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Spinosad high-yield genetically engineered bacteria and its construction method and application

A technology of genetically engineered bacteria and spinosyn, applied in the field of genetic engineering, can solve the problems of long cycle, heavy screening workload, and small yield fluctuation, and achieve the effects of increasing the yield and utilization of spinosyn

Active Publication Date: 2020-03-31
ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, traditional physical and chemical mutagenesis methods are basically used to increase the fermentation yield of spinosyns. This random mutation has a certain degree of blindness, and the screening workload is large, the cycle is long, and the yield does not fluctuate much after multiple mutagenesis.

Method used

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  • Spinosad high-yield genetically engineered bacteria and its construction method and application
  • Spinosad high-yield genetically engineered bacteria and its construction method and application
  • Spinosad high-yield genetically engineered bacteria and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of Saccharopolyspora spinosa Fatty Acid Metabolic Pathway Enhanced Strain

[0042] 1. Obtaining the starting strain

[0043] NTG and 60 Co alternate mutagenesis, the mutant strain of Saccharopolyspora spinosa obtained by mutagenesis, that is, the starting strain;

[0044] 2. PCR amplification of the coding gene fadD1 and fadE fragments of Streptomyces coelicolor acyl-CoA synthetase and acyl-CoA dehydrogenase

[0045] 2.1 Primer design

[0046] According to the DNA sequences of the coding regions of acyl-CoA synthetase (fadD1, GeneID: 1101636) and acyl-CoA dehydrogenase (fadE, GeneID: 1098484) involved in fatty acid metabolism in Streptomyces coelicolor, two pairs of primers were designed for respectively Amplify it. In the process of primer synthesis, the promoter kasOp* and RBS sequence were introduced into the upstream of the fadD1 fragment, and at the same time, the same RBS sequence was introduced upstream of the fadE coding frame fragment...

Embodiment 2

[0069] Example 2 Comparison of Shake Flask Fermentation of Fatty Acid Metabolic Pathway Enhanced Strains (Engineering Strains) and Starting Strains

[0070] 1. Shake flask fermentation of starting strains and engineering strains

[0071]Spread the Saccharopolyspora spinosa starting strain and the engineering strain spores on the solid medium (glucose 4g, yeast extract 10g, malt extract 10g, calcium carbonate 2g, agar 15g, deionized water supplemented to 1 liter). After culturing at 28°C for 168h, the spores were collected by hanging with 30% sterile glycerol to prepare a spore suspension, which was stored at -80°C for subsequent experiments.

[0072] Saccharopolyspora spinosa seed culture: Inoculate the Saccharopolyspora spinosa spore suspension at 1% in a 250mL Erlenmeyer flask with 30mL seed medium, and cultivate it for 45-48h at 28°C and 240rpm to obtain Saccharomyces spinosa Spore seed liquid. Among them, the seed medium is: 4g yeast extract, 4g peptone, 4g casein hydrol...

Embodiment 3

[0081] Example 3 The enhancement of the fatty acid metabolic pathway causes the change of the oxygen stress condition and then affects the production of spinosad

[0082] 1. During the fermentation process of engineering bacteria and starting bacteria, H 2 o 2 production testing

[0083] Reduced flavin dinucleotide (FADH) produced during β-oxidation of saturated and unsaturated fatty acids 2 ) amount is different, at the same time, fatty acyl-coenzyme dehydrogenase (FadE) and oxygen transfer flavoprotein are the organisms to form endogenous hydrogen peroxide (H 2 o 2 ) factor, while H 2 o 2 It also stimulates the secondary metabolism of actinomycetes. Therefore, for the H of the S. spinosa fermentation process 2 o 2 to test.

[0084] Such as Figure 4 As shown, in the case of no oil added, the H measured in the starting strain and the engineering strain 2 o 2 No difference, in case of added grease, H 2 o 2 Yield increased significantly. H 2 o 2 The amount was ...

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Abstract

The invention discloses a spinosad high-yield gene engineering bacterium. The preservation number of the gene engineering bacterium is CGMCC No. 14193. The invention further discloses a method by enhancing a fatty acid metabolism pathway to construct the spinosad high-yield gene engineering bacterium and application of the engineering bacterium to produce spinosad. According to the construction method, by conducting expression enhancement on the oxidation pathway of fatty acid Beta of saccharopolyspora spinosa, supply for the spinosad to biosynthesize the important precursor of acetyl coenzyme A is improved, and compared with original strains, the yield of the spinosad of obtained gene engineering strains is improved obviously.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, it relates to a genetic engineering bacterium with high spinosad production and its construction method and application. Background technique [0002] Spinosyn (spinosad) is an insecticide with a macrocyclic lactone structure produced by the aerobic fermentation of the actinomycete Saccharopolyspora spinosa, which can effectively control a variety of Lepidoptera pests. The mechanism of action on pests is to stimulate the nervous system, resulting in non-functional muscle contraction, failure, accompanied by tremors and paralysis, resulting in insect death. Spinosyn has both the safety of biological pesticides and the quick-acting properties of chemically synthesized pesticides, and has low toxicity, low residue, safety to insect natural enemies, fast natural decomposition, and no cross-resistance with other pesticides. It is widely used in agriculture and animal husbandry I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/62C12N15/01C12R1/01
CPCC12N9/001C12N9/1029C12N15/01C12P19/62C12Y103/01008C12Y203/01086
Inventor 赵晨黄颖张晓琳王超印铁
Owner ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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