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Construction and application of bacillus subtilis recombinant bacteria for producing L-lysine

A technology of Bacillus subtilis and recombinant bacteria, applied in the field of genetic engineering

Pending Publication Date: 2022-07-22
JIANGSU XINGHAI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the metabolic engineering of B. subtilis to synthesize L-lysine

Method used

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  • Construction and application of bacillus subtilis recombinant bacteria for producing L-lysine
  • Construction and application of bacillus subtilis recombinant bacteria for producing L-lysine
  • Construction and application of bacillus subtilis recombinant bacteria for producing L-lysine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Plasmid pHT01-Cas9, pBE980b-hom 59 , pBE980b-lysC 311 , pBE980b-zwf 234 , pBE980b-gnd 361 and construction of pBE980b-ddh

[0039] Using the genome of Streptococcus pyogenes as a template, the gene Spcas9 was amplified by PCR using the primers Spcas9-F and Spcas9-R, and then the plasmid pHT01 and the fragment Spcas9 were double digested and ligated with restriction enzymes XbaI and XamI. , to obtain the target recombinant plasmid pHT01-Cas9.

[0040] pBE980b-hom 59 , pBE980b-lysC 311 , pBE980b-zwf 234 , pBE980b-gnd 361 and the construction of pBE980b-ddh using plasmid pBE980b as a template, using primers hom-F, hom-R, lysC-F, lysC-R, zwf-F, zwf-R, gnd-F, gnd-R, pksD-F and pksD-R seamlessly ligated 20bp sgRNA to plasmid pBE980b by inverse PCR amplification technology to obtain five plasmids with targeting sites.

[0041] Using B.subtilis ACCC11025 genome as a template, using primers hom-L-F, hom-L-R, hom-R-F, hom-R-R, lysC-L-F, lysC-L-R, lysC-R-F, lys...

Embodiment 2

[0042] Example 2: Screening and identification of target recombinant strains

[0043] The recombinant plasmids pHT01-Cas9, pBE980b-hom 59 , pBE980b-lysC 311 , pBE980b-zwf 234 , pBE980b-gnd 361 Double-enzyme digestion or PCR verification was performed with pBE980b-ddh to determine the target recombinant plasmid. from figure 2 A shows that the selected plasmids all carry the target fragment and are the target recombinant plasmids. Subsequently, the plasmid pHT01-Cas9 was electroporated into B. subtilis ACCC11025, followed by the target recombinant plasmid pBE980b-hom 59 , pBE980b-lysC 311 , pBE980b-zwf 234 , pBE980b-gnd 361 and pBE980b-ddh were electroporated into B.subtilisACCC11025 competent cells with Cas9 protein. After PCR verification, the target heavy strain was confirmed ( figure 2 B).

Embodiment 3

[0044] Example 3: Growth, G6PD and 6GPD enzyme activities, and intracellular NADH concentration determination in recombinant bacteria and starting strains

[0045] Growth determination: take 200 μL of fermentation broth regularly, and dilute it to 5 mL with 0.25 mol / L dilute hydrochloric acid solution, then measure the absorbance at 562 nm with a UV spectrophotometer (ie: OD). 562 ).

[0046] Recombinant strain B. subtilis XH1 (ie: XH0 thrD::lysC 311 ) grew normally in LB solid medium supplemented with L-lysine structural analog S-(2-aminoethyl)-L-cysteine ​​(AEC), while the starting strain B.subtilisXH0 could not grow ( image 3 B). These results suggest that replacing the thrD gene with lysC in B. subtilis 311The feedback regulation effect of L-lysine on AK III was successfully achieved.

[0047] It should be pointed out that compared with the starting strain B.subtilis XH0, the bacterial growth of the recombinant strain B.subtilis XH1 was not significantly inhibited, an...

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Abstract

The invention discloses construction and application of bacillus subtilis recombinant bacteria for producing L-lysine, and belongs to the technical field of genetic engineering. According to the invention, a genetic engineering method is applied, and systematic metabolic engineering transformation is carried out on the probiotics B.subtilis ACCC11025 commonly used in the feed industry. It is found that lysC311, zwf243 and gnd361 from C.gutamicum are used for replacing thrD, zwf and gnd (namely, recombinant bacteria B.subtilis XH4) in B.subtilis, synthesis of the L-lysine is facilitated, and the yield of the L-lysine reaches 20.3 + / -1.9 g / L. The method has the advantages that the yield of the L-lysine is increased, and the yield of the L-lysine is increased to 20.3 + / -1.9 g / L; in addition, hom in B. subtilis is replaced by hom 59 (namely, recombinant bacterium B. subtilis XH5) in C.glutamicum, so that the yield of byproducts can be remarkably reduced, the yield of L-lysine reaches 23.2 + / -1.7 g / L, and the growth of thalli is not influenced. DapDH in C.glutamicum is introduced into the recombinant bacterium B.subtilis XH5, so that carbon distribution in a diaminopimelic acid pathway can be changed, synthesis of L-lysine is promoted, and the yield of L-lysine in the target recombinant bacterium XH6 reaches 25.6 + / -2.3 g / L.

Description

technical field [0001] The invention relates to the construction and application of a Bacillus subtilis recombinant bacteria producing L-lysine, and belongs to the technical field of genetic engineering. Background technique [0002] L-Lysine is an amino acid that animals and humans cannot synthesize by themselves, and is one of the eight essential amino acids. Since lysine is easily destroyed during cereal processing and the yield of L-lysine is extremely low, L-lysine is called the first limiting amino acid. Therefore, L-Lysine is widely used in animal feed, which can balance the amino acid composition in the feed, improve the intake and metabolism of animal precious amino acids, and promote the growth and development of livestock, poultry and fish, thereby improving the protein content of feed. Utilization rate, saving production costs and reducing environmental pollution. The global L-lysine market is currently estimated at 2.2 million tons per year and growing at a ra...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N15/54C12N15/53C12P13/08A23K20/142C12R1/125
CPCC12N15/75C12N9/1217C12N9/0006C12N9/0016C12P13/08C12Y207/02004C12Y101/01049C12Y101/01044C12Y101/01003C12Y104/01016A23K20/142
Inventor 康春涛朱枝群陈胜玲张晓霞
Owner JIANGSU XINGHAI BIOLOGICAL TECH
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