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Corynebacterium glutamicum capable of increasing lysine yield and constructing method of corynebacterium glutamicum

A Corynebacterium glutamicum, construction method technology, applied in the directions of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve NADH deficiency, limit bacterial growth sugar utilization L-lysine Acid production efficiency, insufficient content, etc.

Inactive Publication Date: 2018-08-24
JIANGNAN UNIV
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Problems solved by technology

[0005] The low production of L-lysine in the existing Corynebacterium glutamicum is mainly due to the insufficient content of NADPH, which limits the further increase of L-lysine. This laboratory tries to make NAD in the strain + -dependent glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase, NAD-GADPH) was replaced by NADP-GADPH derived from Clostridium acetobutylicum (Clostridium acetobutylicum), although the obtained recombinant strain was in Increase the production of L-lysine to a certain extent, but due to insufficient intracellular NADH and excessive accumulation of NADPH, the growth of bacteria, the utilization rate of sugar and the production efficiency of L-lysine are limited

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  • Corynebacterium glutamicum capable of increasing lysine yield and constructing method of corynebacterium glutamicum
  • Corynebacterium glutamicum capable of increasing lysine yield and constructing method of corynebacterium glutamicum
  • Corynebacterium glutamicum capable of increasing lysine yield and constructing method of corynebacterium glutamicum

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Experimental program
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Embodiment 1

[0028] Embodiment 1: Construction of recombinant bacterial strain C.glutamicum RGI

[0029] According to literature reports, NAD-IDH exists in a small number of prokaryotes such as bacteria and archaea, such as Hydrogenobacter thermophilus, A.thiooxidans, Streptococcus suis, motility fermentation Single cell bacteria (Z.mobilis) and mutans streptococcus (S.mutans). Therefore, by comparing the affinity of NAD-IDH to the cofactor NAD+ and NADP+ in the above five strains, we found that the NAD-IDH from S. + The affinity was significantly lower than that of NAD-IDH in the other four strains. Therefore, we chose NAD-IDH derived from S.mutans as a later experimental factor.

[0030] Using gene synthesis method to obtain NAD-IDH coding gene icd from S.mutans Sm , and then use the novel integrative vector pK18-MBPMT carrying antibiotic resistance and conditional lethal marker sacB double markers to replace the self-gene icd in C. glutamicum RG by two homologous recombination Cg , ...

Embodiment 2

[0039] Example 2: Determination of IDH enzyme activity of the starting bacterium C.glutamicum RG and the recombinant strain C.glutamicum RGI

[0040] Inoculate the strains preserved in the frozen tube containing 0.25g·L -1 of L-methionine and 40g·L -1 In the CgXII medium of glucose (i.e. CgXIIMG medium), shake culture at 30°C overnight, and at 10000r·min -1 Bacteria were collected by centrifugation. Subsequently, the cells were suspended in Tris-HCl buffer (pH 8.0) and subjected to ultrasonication to prepare crude enzyme solution. The crude enzyme solution was assayed by colorimetric method for enzyme activity (A 340nm ). Enzyme reaction system: 20mmol·L -1 Tris-HCl buffer (pH 8.0), 1mmol L -1 DL-trisodium isocitrate, 2mmol·L -1 MgCl 2 , 0.5mmol·L -1 NADP + or 0.5mmol·L -1 NAD + ; Reaction temperature: 30°C; Reaction time: ≥300s. One enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol NADPH or NADH per minute under the assay con...

Embodiment 3

[0043] Example 3: Determination of intracellular cofactors in the starting bacterium C.glutamicum RG and the recombinant strain C.glutamicum RGI

[0044] Take a single colony and inoculate it in CgXIIMG liquid medium, 30°C, 100r min -1 Cultivate on a shaker for about 10 hours, 4°C, 6000r·min-1 The cells were collected by centrifugation, and the cells were washed three times to remove residual extracellular metabolites. Subsequently, acidic extract (0.5mol·L -1 HCl) to extract oxidized pyridine nucleotides (NAD + and NADP + ), with alkaline extract (0.5mol L - 1 NaOH) to extract reduced pyridine nucleotides (NADH and NADPH). Subsequently, with the help of the quantitative analysis kit purchased from BioVision, NAD(P) was determined by enzymatic cycle method + and NAD(P)H concentrations and calculate NADH / NAD + and NADPH / NADP + , where NAD is specifically detected with the NAD / NADH Quantification Colorimeteric Kit + and NADH, specifically detect NADP with NADP / NADPH Qua...

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Abstract

The invention discloses corynebacterium glutamicum capable of increasing lysine yield and a constructing method of the corynebacterium glutamicum, and belongs to the technical field of genetic engineering. A genetic engineering method is adopted for substituting IDH (isocitrate dehydrogenase) coding gene icdCg in corynebacterium glutamicum RG for IDH coding gene icdSm in streptococcus mutans, andaccordingly, affinity of IDH to different redox cofactors is regulated, the problem of intracellular redox imbalance in the synthesis process of L-lysine is solved, and the L-lysine accumulation capacity of the strain is improved. Tank (5L fermentation tank) experiments show that L-lysine accumulation amount of the recombinant bacterium reaches 121.4 g / L. Redox cofactor affinity of IDH in corynebacterium glutamicum is successfully changed, problem of intracellular redox imbalance during synthesis of L-lysine is solved, and a new idea for breeding the L-Lysine high-yield strain is provided.

Description

technical field [0001] The invention relates to a Corynebacterium glutamicum with improved lysine yield and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-Lysine is one of the eight essential amino acids that are necessary for humans and animals and cannot be synthesized by themselves. Because L-lysine has a variety of physiological functions, such as balancing amino acid composition, regulating internal metabolic balance, improving the body's absorption and utilization of cereal protein, and promoting body growth and development, it is widely used in feed industry, pharmaceutical industry and food in industry. There are three main production methods for L-lysine: proteolysis, chemical synthesis and microbial fermentation. Microbial fermentation has the advantages of low production cost, high production intensity, high specificity and less environmental pollution, and has become the current industrial ...

Claims

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Application Information

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IPC IPC(8): C12P13/08C12N1/21C12N15/77C12N15/53C12R1/15
CPCC12N9/0006C12N15/77C12P13/08C12Y101/01041
Inventor 徐建中张伟国
Owner JIANGNAN UNIV
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