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Gene engineering high-yield strain streptomyces diastatochromogenes, method for increasing yield of epsilon-polylysine and application of streptomyces diastatochromogenes

A technology of Streptomyces chromogenes and polylysine, applied in the direction of genetic engineering, microorganism-based methods, applications, etc., can solve the problems of improving ε-polylysine, achieve production, and enhance diaminopimelic acid The effect of the pathway

Active Publication Date: 2020-09-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on improving the production of ε-polylysine by genetic engineering, especially by overexpressing the aspartokinase gene ask and the dihydrodipicolinate synthase gene dhdps

Method used

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  • Gene engineering high-yield strain streptomyces diastatochromogenes, method for increasing yield of epsilon-polylysine and application of streptomyces diastatochromogenes
  • Gene engineering high-yield strain streptomyces diastatochromogenes, method for increasing yield of epsilon-polylysine and application of streptomyces diastatochromogenes
  • Gene engineering high-yield strain streptomyces diastatochromogenes, method for increasing yield of epsilon-polylysine and application of streptomyces diastatochromogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] A genetically engineered high-yield amylase strain Streptomyces chromogenes, the construction steps are as follows:

[0127] 1) Acquisition of aspartokinase gene (ask):

[0128] Using the amylase Streptomyces chromogenes genomic DNA as a template, use the primers ask-F / ask-R to amplify the full length of ask by PCR, and add an XbaI restriction site at the 5' end of the ask, and an EcoRI restriction site at the 3' end point. The gene size is 1272bp.

[0129] Where: ask-F:5'-GC TCTAGA GTGGGCCTTGTCGTGCAGAAGTACGG-3' horizontal line is the XbaI restriction site, and the boldface is the start codon;

[0130] ask-R: 5'-CCG GAATTC TCATCGCCCGGTGCCGCCGATG-3' horizontal line is the EcoRI restriction site, bold is the stop codon.

[0131] PCR reaction system and conditions: 5×PrimeSTAR GXLBuffer 10μL, dNTP mixture (2.5mM) 4μL, template (20ng / ul) 1μL, upstream and downstream primers ((10μM)) 2μL each, DMSO 2μL, PrimeSTAR GXLDNAPolymerase 1μL, supplemented with ultrapure wate...

Embodiment 2

[0140] A genetically engineered high-yield amylase strain Streptomyces chromogenes, the construction steps are as follows:

[0141] 1) Acquisition of dihydrodipicolinate synthase gene (dhdps):

[0142] Using amylase Streptomyces chromogenes genomic DNA as a template, use primers dhdps-F / dhdps-R to amplify the full length of dhdps by PCR, and add XbaI restriction site at the 5' end of dhdps, and EcoRI restriction enzyme at the 3' end location. The gene size is 897bp.

[0143] Where: dhdps-F:5’-GGC TCTAGA ATGGTTGATCGCACCCCCCTGGAGG-3' horizontal line is the XbaI restriction site, and the boldface is the start codon;

[0144] dhdps-R: 5'-CCG GAATTC The horizontal line at TCAGCCCAGGGCTGCCAGGTGC-3' is the EcoRI restriction site, and the stop codon is in bold.

[0145] PCR reaction system and conditions: 5×PrimeSTAR GXLBuffer 10μL, dNTP mixture (2.5mM) 4μL, template (20ng / ul) 1μL, upstream and downstream primers ((10μM)) 2μL each, DMSO 2μL, PrimeSTAR GXL DNAPolymerase 1μL, su...

Embodiment 3

[0154] Verification of genetically engineered strains:

[0155] The genomic DNA of the genetically engineered Streptomyces was extracted, the primers aac-F / aac-R were designed and verified, and the apramycin resistance gene aac(3)IV was amplified with the genomic DNA of the genetically engineered Streptomyces as a template. The original strain genomic DNA was used as a negative control, and the plasmid pIMEP was used as a positive control. The results are shown in image 3 .

[0156] Among them, SEQ No.7aac-F: 5'-GTGCAATACGAATGGCGAAAAG-3'

[0157] SEQ No.8aac-R: 5'-TCAGCCAATCGACTGGCG-3'

[0158] PCR reaction system and conditions: 10X PCR Buffer 5μL, dNTPmixture (2.5mM) 4μL, template (20ng / ul) 1μL, upstream and downstream primers ((10μM)) 2μL, DMSO 2μL, TaKaRa Taq 1μL, add ultrapure water to 50 μL. PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 60 s, 30 cycles in total, extension...

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Abstract

The invention relates to a gene engineering strain streptomyces diastatochromogenes for high yielding of epsilon-polylysine. The construction of the gene engineering strain comprises the following steps of: step 1, constructing plasmids for expressing the aspartokinase gene ask or the dihydropyridine dicarboxylic acid synthetase gene dhdps, wherein the gene ask or dhdps is controlled by an erythromycin promoter ermE * on pIMEP plasmids; and step 2, obtaining a strain for expressing the ask or dhdps gene, namely the gene engineering strain streptomyces diastatochromogenes for high yielding of epsilon-polylysine. Experiments prove that the epsilon-polylysine production capacity of the streptomyces gene engineering strain is remarkably improved by 17.2%-25.8% compared with that of the original strain streptomyces diastatochromogenes TUST under the same condition, and an excellent strain is provided for epsilon-polylysine production.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to a gene engineering high-yield strain Streptomyces chromogenes and a method for increasing the yield of ε-polylysine and its application. Background technique [0002] ε-polylysine is one of the two natural amino acid homopolymers discovered so far (the other is γ-polyglutamic acid). After the discovery of the first strain of ε-polylysine-producing bacteria, ε-polylysine was obtained through soil screening. -The polylysine-producing bacteria all belong to the genera Streptomyces, Streptoverticillum, Kitasatospora, and Cinnamomum . The distribution of ε-polylysine producers was mainly restricted to the filamentous bacteria Streptomycesaceae and ergot fungi. ε-polylysine has a broad antibacterial spectrum, and has inhibitory effects on Gram-positive bacteria, Gram-negative bacteria, fungi and some viruses. It has good thermal stability and can be directly added to food for pr...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N15/66C12N15/54C12N15/60C12N1/21C12P13/02C12R1/525
CPCC12N15/76C12N15/66C12N9/1217C12N9/88C12P13/02C12Y207/02004C12Y402/01Y02A50/30
Inventor 谭之磊魏希庆贾士儒侯颖崔建东王贺丽王鑫宇闫佳佳
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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