pathway, effectively improves electron supply during fermentation, thereby increasing glucose utilization efficiency and ultimately increasing L-lysine production.

[0006] To address the aforementioned technical problems, the present invention provides an expression cassette comprising the following components: a promoter; and a transaldolase gene and / or a transketoolase gene.

[0007] In the expression cassette, the sequence of the transaldolase gene is preferably as shown in SEQ ID NO:2.

[0008] In the expression cassette, the sequence of the transketolase gene is preferably as shown in SEQ ID NO:1.

[0009] In the expression cassette, the promoter is preferably the Peftu promoter, and the sequence of the Peftu promoter is preferably as shown in SEQ ID NO:3.

[0010] Another aspect of the present invention provides an isolated nucleic acid comprising an expression cassette as described in the present invention.

[0011] Another aspect of the present invention provides a recombinant expression vector, which includes the expression cassette described in the present invention, or includes the nucleic acid described in the present invention.

[0012] In a preferred embodiment, the backbone plasmid of the recombinant expression vector is preferably pK18mob.

[0013] For detailed information about pK18mob, please refer to the following webpage:

[0014] http: / / www.biovector.net / product / 1089.html

[0015] Another aspect of the present invention provides a genetically engineered bacterium, which is formed by transferring an expression cassette as described in the present invention, or a nucleic acid as described in the present invention, or a recombinant expression vector as described in the present invention into the starting bacterium.

[0016] In the genetic engineering, the starting bacterium is preferably Corynebacterium glutamicum, such as C. glutamicum B253 or C. glutamicum CathS141.

[0017] In the genetic engineering process, the expression cassette is preferably integrated into the genome of the originating bacterium via homologous recombination, or exists in the originating bacterium in a non-integrated form.

[0018] The genetically engineered bacteria preferably do not express lactate dehydrogenase, for example, the ldh gene is knocked out.

[0019] In a preferred embodiment, the expression cassette is integrated into the site of the ldh gene.

[0020] The ldh gene is preferably accessed in GenBank with accession number AJE68497.1.

[0021] The starting strain described in this invention can be a wild-type strain or a modified strain.

[0022] Another aspect of the present invention provides a method for preparing L-lysine, the method comprising fermenting genetically engineered bacteria as described in the present invention in a fermentation medium.

[0023] In a preferred embodiment, the fermentation medium contains not less than 25 g / L of glucose, for example, 80-150 g / L of glucose.

[0024] The fermentation temperature is preferably 28–32°C, for example 30°C.

[0025] The aeration rate for the fermentation is preferably 1.0 to 1.7 vvm, for example 1.4 vvm.

[0026] The pH of the fermentation is preferably 6 to 8, for example 7;

[0027] The fermentation is preferably carried out by stirring, with the stirring speed being 400-800 rpm, preferably 600 rpm.

[0028] In a preferred embodiment, the fermentation medium is a lignocellulose hydrolysate, such as a straw hydrolysate.

[0029] The straw hydrolysate is preferably formed from crop straw after enzymatic hydrolysis and saccharification.

[0030] In a preferred embodiment, 15–25 g / L ammonium sulfate, 2–8 g / L methionine, and 2–8 g / L threonine are added to the straw hydrolysate.

[0031] In a preferred embodiment, the straw hydrolysate is pretreated before enzymatic saccharification. The pretreatment includes sieving, impurity removal, acid pretreatment, and / or detoxification. Pretreatment can improve the saccharification efficiency of crop straw. The detoxification treatment can reduce the content of toxic inhibitors such as acetic acid, furfural, 5-hydroxybenzaldehyde, furfural, hydroxymethylfurfural, 4-hydroxybenzaldehyde, and levulinic acid.

[0032] Another aspect of the present invention provides an expression cassette as described in the present invention, a nucleic acid as described in the present invention, a recombinant expression vector as described in the present invention, or the application of genetically engineered bacteria as described in the present invention in the preparation of L-lysine.

[0033] A method for preparing genetically engineered bacteria, wherein the genetically engineered bacteria are obtained by introducing an expression cassette as described in this invention into a starting bacterium as defined in this invention.

[0034] The preparation method preferably further includes: blocking the expression of lactate dehydrogenase in the genetically engineered bacteria, for example, knocking out the ldh gene.

[0035] In the preparation method, preferably, the expression cassette is integrated into the site of the ldh gene.

[0036] Preferably, the ldh gene has the accession number AJE68497.1 in GenBank.

[0037] In a preferred embodiment, the accession number of C. glutamicum CathS141 described in this invention is CCTCCNO:M 20211495.

[0038] The C. glutamicum B253 described in this invention was purchased from the Shanghai Institute of Industrial Microbiology.

[0039] The transaldolase gene described in this invention is also called cgl1575 (the amino acid sequence of the transaldolase it encodes can be the sequence corresponding to GenBank accession number BAB98968.1); the transketolase gene described in this invention is also called cgl1574 (the amino acid sequence of the transketolase it encodes can be the sequence corresponding to GenBank accession number BAB98967.1).

[0040] The starting strain described in this invention can be a wild-type strain or a modified strain.

[0041] Although the starting strain selected in the embodiments of the present invention is a strain resistant to straw hydrolysate, Corynebacterium glutamicum CathS141, it can be seen from Example 5 that when the culture medium is a prepared glucose medium, the genetically engineered bacteria of the present invention are also better at producing L-lysine than the starting strain. That is, resistance to the toxicity of the inhibitor of straw hydrolysate is not a necessary condition for the starting strain.

[0042] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0043] The reagents and raw materials used in this invention are all commercially available.

