CN117987444A - Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production - Google Patents

Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production Download PDF

Info

Publication number
CN117987444A
CN117987444A CN202211380895.7A CN202211380895A CN117987444A CN 117987444 A CN117987444 A CN 117987444A CN 202211380895 A CN202211380895 A CN 202211380895A CN 117987444 A CN117987444 A CN 117987444A
Authority
CN
China
Prior art keywords
genetically engineered
expression cassette
engineered bacterium
lysine
corynebacterium glutamicum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211380895.7A
Other languages
Chinese (zh)
Inventor
张明明
刘修才
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cathay R&D Center Co Ltd
CIBT America Inc
Original Assignee
Cathay R&D Center Co Ltd
CIBT America Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cathay R&D Center Co Ltd, CIBT America Inc filed Critical Cathay R&D Center Co Ltd
Priority to CN202211380895.7A priority Critical patent/CN117987444A/en
Publication of CN117987444A publication Critical patent/CN117987444A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01009—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses an expression cassette, genetically engineered bacteria containing the same and application of the genetically engineered bacteria in L-lysine production. The gene expression cassette comprises an NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene, and the nucleotide sequences of the NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene are respectively shown in SEQ ID NO. 1-5. The genetic engineering bacteria which are constructed by the invention and take the corynebacterium glutamicum as a starting strain and comprise the NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene can effectively utilize agricultural wastes such as straws and the like for fermentation, obviously improves the yield of L-lysine, and has good application prospect.