[0044] The positive and progressive effects of this invention are as follows:

[0045] The expression cassette provided by this invention, after being introduced into the starting bacteria, enhances the expression of transketolase and transaldolase, strengthens the pentose phosphate pathway, and effectively improves electron supply during fermentation, thereby increasing glucose utilization efficiency and consequently increasing L-lysine production. When using straw hydrolysate, the lysine production of the genetically engineered bacteria of this invention is significantly higher than that of the starting bacteria. The genetically engineered bacteria provided by this invention can effectively utilize agricultural waste such as straw for fermentation and has promising application prospects.

[0046] Information on the preservation of biological materials

[0047] The Corynebacterium glutamicum strain CathS141 of this invention was deposited on November 29, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC NO: M20211495. The culture name is CathS141, and the taxonomic name is *Corynebacterium glutamicum* CathS141. Detailed Implementation

[0048] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0049] I. Strains used in this invention

[0050] E. coli DH5α was used for the construction of expression plasmids and knockout plasmids. Corynebacterium glutamicum CathS141 is a strain used for the production of lysine. In this experiment, C. glutamicum CathS141 was mainly used as the starting strain.

[0051] II. Reagents and Culture Media

[0052] Cellulase CTec 2.0, used for hydrolyzing cellulose and hemicellulose in lignocellulose, was purchased from Novozymes (China) Co., Ltd., Beijing, China. The filter paper activity of cellulase was measured to be 203.2 FPU / mL, and the cellobiase activity was 4900.0 CBU / mL, according to the NREL LAP-006 guideline. The protein concentration was measured to be 87.3 mg / mL according to the Bradford method. Restriction endonucleases, used to cut plasmids or gene fragments to generate sticky ends, were purchased from Thermo Scientific (Wilmington, DE, USA). DNA polymerase was used to amplify gene fragments, and DNA ligase was used to ligate the digested gene fragments and plasmid vectors; both enzymes were purchased from Takara (Otsu, Japan). A seamless cloning kit was used to ligate gene fragments containing homologous fragments and plasmid vectors; this kit was purchased from Hanheng Biotechnology Co., Ltd. (Nanjing, China). Plasmid extraction kits, PCR product purification and recovery kits, and gel extraction kits were purchased from Shanghai Jierui Biotechnology Co., Ltd. (Shanghai, China). Other reagents were purchased from local suppliers.

[0053] The culture medium used to culture Escherichia coli was Luria-Bertani (LB) medium, with the following components: 10.0 g / L sodium chloride, 10.0 g / L peptone, and 5.0 g / L yeast extract.

[0054] The specific components of the culture medium used to culture Corynebacterium glutamicum are as follows:

[0055] (1) Seed culture medium: 25 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 2.5 g / L urea and 0.6 g / L magnesium sulfate, 25 g / L corn steep liquor.

[0056] (2) Fermentation medium: 1 g / L potassium dihydrogen phosphate, 3 g / L urea, 0.6 g / L magnesium sulfate, 20 g / L corn steep liquor, and glucose as a carbon source as needed.

[0057] Example 1: Obtaining the starting bacterium Corynebacterium glutamicum CathS141

[0058] Soil samples were collected from Wusu, Xinjiang. Each 1g of soil sample was added to 10mL of sterile water, vigorously mixed for 1 minute, and then allowed to settle for a period of time. The original samples were then diluted to 10 mL. -3 10 -4 10 -5Spread the culture onto LB agar plates containing 100 mg / L nystatin and incubate at 30°C. After repeated streak isolation cultures, purified single colonies were obtained.

[0059] A strain capable of producing L-lysine was obtained through isolation and purification. The strain's cell characteristics and physiological and biochemical properties were determined according to the *Handbook of Systematic Identification of Common Bacteria*. The strain exhibited the following morphological characteristics: moist, round colonies with smooth surfaces, regular edges, and a light yellow color. DNA was extracted from the strain using a bacterial genomic DNA extraction kit. Using this DNA as a template, PCR amplification was performed using universal bacterial primers 27F and 1492R, and 16S rDNA was used. The 16S rDNA sequence was obtained and compared with the GenBank database, identifying the strain as *Corynebacterium glutamicum*.

[0060] Wheat straw was pretreated with acid, detoxified, and enzymatically hydrolyzed to obtain wheat straw hydrolysate. Using the *Corynebacterium glutamicum* strain isolated and purified above as the starting strain, stable strains capable of normal growth and lysine production were obtained through multiple individual and combined mutagenesis using ultraviolet light, nitrosoguanidine, 5-fluorouracil, and ARTP. This strain was named CathS141 and is currently deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, accession number CCTCC NO: M 20211495, deposited on November 29, 2021.

[0061] Example 2: Construction of the Peftu_cgl1574 expression box

[0062] First, an integration plasmid for the transketolase cgl1574 gene was constructed. The specific construction method is as follows: Using the genome of C. glutamicum CathS141 as a template, the Peftu promoter (as shown in SEQ ID NO:3) was amplified by PCR using primers Peftu-F (as shown in SEQ ID NO:4) and Peftu-R (as shown in SEQ ID NO:5); using the genome of C. glutamicum CathS141 as a template, the cgl1574 fragment (as shown in SEQ ID NO:1) was amplified by PCR using primers cgl1574-F (as shown in SEQ ID NO:6) and cgl1574-R (as shown in SEQ ID NO:7); using Peftu and cgl1574 as templates, the Peftu_cgl1574 fusion fragment (as shown in SEQ ID NO:8) was obtained by overlapping extension PCR using primers Peftu-F and cgl1574-R.

[0063] Example 3: Integrating the Peftu_cgl1574 expression box

[0064] Using the genome of *C. glutamicum* CathS141 as a template, the ldh-up fragment (as shown in SEQ ID NO:11) was amplified by PCR using primers ldh-up-F (as shown in SEQ ID NO:9) and ldh-up-R (as shown in SEQ ID NO:10); the ldh-down fragment (as shown in SEQ ID NO:13) was amplified by PCR using primers ldh-down-F (as shown in SEQ ID NO:12) and ldh-down-R (as shown in SEQ ID NO:13). (As shown in NO:14); Using the ldh-up fragment, Peftu_cgl1574, and ldh-down fragment as templates, the Δldh::cgl1574 fusion fragment was obtained by overlapping extension PCR using ldh-up-F and ldh-down-R primers. This fragment was then treated with EcoRI and HindIII restriction enzymes, and subsequently inserted into the pK18mob plasmid (available for purchase at http: / / www.biovector.net / product / 1089.html) using T4 ligase, resulting in the pK18-Δldh::cgl1574 plasmid. During this process, successfully ligated plasmids can be screened using seed culture plates containing kanamycin resistance.

[0065] Next, the integration plasmid pK18-Δldh::cgl1574 was transformed into C. glutamicum CathS141 by electroporation. Then, the strain that underwent correct homologous recombination was screened by PCR verification to obtain recombinant Corynebacterium glutamicum, named cg1574.

[0066] Example 4: Construction and integration of the Peftu_cgl1575 expression box

[0067] Referring to Example 2, the primers used were cgl1575-F (as shown in SEQ ID NO:15) and cgl1575-R (as shown in SEQ ID NO:16) to construct the Peftu_cgl1575 expression cassette (as shown in SEQ ID NO:17). Referring to Example 3, a recombinant Corynebacterium glutamicum integrating the Peftu_cgl1575 expression cassette at the ldh position was obtained and named cg1575.

[0068] Example 5: Construction and integration of the cgl1574-cgl1575 combined expression box

[0069] Referring to Examples 2 and 4, the obtained single expression cassettes Peftu_cgl1574 and Peftu_cgl1575 were fused to obtain the combined expression cassette cgl1574-cgl1575 (as shown in SEQ ID NO:18). Referring to Example 3, the obtained combined expression was integrated into the ldh chromosome location via electroporation, and the correct recombinant Corynebacterium glutamicum was named cg1574&cg1575.

[0070] Example 6 Fermentation of different recombinant Corynebacterium glutamicum

[0071] Genetically engineered strains and the parent strain *Corynebacterium glutamicum*, *C. glutamicum* Cath S141, were inoculated into seed culture medium for two rounds of activation, and then transferred to fermentation medium. The fermentation medium consisted of 1 g / L potassium dihydrogen phosphate, 3 g / L urea, 0.6 g / L magnesium sulfate, 20 g / L corn steep liquor, and 80 g / L glucose as a carbon source. Fermentation was carried out in 250 mL shake flasks at 30°C and 200 rpm for 48 hours. The fermentation results are shown in Table 1: Enhancing the performance of transketolase and transaldolase improved the L-lysine conversion efficiency of *Corynebacterium glutamicum*. It was also found that this enhancement was most effective when both transketolase and transaldolase were enhanced simultaneously.

[0072] Table 1 Fermentation of different recombinant Corynebacterium glutamicum

[0073] C. glutamicum CathS141 23.55±0.52 Cg1574 26.69±0.30 Cg1575 24.32±0.26 Cg1574&Cg1575 29.29±0.47

[0074] Example 7: Fermentation of recombinant Corynebacterium glutamicum in lignocellulose hydrolysate

[0075] Wheat straw was crushed and then sieved through a 10 mm diameter sieve. The sieved straw was then washed to remove impurities such as mud, stones, and metal. After drying to constant weight in a 105℃ oven, it was stored in sealed plastic bags for later use. Following acid pretreatment, biological detoxification, and enzymatic saccharification, a wheat straw hydrolysate containing 95.4 g / L glucose was obtained. 20 g / L ammonium sulfate and 5 g / L methionine and threonine were added to the hydrolysate. The modified strains cg1574 & cg1575 and the original strain, *Corynebacterium glutamicum* CathS141, were cultured in the wheat straw hydrolysate for comparative fermentation. The fermentation temperature was 30℃, the pH was controlled at 7.0 with ammonia, the aeration rate was 1.4 vvm, and the fermentation speed was 600 rpm. Fermentation was terminated when glucose was exhausted.

[0076] The results showed that when wheat straw hydrolysate was used as the culture medium, there was a significant difference in lysine production between the two strains. The recombinant strain produced 25.84 g / L of lysine, while the control strain produced 19.93 g / L. Also noteworthy was the fermentation time required for the control strain and the recombinant strain Cg1574 & cg1575, which were 72 and 62 hours, respectively. Therefore, the two strains showed a significant difference in production intensity, with the recombinant strain Cg1574 & cg1575 exhibiting a significantly higher production intensity (lysine production per unit time) than the control strain. Furthermore, when using *Corynebacterium glutamicum* B253 as the starting strain and modifying it using the same method as strains Cg1574 & cg1575, a strain named B253-2 was obtained. Verifying its fermentation effect under the same conditions revealed that the final lysine production of B253-2 was 11.42% higher than that of the control strain B253. Moreover, strain B253-2 completed fermentation 9.5 hours earlier than strain B253.

[0077] Therefore, the recombinant strain obtained by this invention has a high lysine production capacity in real material hydrolysate, and thus has good application prospects.