Description

Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an expression cassette, genetically engineered bacteria containing the expression cassette and application of the genetically engineered bacteria in L-lysine production.
Background
Lysine (lysine), also known as 2, 6-diaminocaproic acid, belongs to the basic amino acid. Lysine has important nutrition physiological functions and is widely applied in the industries of medicine, food and feed. At the same time, it can also be used as a precursor substance for synthesizing nylon polymer materials. The production path of lysine mainly comprises three methods of a protein hydrolysis method, a chemical synthesis method and a fermentation method, wherein the microbial fermentation method has the characteristics of low production cost, high production strength, high specificity, small environmental pollution and the like, and becomes the method with the widest application of industrial production of lysine.
The prokaryotic microorganism for producing lysine mainly includes coryneform bacteria, brevibacterium, nocardia, pseudomonas, escherichia, bacillus, and the like. Corynebacterium glutamicum (c.glutamicum) is the most important and safe strain for fermentative production of amino acids, and improving its ability to excessively synthesize various amino acids by metabolic engineering has been a research hotspot. For example, overexpression of a gene involved in lysine synthesis pathway and a gene involved in desensitization of feedback inhibition, or enhancement of energy supply pathway from glucose metabolism and optimization of lysine transporter on cell membrane, etc. are effective in improving productivity of lysine.
In addition to constantly enhancing metabolic pathways for lysine production, fermentation methods that open other bypasses to non-lysine metabolism are also worth the deep excavation. Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is the most important redox carrier in cellular metabolism, and is considered as an important cofactor, which not only serves as an electron acceptor catalyzing substrate catabolism, but also provides reducing power for energy-dependent redox reactions, and is a main driving force in the synthesis of various metabolites including lysine. At present, some researches on improving lysine yield by regulating NADPH through glyceraldehyde 3-phosphate dehydrogenase exist, but no report on efficient production of lysine by optimally breeding the enzyme exists.
Disclosure of Invention
In order to solve the problem of low yield of L-amino acid in the prior art, the invention provides an expression cassette for preparing L-lysine, genetically engineered bacteria and application thereof. Specifically, the invention provides an expression cassette containing NADP dependent glyceraldehyde 3-phosphate dehydrogenase and a genetic engineering bacterium containing the expression cassette, wherein the genetic engineering bacterium can effectively utilize glucose in straws in straw hydrolysate. The invention screens the multifunctional glyceraldehyde 3-phosphate dehydrogenase and obviously improves the yield of L-lysine by regulating and controlling the supply of intracellular cofactor and the metabolism angle of acyl-CoA.
To solve the above technical problem, a first aspect of the present invention provides an expression cassette, which comprises the following components: the nucleotide sequences of the NADP dependent glyceraldehyde 3-phosphate dehydrogenase encoding genes are shown as SEQ ID NO. 1-5 respectively.
In a second aspect the present invention provides a recombinant expression vector comprising an expression cassette according to the first aspect of the invention or comprising a nucleotide sequence as defined in the first aspect of the invention.
In a preferred embodiment, the backbone plasmid of the recombinant expression vector is pK18mob.
The specific information of the pK18mob can be referred to as the following web page:
http://www.biovector.net/product/1089.html。
In a third aspect, the invention provides a genetically engineered bacterium transformed into an expression cassette according to the first aspect of the invention, or a nucleotide sequence as defined in the first aspect of the invention, or a recombinant expression vector according to the second aspect of the invention.
In the genetically engineered bacterium, the starting bacterium is corynebacterium glutamicum (Corynebacterium glutamicum), for example, c.glutamicum B253 or c.glutamicum CathS.
In the genetically engineered bacterium, the expression cassette is preferably integrated in the genome of the starting bacterium by homologous recombination or is present in the starting bacterium in non-integrated form.
In the genetically engineered bacterium, it is preferable that the glyceraldehyde 3-phosphate dehydrogenase (gapdh) of the original strain is not expressed, and for example, the gapdh gene (NCBI GeneID of CP 025533.1) is knocked out.
In a preferred embodiment, the expression cassette is integrated into the gapdh gene at a site.
The starting strain can be a wild strain or a modified strain.
In a fourth aspect, the present invention provides a process for producing L-lysine, which comprises fermentatively culturing the genetically engineered bacterium according to the third aspect of the present invention, and recovering L-lysine from the culture broth of the fermentative culture.
In a preferred embodiment, the fermentation culture is performed in a fermentation medium suitable for the growth of the genetically engineered bacterium.
In a preferred embodiment, the fermentation medium contains not less than 25g/L glucose, for example 80-150g/L glucose. In addition to glucose, the fermentation medium contains other components for the growth, propagation or synthesis of L-lysine by the genetically engineered bacteria, such as potassium dihydrogen phosphate, and/or urea, and/or magnesium sulfate, and/or methionine, and/or corn steep liquor, and/or yeast extract.
In a preferred embodiment, the fermentation temperature is 28 to 37 ℃, for example 35 ℃.
In a preferred embodiment, the pH of the fermentation is from 6 to 8, for example 7.
In a preferred embodiment, the fermentation medium comprises a hydrolysate of lignocellulosic material.
In a preferred embodiment, the carbon source in the fermentation medium is derived from a hydrolysate of lignocellulosic material. The carbon source is preferably glucose.
The hydrolysate of the lignocellulose material is formed by enzymolysis and saccharification of the lignocellulose material. The enzymolysis uses cellulase.
The lignocellulose material comprises at least one of, but not limited to, straw, rice husk, bagasse, wood chips. The straw preferably comprises corn straw, wheat straw and cotton straw.