[0078] The above description of operational examples of the technical solution of the present invention is not intended to limit the application of the present invention. All equivalent substitutions of operational conditions are within the protection scope of the present invention. SEQUENCE LISTING <110> Shanghai Kaisai Biotechnology Co., Ltd. CIBT USA <120> Expression cassettes, genetically engineered bacteria, and their applications for the preparation of L-lysine. <130> P21018728C <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 2031 <212> DNA <213> Artificial Sequence <220> <223> cgl1574 <400> 1 gtggacacca aggctgtaga cactgttcgt gtcctcgctg cagacgctgt agaaaactgt 60 ggctccggcc acccaggcac cgcaatgagc ctggctcccc ttgcatacac cttgtaccag 120 cgggttatga acgtagatcc acaggacacc aactgggcag gccgtgaccg cttcgttctt 180 tcttgtggcc actcctcttt gacccagtac atccagcttt acttgggtgg attcggcctt 240 gagatggatg acctgaaggc tctgcgcacc tgggattcct tgaccccagg acaccctgag 300 taccgccaca ccaagggcgt tgagatcacc actggccctc ttggccaggg tcttgcatct 360 gcagttggta tggccatggc tgctcgtcgt gagcgtggcc tattcgaccc aaccgctgct 420 gagggcgaat ccccattcga ccaccacatc tacgtcattg cttctgatgg tgacctgcag 480 gaaggtgtca cctctgaggc atcctccatc gctggcaccc agcagctggg caacctcatc 540 gtgttctggg atgacaaccg catctccatc gaagacaaca ctgagatcgc tttcaacgag 600 gacgttgttg ctcgttacaa ggcttacggc tggcagacca ttgaggttga ggctggcgag 660 gacgttgcag caatcgaagc tgcagtggct gaggctaaga aggacaccaa gcgacctacc 720 ttcatccgcg ttcgcaccat catcggcttc ccagctccaa ctatgatgaa caccggtgct 780 gtgcacggtg ctgctcttgg cgcagctgag gttgcagcaa ccaagactga gcttggattc 840 gatcctgagg ctcacttcgc gatcgacgat gaggttatcg ctcacacccg ctccctcgca 900 gagcgcgctg cacagaagaa ggctgcatgg caggtcaagt tcgatgagtg ggcagctgcc 960 aaccctgaga acaaggctct gttcgatcgc ctgaactccc gtgagcttcc agcgggctac 1020 gctgacgagc tcccaacatg ggatgcagat gagaagggcg tcgcaactcg taaggcttcc 1080 gaggctgcac ttcaggcact gggcaagacc cttcctgagc tgtggggcgg ttccgctgac 1140 ctcgcaggtt ccaacaacac cgtgatcaag ggctcccctt ccttcggccc tgagtccatc 1200 tccaccgaga cctggtctgc tgagccttac ggccgtaacc tgcacttcgg tatccgtgag 1260 cacgctatgg gatccatcct caacggcatt tccctccacg gtggcacccg cccatacggc 1320 ggaaccttcc tcatcttctc cgactacatg cgtcctgcag ttcgtcttgc agctctcatg 1380 gagaccgacg cttactacgt ctggacccac gactccatcg gtctgggcga agatggccca 1440 acccaccagc ctgttgaaac cttggctgca ctgcgcgcca tcccaggtct gtccgtcctg 1500 1560 ggccctaagg gtcttgcact gacccgccag aacgttcctg ttctggaagg caccaaggag 1620 aaggctgctg aaggcgttcg ccgcggtggc tacgtcctgg ttgagggttc caaggaaacc 1680 ccagatgtga tcctcatggg ctccggctcc gaggttcagc ttgcagttaa cgctgcgaag 1740 gctctggaag ctgagggcgt tgcagctcgc gttgtttccg ttccttgcat ggattggttc 1800 caggagcagg acgcagagta catcgagtcc gttctgcctg cagctgtgac cgctcgtgtg 1860 tctgttgaag ctggcatcgc aatgccttgg taccgcttct tgggcaccca gggccgtgct 1920 gtctcccttg agcacttcgg tgcttctgcg gattaccaga ccctgtttga gaagttcggc 1980 atcaccaccg atgcagtcgt ggcagcggcc aaggactcca ttaacggtta a 2031 <210> 2 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> cgl1575 <400> 2 atgtctcaca ttgatgatct tgcacagctc ggcacttcca cttggctcga cgacctctcc 60 cgcgagcgca ttacttccgg caatctcagc caggttattg aggaaaagtc tgtagtcggt 120 gtcaccacca acccagctat tttcgcagca gcaatgtcca agggcgattc ctacgacgct 180 cagatcgcag agctcaaggc cgctggcgca tctgttgacc aggctgttta cgccatgagc 240 atcgacgacg ttcgcaatgc ttgtgatctg ttcaccggca tcttcgagtc ctccaacggc 300 tacgacggcc gcgtgtccat cgaggttgac ccacgtatct ctgctgaccg cgacgcaacc 360 ctggctcagg ccaaggagct gtgggcaaag gttgatcgtc caaacgtcat gatcaagatc 420 cctgcaaccc caggttcttt gccagcaatc accgacgctt tggctgaggg catcagcgtt 480 aacgtcacct tgatcttctc cgttgctcgc taccgcgagg tcatcgctgc gttcatcgag 540 ggcatcaagc aggctgctgc aaacggccac gacgtctcca agatccactc tgtggcttcc 600 ttcttcgtct cccgcgtcga cgttgagatc gacaagcgcc tcgaggcaat cggatccgat 660 gaggctttgg ctctgcgcgg caaggcaggc gttgccaacg ctcagcgcgc ttacgctgtg 720 tacaaggagc ttttcgacgc cgccgagctg cctgaaggtg ccaacactca gcgcccactg 780 tgggcatcca ccggcgtgaa gaaccctgcg tacgctgcaa ctctttacgt ttccgagctg 840 gctggtccaa acaccgtcaa caccatgcca gaaggcacca tcgacgcggt tctggagcag 900 ggcaacctgc acggtgacac cctgtccaac tccgcggcag aagctgacgc tgtgttctcc 960 cagcttgagg ctctgggcgt tgacttggca gatgtcttcc aggtcctgga gaccgagggt 1020 gtggacaagt tcgttgcttc ttggagcgaa ctgcttgagt ccatggaagc tcgcctgaag 1080 tag 1083 <210> 3 <211> 335 <212> DNA <213> Artificial Sequence <220> <223> Peftu Promoter <400> 3 cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60 tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120 ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180 aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240 tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300 agtcgtagcc accacgaagt ccaggaggac ataca 335 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Peftu-F <400> 4 gaaatcagga agtgggatcg aaacgaaaag caatttgctt ttcgacg 47 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Peftu-R <400> 5 tgtatgtcct cctggacttc gtg 23 <210> 6 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> cgl1574-F <400> 6 ccacgaagtc caggaggaca tacagtggac accaaggctg tag 43 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> cgl1574-R <400> 7 ttaaccgtta atggagtcct tgg 23 <210> 8 <211> 2366 <212> DNA <213> Artificial Sequence <220> <223> Peftu_cgl1574 <400> 8 cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60 tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120 ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180 aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240 tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300 agtcgtagcc accacgaagt ccaggaggac atacagtgga caccaaggct gtagacactg 360 ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc cggccaccca ggcaccgcaa 420 tgagcctggc tccccttgca tacaccttgt accagcgggt tatgaacgta gatccacagg 480 acaccaactg ggcaggccgt gaccgcttcg ttctttcttg tggccactcc tctttgaccc 540 agtacatcca gctttacttg ggtggattcg gccttgagat ggatgacctg aaggctctgc 600 gcacctggga ttccttgacc ccaggacacc ctgagtaccg ccacaccaag ggcgttgaga 660 tcaccactgg ccctcttggc cagggtcttg catctgcagt tggtatggcc atggctgctc 720 gtcgtgagcg tggcctattc gacccaaccg ctgctgaggg cgaatcccca ttcgaccacc 780 acatctacgt cattgcttct gatggtgacc tgcaggaagg tgtcacctct gaggcatcct 840 ccatcgctgg cacccagcag ctgggcaacc tcatcgtgtt ctgggatgac aaccgcatct 900 ccatcgaaga caacactgag atcgctttca acgaggacgt tgttgctcgt tacaaggctt 960 acggctggca gaccattgag gttgaggctg gcgaggacgt tgcagcaatc gaagctgcag 1020 tggctgaggc taagaaggac accaagcgac ctaccttcat ccgcgttcgc accatcatcg 1080 gcttcccagc tccaactatg atgaacaccg gtgctgtgca cggtgctgct cttggcgcag 1140 ctgaggttgc agcaaccaag actgagcttg gattcgatcc tgaggctcac ttcgcgatcg 1200 acgatgaggt tatcgctcac acccgctccc tcgcagagcg cgctgcacag aagaaggctg 1260 catggcaggt caagttcgat gagtgggcag ctgccaaccc tgagaacaag gctctgttcg 1320 atcgcctgaa ctcccgtgag cttccagcgg gctacgctga cgagctccca acatgggatg 1380 cagatgagaa gggcgtcgca actcgtaagg cttccgaggc tgcacttcag gcactgggca 1440 agacccttcc tgagctgtgg ggcggttccg ctgacctcgc aggttccaac aacaccgtga 1500 tcaagggctc cccttccttc ggccctgagt ccatctccac cgagacctgg tctgctgagc 1560 cttacggccg taacctgcac ttcggtatcc gtgagcacgc tatgggatcc atcctcaacg 1620 gcatttccct ccacggtggc acccgcccat acggcggaac cttcctcatc ttctccgact 1680 acatgcgtcc tgcagttcgt cttgcagctc tcatggagac cgacgcttac tacgtctgga 1740 cccacgactc catcggtctg ggcgaagatg gcccaaccca ccagcctgtt gaaaccttgg 1800 ctgcactgcg cgccatccca ggtctgtccg tcctgcgtcc tgcagatgcg aacgagaccg cccaggcttg ggctgcagca cttgagtaca aggaaggccc taagggtctt gcactgaccc gccagaacgt tcctgttctg gaaggcacca aggagaaggc tgctgaaggc gttcgccgcg gtggctacgt cctggttgag ggttccaagg aaaccccaga tgtgatcctc atgggctccg gctccgaggt tcagcttgca gttaacgctg cgaaggctct ggaagctgag ggcgttgcag ctcgcgttgt ttccgttcct tgcatggatt ggttccagga gcaggacgca gagtacatcg 2160. agtccgttct gcctgcagct gtgaccgctc gtgtgtctgt tgaagctggc atcgcaatgc cttggtaccg cttcttgggc acccagggcc gtgctgtctc ccttgagcac ttcggtgctt 2280 ctgcggatta ccagaccctg tttgagaagt tcggcatcac caccgatgca gtcgtggcag cggccaagga ctccattaac ggttaac <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ldh-up-F <400> 9 tcccccgggg gc 32 <210> 10 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> ldh-up-R <400> 10 caaattgctt ttcgtttcga tcccacttcc tgatttccct aac 43 <210> 11 <211> 943 <212> DNA <213> Artificial Sequence <220> <223> ldh-up <400> 11 ggaacaccat gcgattaagg tgcgctgctt gaattgcaga attatgcaag atgcgccgca 60 acaaaacgcg atcggccaag gtcaaagtgg tcaatgtaat gaccgaaacc gctgcgatga 120 aactaatcca cggcggtaaa aacctctcaa ttaggagctt gacctcatta atgctgtgct 180 gggttaattc gccggtgatc agcagcgcgc cgtaccccaa ggtgccgaca ctaatgcccg 240 cgatcgtctc cttcggtcca aaattcttct gcccaatcag ccggatttgg gtgcgatgcc 