In a preferred embodiment, the lignocellulosic material is subjected to a pretreatment prior to the enzymatic saccharification, the pretreatment comprising screening, impurity removal, dry acid pretreatment and/or detoxification treatment of the lignocellulosic material.
In some embodiments, the lignocellulosic material has a particle size of 2 to 20mm, e.g., 5mm, 10mm.
In a fifth aspect, the present invention provides the use of an expression cassette according to the first aspect of the invention, a nucleotide sequence as defined in the first aspect of the invention, a recombinant expression vector according to the second aspect of the invention, or a genetically engineered bacterium according to the third aspect of the invention for the preparation of L-lysine.
The sixth aspect of the invention provides the use of the expression cassette according to the first aspect of the invention and the recombinant expression vector according to the second aspect of the invention in the preparation of corynebacterium glutamicum engineering bacteria.
In a seventh aspect, the present invention provides a method for preparing a genetically engineered bacterium obtained by introducing an expression cassette according to the present invention into a starting bacterium as defined in the present invention.
The preparation method preferably further comprises: blocking the expression of lactate dehydrogenase in the genetically engineered bacterium, e.g., knocking out gapdh gene.
In the preparation method, preferably, the expression cassette is integrated into the gapdh gene at a site.
In a preferred embodiment, the starting bacterium is C.glutamicum CathS141,141, and the preservation number is CCTCC NO: M20211495.
In a preferred embodiment, the starting bacterium of the present invention is C.glutamicum B253, purchased from Shanghai Industrial microorganism.
The NADP dependent glyceraldehyde 3-phosphate dehydrogenase is respectively from Clostridium sp.NJ4,Chryseobacterium joostei,Streptococcus mutans serotype c,Chryseobacterium piscicola and Pedobacter alluvionis strains, and the nucleotide sequences of the NADP dependent glyceraldehyde 3-phosphate dehydrogenase are respectively shown in SEQ ID NO. 1-5.
The starting strain can be a wild strain or a modified strain.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
After the expression cassette provided by the invention is introduced into the starting bacteria, the electron supply and the like in the fermentation process are effectively improved, so that the glucose utilization efficiency is improved, and the yield of L-lysine is further improved. When the lignocellulose material hydrolysate is used, the lysine yield of the genetically engineered bacterium is obviously improved compared with that of the starting bacterium. The genetically engineered bacterium provided by the invention can effectively utilize agricultural wastes such as straw and the like for fermentation, has good application prospect, and is also beneficial to the efficient production of lysine derivatives such as pentanediamine.
Biological material preservation information
The corynebacterium glutamicum CathS141,141 of the present invention has been preserved in China Center for Type Culture Collection (CCTCC) at 11/29 of 2021, and the preservation address is: chinese university of Wuhan, post code 430072, deposit number: CCTCC NO: M20211495, culture designation CathS, classification designation Corynebacterium glutamicum (Corynebacterium glutamicum).
Paecilomyces variotii CATHTD891 of the invention is preserved in China Center for Type Culture Collection (CCTCC) at the year 2021, 11 and 4, and the preservation address is as follows: chinese university of Wuhan, post code 430072, deposit number: CCTCC NO: M20211366, culture name CATHTD891, classification name Paecilomyces variotii (Paecilomyces variotii Bainier).
Detailed Description
1. Strains used in the present invention
Coli E.coli DH 5. Alpha. Was used for construction of expression plasmid and knock-out plasmid, C.glutamicum CathS141 was a strain for lysine production, and C.glutamicum CathS141 was used mainly as a starting strain in this experiment.
2. Reagent and culture medium
Cellulase CTec 2.0.0 was used to hydrolyze cellulose and hemicellulose in lignocellulose and was purchased from novelin (china) corporation of beijing, china. The cellulase enzyme activity was 203.2FPU/mL according to the method in the NREL LAP-006 guide, the cellobiase activity was 4900.0CBU/mL, and the protein concentration was 87.3mg/mL according to the Bradford method. Restriction enzymes are used to cleave plasmids or gene fragments to produce cohesive ends, available from Thermo Scientific (Wilmington, DE, USA). DNA polymerase is used to amplify the gene fragment, DNA ligase is used to ligate the digested gene fragment and plasmid vector, both of which are available from Takara (Otsu, japan). The seamless cloning kit was used to ligate gene fragments containing homologous fragments to plasmid vectors available from hantao biotechnology company (Nanjing, china). Plasmid extraction kits, PCR product purification recovery kits and gel recovery kits were all purchased from Shanghai swirley biotechnology company (Shanghai, china). Other reagents were purchased from local suppliers.
The culture medium used for culturing the escherichia coli is Luria-Bertani (LB) culture medium, and the specific components are as follows: 10.0g/L sodium chloride, 10.0g/L peptone and 5.0g/L yeast extract.
The specific components of the culture medium used for culturing the corynebacterium glutamicum are as follows:
(1) Seed culture medium: 25g/L glucose, 1.5g/L potassium dihydrogen phosphate, 2.5g/L urea and 0.6g/L magnesium sulfate, 25g/L corn steep liquor.
(2) Fermentation medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and optionally glucose as a carbon source.
Example 1: acquisition of Corynebacterium glutamicum CathS141 as a starting bacterium
Soil samples of Sinkiang urso were collected, added to 10mL of sterile water per 1g of soil sample, vigorously mixed for 1min, allowed to stand for precipitation for a period of time, and the samples were diluted to 10 -3、10-4、10-5 respectively, spread on LB agar plates containing 100mg/L nystatin, and incubated at 30 ℃. And obtaining a purified single colony through repeated streak separation culture. The strain was identified as Corynebacterium glutamicum by 16S rDNA sequence alignment (Corynebacterium glutamicum).
The wheat straw is subjected to acid pretreatment, detoxification and enzymatic hydrolysis to obtain wheat straw hydrolysate, the corynebacterium glutamicum obtained by separation and purification is used as an initial strain, and ultraviolet rays, nitrosoguanidine, 5-fluorouracil, ARTP and the like are used for multiple independent mutagenesis and composite mutagenesis to obtain a stable strain capable of resisting normal growth and lysine production of toxicity inhibitors (acetic acid, furfural, 5-hydroxybenzaldehyde and the like) in the straw hydrolysate. The strain is named CathS141,141 and is now deposited in China center for type culture Collection, address: chinese university of Wuhan, post code 430072, preservation number CCTCC NO: M20211495, preservation date 2021, 11 months and 29 days.
Example 2: construction of the starting Strain gapdh knockout vector
Taking the genome of C.glutamicum CathS141,141 as a template, and knocking out the glyceraldehyde 3-phosphate dehydrogenase gapdh gene (NCBI GeneID of CP 025533.1) of the original strain by designing primers, PCR amplification and other modes by the conventional technical means in the field to obtain a delta-gapdh fragment. The pK18mob plasmid was treated with EcoRI and XbaI endonucleases and then ligated with the delta-gapdh fragment by Gibson and transferred into E.coli by conventional chemical transformation methods. During this time, successfully ligated plasmids, designated pcg0, were selected using seed culture plates containing kanamycin resistance.
Example 3: construction of engineering bacteria containing NADP-dependent glyceraldehyde 3-phosphate dehydrogenase
The glyceraldehyde 3-phosphate dehydrogenase editing genes in reference Clostridium sp.NJ4,Chryseobacterium joostei,Streptococcus mutans serotype c,Chryseobacterium piscicola and Pedobacter alluvionis are synthesized in Huada genes respectively to obtain gapdh1, gapdh2, gapdh3, gapdh4 and gapdh5, and the nucleotide sequences of the gapdh1 to the gapdh5 are shown in SEQ ID NOs. Then, the gene is used as a template, a primer is designed, PCR amplification and other modes are carried out to obtain fragments, and then the fragments are connected into a pcg0 vector which is digested with BamHI by a Gibson method, and the obtained plasmids are named pcg1, pcg2, pcg3, pcg4 and pcg5 in sequence.
The plasmids pcg1, pcg2, pcg3, pcg4 and pcg5 were then transferred into C.glutamicum CathS141, respectively, by electrotransformation. Strains with correct homologous recombination are selected by a PCR method to obtain recombinant corynebacterium glutamicum, and the recombinant corynebacterium glutamicum is named cg1, cg2, cg3, cg4 and cg5 in sequence.
EXAMPLE 4 fermentation of different recombinant Corynebacterium glutamicum
The genetically engineered strain and the original strain Corynebacterium glutamicum CathS were inoculated into a seed medium for two rounds of activation and then transferred into a fermentation medium. 80g/L glucose was added as a carbon source to the fermentation medium. The fermentation was carried out in 250mL shake flasks at an inoculum size of 10% (v/v), at 35℃and 200rpm, and at a pH of 6.5, with glucose consumption as the fermentation termination time. The fermentation results are shown in Table 1: by replacing gapdh of the starting strain with NADP dependent glyceraldehyde 3-phosphate dehydrogenase, the glucose-lysine conversion rate is improved, which shows that the regulation of cofactor angle is an effective means for improving lysine production. Meanwhile, compared with 5 engineering bacteria, the cg4 lifting effect can be found to be most remarkable. In the course of the actual study, it was found that the insertion of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase without destroying gapdh of the starting strain also had an increasing effect, but was not as remarkable as the knockout effect.
TABLE 1 fermentation of different recombinant Corynebacterium glutamicum
Strain Glucose-lysine conversion (%)
CathS141 23.45±0.32
Cg1 24.89±0.23
Cg2 26.32±0.21
Cg3 25.28±0.47
Cg4 29.81±0.34
Cg5 27.29±0.15
As can be seen from example 4, the genetically engineered bacterium of the present invention produced L-lysine with better effect than the starting strain.
Example 5: fermentation of recombinant corynebacterium glutamicum in lignocellulose material hydrolysate
The wheat straw is crushed and sieved, the grain diameter is 10mm, then the impurities such as soil, stones and metal are removed by washing, and the wheat straw is dried to constant weight in a drying oven at 105 ℃. Adding 8wt% sulfuric acid solution into the dried wheat straw, controlling the solid-to-liquid ratio (g/g) to be 7:3, heating for 10min by saturated steam at 180 ℃, and then carrying out enzymolysis at 50 ℃ by using cellulase to obtain straw hydrolysate. The straw hydrolysate is inoculated with a detoxified bacterial strain Paecilomyces variotii (Paecilomyces variotii Bainier) CATHTD891 (with the preservation number of CCTCC NO: M20211366), biological detoxication is carried out for 36 hours at the temperature of 33 ℃, and then centrifugation is carried out, and the supernatant is collected, so that the detoxified straw hydrolysate which contains 95.4g/L glucose is obtained to provide a carbon source. And (3) adding a fermentation medium into the straw hydrolysate, inoculating the transformed strain cg4 to the original strain CathS, performing fermentation comparison, and performing fermentation in a 250mL shaking flask, wherein the inoculum size is 10% (v/v), the culture condition is 35 ℃, the rpm is 200, the pH value is controlled to be 6.5, and the glucose consumption is taken as the fermentation termination time.
The results show that when the fermentation medium carbon source is provided by the wheat straw hydrolysate, the two strains have a significant difference in lysine yield, wherein the final lysine yield of the recombinant strain is 26.11g/L, and the yield of the original strain is 19.85g/L. Meanwhile, the fermentation time of the engineering bacteria cg4 is 61h, which is shortened by 11h compared with that of the original strain. Thus, the two strains exhibited a significant difference in production intensity, with the production intensity (lysine production per unit time) of the recombinant strain being significantly higher than that of the control strain. In addition, when Corynebacterium glutamicum C.glutamicum B253 was used as a starting strain, the strain was obtained by modification in the same modification method as the strain cg4 and designated as Bcg4, and the fermentation effect of the fermentation under the same fermentation conditions described above was confirmed, it was found that the final lysine yield of Bcg4 was increased by 12.21% as compared with the control strain B253. And strain Bcg4 ended fermentation 10h earlier than strain B253.
Therefore, the recombinant strain obtained by the invention has high-efficiency lysine production capacity in real lignocellulose material hydrolysate, and thus has good application prospect.
The foregoing specifically describes an operation example of the technical solution of the present invention, and is not to be construed as limiting the application of the present invention. All equivalent substitutions of operating conditions are within the scope of the present invention.