300 tgatcaatcc cacaaccgtg gtggtcaacg tgatggcacc agttgcgatg tgggtggcgt 360 tgtaaatttt cctggatacc cgccggttgg ttctggggag gatcgagtgg attcccgtcg 420 ctgacgcatg ccccaccgct tgtaaaacag ccaggttagc agccgtaacc caccacggtt 480 tcggcaacaa tgacggcgag agagcccacc acattgcgat ttccgctccg ataaagccag 540 cgcccatatt tgcagggagg attcgcctgc ggtttggcga cattcggatc cccggaacca 600 gctctgcaat cacctgcgcg ccgagggaag cgaggtgggt ggcaggtttt agtgcgggtt 660 taagcgttgc caggcgagtg gtgagcagag acgctagtct ggggagcgaa accatattga 720 gtcatcttgg cagagcatgc acaattctgc agggcataga ttggttttgc tcgatttaca 780 atgtgatttt ttcaacaaaa ataacacatg gtctgaccac attttcggac ataatcgggc 840 ataattaaag gtgtaacaaa ggaatccggg cacaagctct tgctgatttt ctgagctgct 900 ttgtgggttg tccggttagg gaaatcagga agtgggatcg aaa 943 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> ldh-down-F <400> 12 caaggactcc attaacggtt aaatctttgg cgcctagttg gc 42 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ldh-down-R <400> 13 gtaagcttgt ctgggacgtt gatgacgctg 30 <210> 14 <211> 959 <212> DNA <213> Artificial Sequence <220> <223> ldh-down <400> 14 atctttggcg cctagttggc gacgcaagtg tttcattgga acacttgcgc tgccaacttt 60 ttggtttacg ggcaaaatga aactgttgga tggaatttaa agtgtttgta gcttaaggag 120 ctcaaatgaa tgagtttgac caggacattc tccaggagat caagactgaa ctcgacgagt 180 taattctaga acttgatgag gtgacacaaa ctcacagcga ggccatcggg caggtctccc 240 caacccatta cgttggtgcc cgcaacctca tgcattacgc gcatcttcgc accaaagacc 300 tccgtggcct gcagcaacgc ctctcctctg tgggagctac ccgcttgact accaccgaac 360 cagcagtgca ggcccgcctc aaggccgccc gcaatgttat cggagctttc gcaggtgaag 420 gcccacttta tccaccctca gatgtcgtcg atgccttcga agatgccgat gagattctcg 480 acgagcacgc cgaaattctc cttggcgaac ccctaccgga tactccatcc tgcatcatgg 540 tcaccctgcc caccgaagcc gccaccgaca ttgaacttgt ccgtggcttc gccaaaagcg 600 gcatgaatct agctcgcatc aactgtgcac acgacgatga aaccgtctgg aagcagatga 660 tcgacaacgt ccacaccgtt gcagaagaag ttggccggga aatccgcgtc agcatggacc 720 ttgccggacc aaaagtacgc accggcgaaa tcgccccagg cgcagaagta ggtcgcgcac 780 gagtaacccg cgacgaaacc ggaaaagtac tgacgcccgc aaaactgtgg atcaccgccc 840 acggctccga accagtccca gcccccgaaa gcctgcccgg tcgccccgct ctgccgattg 900 aagtcacccc agaatggttc gacaaactag aaatcggcag cgtcatcaac gtcccagac 959 <210> 15 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> cgl1575-F <400> 15 ccacgaagtc caggaggaca tacaatgtct cacattgatg atcttgc 47 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> cgl1575-R <400> 16 ctacttcagg cgagcttcc 19 <210> 17 <211> 1418 <212> DNA <213> Artificial Sequence <220> <223> Peftu_cgl1575 <400> 17 cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60 tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120 ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180 aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240 tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300 agtcgtagcc accacgaagt ccaggaggac atacaatgtc tcacattgat gatcttgcac 360 agctcggcac ttccacttgg ctcgacgacc tctcccgcga gcgcattact tccggcaatc 420 tcagccaggt tattgaggaa aagtctgtag tcggtgtcac caccaaccca gctattttcg 480 cagcagcaat gtccaagggc gattcctacg acgctcagat cgcagagctc aaggccgctg 540 gcgcatctgt tgaccaggct gtttacgcca tgagcatcga cgacgttcgc aatgcttgtg 600 atctgttcac cggcatcttc gagtcctcca acggctacga cggccgcgtg tccatcgagg 660 ttgacccacg tatctctgct gaccgcgacg caaccctggc tcaggccaag gagctgtggg 720 caaaggttga tcgtccaaac gtcatgatca agatccctgc aaccccaggt tctttgccag 780 caatcaccga cgctttggct gagggcatca gcgttaacgt caccttgatc ttctccgttg 840 ctcgctaccg cgaggtcatc gctgcgttca tcgagggcat caagcaggct gctgcaaacg 900 gccacgacgt ctccaagatc cactctgtgg cttccttctt cgtctcccgc gtcgacgttg 960 agatcgacaa gcgcctcgag gcaatcggat ccgatgaggc tttggctctg cgcggcaagg 1020 caggcgttgc caacgctcag cgcgcttacg ctgtgtacaa ggagcttttc gacgccgccg 1080 agctgcctga aggtgccaac actcagcgcc cactgtgggc atccaccggc gtgaagaacc 1140 ctgcgtacgc tgcaactctt tacgtttccg agctggctgg tccaaacacc gtcaacacca 1200 tgccagaagg caccatcgac gcggttctgg agcagggcaa cctgcacggt gacaccctgt 1260 ccaactccgc ggcagaagct gacgctgtgt tctcccagct tgaggctctg ggcgttgact 1320 tggcagatgt cttccaggtc ctggagaccg agggtgtgga caagttcgtt gcttcttgga 1380 gcgaactgct tgagtccatg gaagctcgcc tgaagtag 1418 <210> 18 <211> 3784 <212> DNA <213> Artificial Sequence <220> <223> cgl1574-cgl1575 <400> 18 cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60 tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120 ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180 aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240 tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300 agtcgtagcc accacgaagt ccaggaggac atacaatgga caccaaggct gtagacactg 360 ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc cggccaccca ggcaccgcaa 420 tgagcctggc tccccttgca tacaccttgt accagcgggt tatgaacgta gatccacagg 480 acaccaactg ggcaggccgt gaccgcttcg ttctttcttg tggccactcc tctttgaccc 540 agtacatcca gctttacttg ggtggattcg gccttgagat ggatgacctg aaggctctgc 600 gcacctggga ttccttgacc ccaggacacc ctgagtaccg ccacaccaag ggcgttgaga 660 tcaccactgg ccctcttggc cagggtcttg catctgcagt tggtatggcc atggctgctc 720 gtcgtgagcg tggcctattc gacccaaccg ctgctgaggg cgaatcccca ttcgaccacc 780 acatctacgt cattgcttct gatggtgacc tgcaggaagg tgtcacctct gaggcatcct 840 ccatcgctgg cacccagcag ctgggcaacc tcatcgtgtt ctgggatgac aaccgcatct 900 ccatcgaaga caacactgag atcgctttca acgaggacgt tgttgctcgt tacaaggctt 960 acggctggca gaccattgag gttgaggctg gcgaggacgt tgcagcaatc gaagctgcag 1020 tggctgaggc taagaaggac accaagcgac ctaccttcat ccgcgttcgc accatcatcg 1080 gcttcccagc tccaactatg atgaacaccg gtgctgtgca cggtgctgct cttggcgcag 1140 ctgaggttgc agcaaccaag actgagcttg gattcgatcc tgaggctcac ttcgcgatcg 1200 acgatgaggt tatcgctcac acccgctccc tcgcagagcg cgctgcacag aagaaggctg 1260 catggcaggt caagttcgat gagtgggcag ctgccaaccc tgagaacaag gctctgttcg 1320 atcgcctgaa ctcccgtgag cttccagcgg gctacgctga cgagctccca acatgggatg 1380 cagatgagaa gggcgtcgca actcgtaagg cttccgaggc tgcacttcag gcactgggca 1440 agacccttcc tgagctgtgg ggcggttccg ctgacctcgc aggttccaac aacaccgtga 1500 tcaagggctc cccttccttc ggccctgagt ccatctccac cgagacctgg tctgctgagc 1560 cttacggccg taacctgcac ttcggtatcc gtgagcacgc tatgggatcc atcctcaacg 1620 gcatttccct ccacggtggc acccgcccat acggcggaac cttcctcatc ttctccgact 1680 acatgcgtcc tgcagttcgt cttgcagctc tcatggagac cgacgcttac tacgtctgga 1740 cccacgactc catcggtctg ggcgaagatg gcccaaccca ccagcctgtt gaaaccttgg 1800 ctgcactgcg cgccatccca ggtctgtccg tcctgcgtcc tgcagatgcg aacgagaccg 1860 cccaggcttg ggctgcagca cttgagtaca aggaaggccc taagggtctt gcactgaccc gccagaacgt tcctgttctg gaaggcacca aggagaaggc tgctgaaggc gttcgccgcg gtggctacgt cctggttgag ggttccaagg aaaccccaga tgtgatcctc atgggctccg gctccgaggt tcagcttgca gttaacgctg cgaaggctct ggaagctgag ggcgttgcag ctcgcgttgt ttccgttcct tgcatggatt ggttccagga gcaggacgca gagtacatcg 2160. agtccgttct gcctgcagct gtgaccgctc gtgtgtctgt tgaagctggc atcgcaatgc cttggtaccg cttcttgggc acccagggcc gtgctgtctc ccttgagcac ttcggtgctt 2280 ctgcggatta ccagaccctg tttgagaagt tcggcatcac caccgatgca gtcgtggcag cggccaagga ctccattaac ggttaacgaa aagcaatttg cttttcgacg ccccaccccg cgcgttttag cgtgtcagta ggcgcgtagg gtaagtgggg tagcggcttg ttagatatct 2460 tgaaatcggc tttcaacagc attgatttcg atgtatttag ctggccgtta ccctgcgaat gtccacaggg tagctggtag tttgaaaatc aacgccgttg cccttaggat tcagtaactg gcacattttg taatgcgcta gatctgtgtg ctcagtcttc caggctgctt atcacagtga 2640 aagcaaaacc aattcgtggc tgcgaaagtc gtagccacca cgaagtccag gaggacatac 2700 aatgtctcac attgatgatc ttgcacagct cggcacttcc acttggctcg acgacctctc 2760 ccgcgagcgc attacttccg gcaatctcag ccaggttatt gaggaaaagt ctgtagtcgg 2820 tgtcaccacc aacccagcta ttttcgcagc agcaatgtcc aagggcgatt cctacgacgc 2880 tcagatcgca gagctcaagg ccgctggcgc atctgttgac caggctgttt acgccatgag 2940 catcgacgac gttcgcaatg cttgtgatct gttcaccggc atcttcgagt cctccaacgg 3000 ctacgacggc cgcgtgtcca tcgaggttga cccacgtatc tctgctgacc gcgacgcaac 3060 cctggctcag gccaaggagc tgtgggcaaa ggttgatcgt ccaaacgtca tgatcaagat 3120 ccctgcaacc ccaggttctt tgccagcaat caccgacgct ttggctgagg gcatcagcgt 3180 taacgtcacc ttgatcttct ccgttgctcg ctaccgcgag gtcatcgctg cgttcatcga 3240 gggcatcaag caggctgctg caaacggcca cgacgtctcc aagatccact ctgtggcttc 3300 cttcttcgtc tccgcgtcg acgttgagat cgacaagcgc ctcgaggcaa tcggatccga 3360 tgaggctttg gctctgcgcg gcaaggcagg cgttgccaac gctcagcgcg cttacgctgt 3420 gtacaaggag cttttcgacg ccgccgagct gcctgaaggt gccaacactc agcgccccact 3480 gtgggcatcc accggcgtga agaaccctgc gtacgctgca actctttacg tttccgagct 3540 ggctggtcca aacaccgtca acaccatgcc agaaggcacc atcgacgcgg ttctggagca 3600 gggcaacctg cacggtgaca ccctgtccaa ctccgcggca gaagctgacg ctgtgttctc 3660 ccagcttgag gctctgggcg ttgacttggc agatgtcttc caggtcctgg agaccgaggg 3720 tgtggacaag ttcgttgctt cttggagcga actgcttgag tccatggaag ctcgcctgaa 3780 gtag 3784