Claims (10)

1. The gene expression cassette is characterized by comprising an NADP-dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene, wherein the nucleotide sequences of the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase encoding genes are shown as SEQ ID NO. 1-5 respectively.
2. A recombinant expression vector comprising the gene expression cassette of claim 1.
3. The recombinant expression vector of claim 2, wherein the backbone plasmid of the recombinant expression vector is pK18mob.
4. A genetically engineered bacterium, comprising the gene expression cassette of claim 1 or the recombinant expression vector of claim 2 or 3; the starting strain of the genetically engineered bacterium is corynebacterium glutamicum (Corynebacterium glutamicum);
preferably, the gene expression cassette is integrated in the genome of the corynebacterium glutamicum by homologous recombination or is present in non-integrated form in the starting organism.
5. The genetically engineered bacterium of claim 4, wherein the corynebacterium glutamicum is c.glutamicum B253 or c.glutamicum CathS141 with a preservation number of cctccc No. M20211495;
Preferably, the genetically engineered bacterium weakly expresses or does not express glyceraldehyde 3-phosphate dehydrogenase of the original strain;
more preferably, the gene expression cassette is integrated into the site of the gene encoding the glyceraldehyde 3-phosphate dehydrogenase.
6. A method for producing L-lysine, comprising fermentatively culturing the corynebacterium glutamicum engineering bacterium according to claim 4 or 5, and recovering L-lysine from the culture broth of the fermentative culture.
7. The method according to claim 6, wherein the fermentation culture is at a temperature of 28 to 37℃and/or the fermentation culture has a pH of 6 to 8.
8. The method according to claim 6, wherein the carbon source in the fermentation medium used in the fermentation culture is derived from a hydrolysate of lignocellulosic material; the lignocellulose material comprises at least one of straw, rice husk, bagasse, wood and wood chips.
9. Use of the gene expression cassette of claim 1 or the recombinant expression vector of claim 2 or 3 in the preparation of genetically engineered bacteria; preferably, the starting strain of the genetically engineered bacterium is corynebacterium glutamicum (Corynebacterium glutamicum); more preferably C.glutamicum B253 or C.glutamicum CathS with the preservation number of CCTCC NO: M20211495.
10. Use of the gene expression cassette of claim 1, the recombinant expression vector of claim 2 or 3 or the genetically engineered bacterium of claim 4 or 5 in the production of L-lysine.
CN202211380895.7A 2022-11-04 2022-11-04 Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production Pending CN117987444A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211380895.7A CN117987444A (en) 2022-11-04 2022-11-04 Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211380895.7A CN117987444A (en) 2022-11-04 2022-11-04 Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production