Claims

1. A method for preparing L-lysine from glucose, characterized in that, The method includes fermenting genetically engineered bacteria in a fermentation medium; the genetically engineered bacteria are expressed by transferring an expression cassette into a starting bacteria, or containing nucleic acid of the expression cassette, or a recombinant expression vector containing the expression cassette or the nucleic acid. The expression box includes the following components: promoter; and, Transaldolase gene and transketolase gene; The sequence of the transaldolase gene is shown in SEQ ID NO: 2; the sequence of the transketoolase gene is shown in SEQ ID NO: 1; The expression box is integrated into ldh At the gene locus; the originating bacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ).

2. The method as described in claim 1, characterized in that, The promoter is the Peftu promoter.

3. The method according to claim 2, characterized in that, The sequence of the Peftu promoter is shown in SEQ ID NO:

3.

4. The method according to claim 1, characterized in that, The backbone plasmid of the recombinant expression vector is pK18mob.

5. The method as described in claim 1, characterized in that, The originating bacteria is C. glutamicum B253 or collection number CCTCC NO: M 20211495 C. glutamicum CathS141.

6. The method as described in claim 1, characterized in that, The fermentation medium contains not less than 25 g / L of glucose.

7. The method as described in claim 6, characterized in that, The fermentation meets one or more of the following conditions: The fermentation temperature is 28~32℃; The aeration rate for the fermentation is 1.0~1.7 vvm; The pH of the fermentation is 6-8; The fermentation is carried out by stirring at a speed of 400-800 rpm.

8. The method as described in claim 7, characterized in that, The fermentation temperature is 30°C.

9. The method as described in claim 7, characterized in that, The aeration rate for the fermentation was 1.4 vvm.

10. The method as described in claim 7, characterized in that, The pH of the fermentation was 7.

11. The method as described in claim 7, characterized in that, The stirring speed is 600 rpm.

12. The method according to any one of claims 1-11, characterized in that, The fermentation medium is a lignocellulose hydrolysate.

13. The method as described in claim 12, characterized in that, The lignocellulose hydrolysate is a straw hydrolysate.

14. The method as described in claim 13, characterized in that, The straw hydrolysate is formed by enzymatic hydrolysis and saccharification of crop straw.

15. The method as described in claim 13 or 14, characterized in that, Add 15-25 g / L ammonium sulfate, 2-8 g / L methionine and 2-8 g / L threonine to the straw hydrolysate, and / or pretreat the straw hydrolysate before enzymatic hydrolysis and saccharification, the pretreatment including sieving, impurity removal, acid pretreatment and / or detoxification treatment.

16. Application of genetically engineered bacteria in the preparation of L-lysine from glucose; wherein the genetically engineered bacteria is an expression cassette transferred into a starting bacterium, or a nucleic acid containing the expression cassette, or a recombinant expression vector containing the expression cassette or the nucleic acid; The expression box includes the following components: promoter; and, Transaldolase gene and transketolase gene; The sequence of the transaldolase gene is shown in SEQ ID NO: 2; the sequence of the transketoolase gene is shown in SEQ ID NO: 1; The expression box is integrated into ldh At the gene locus; the originating bacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ).

17. The application as described in claim 16, characterized in that, The promoter is the Peftu promoter.

18. The application according to claim 17, characterized in that, The sequence of the Peftu promoter is shown in SEQ ID NO:

3.

19. The application according to claim 16, characterized in that, The backbone plasmid of the recombinant expression vector is pK18mob.

20. The application as described in claim 16, characterized in that, The originating bacteria is C. glutamicum B253 or collection number CCTCC NO: M 20211495 C. glutamicum CathS141.