Publications (1)

Publication Number Publication Date
CN117987444A true CN117987444A (en) 2024-05-07

Family

ID=90896235

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211380895.7A Pending CN117987444A (en) 2022-11-04 2022-11-04 Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production

Country Status (1)

Country Link
CN (1) CN117987444A (en)

Similar Documents

Publication Publication Date Title
CN105647844B (en) Recombinant bacterium for producing glycolic acid by using xylose and construction method and application thereof
CN113073074B (en) Genetically engineered bacterium for efficiently synthesizing riboflavin and application thereof
CN112522223B (en) Genetically engineered bacterium for producing L-sarcosine and construction method and application thereof
CN102433292B (en) Recombinant escherichia coli for high yield of cyclic adenosine monophosphate and application thereof
JPWO2020208842A5 (en)
CN113278568A (en) Recombinant escherichia coli for producing L-valine and application thereof
CN102154339A (en) Construction method of gene engineering strain for producing succinic acid escherichia coli
CN102827853B (en) Halogenohydrin dehalogenation enzyme gene mutant and application thereof
CN102864116B (en) Gene engineering bacterium for producing succinic acid and construction and application thereof
CN110551648B (en) Corynebacterium glutamicum for producing succinic acid by fermenting xylose and application thereof
CN101463358A (en) Nitrile hydratase gene cluster and use thereof
CN106701649B (en) L-glutamine producing strain and method for producing L-glutamine
CN116064435A (en) Curcumin reductase Cfcura, coding gene and application thereof
CN111394396B (en) Method for producing 1, 3-propylene glycol by using glycerol fermentation by microorganisms
CN117987444A (en) Expression cassette, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production
CN106884001B (en) Recombinant alkalophilic bacillus, preparation method and application thereof, and method for preparing D-lactic acid
JP4294373B2 (en) New production method of ethanol
CN110499259B (en) Yarrowia lipolytica YW100-1 and application thereof
CN118272397A (en) Gene combination, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production
CN116411007A (en) Expression cassette for preparing L-lysine, genetically engineered bacterium and application thereof
CN117417874B (en) Engineering strain HC6-MT and application thereof in low-temperature production of trehalose
CN116411009A (en) Expression cassette for preparing L-lysine, genetically engineered bacterium and application thereof
CN116411010A (en) Expression cassette and strain for producing L-lysine by fermentation and application of expression cassette and strain
CN116555156B (en) Method for improving L-valine yield and recombinant bacterium used by same
CN113817761B (en) Escherichia coli chassis bacteria without tricarboxylic acid cycle, construction method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination