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A kind of recombinant bacterium, its construction method and the method for improving avermectin output

A construction method and a technology of recombinant bacteria, applied in the biological field, can solve the problems of low yield of abamectin production strains, and achieve the effects of increasing synthesis, increasing yield, and improving supply of reducing power

Active Publication Date: 2018-11-23
内蒙古新威远生物化工有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The invention provides a recombinant bacterium, its construction method and a method for increasing the production of abamectin. The sucD1 gene of the succinyl-CoA synthetase α subunit in Streptomyces avermitilis is knocked out to obtain a high-yield avermectin. Recombinant bacteria, through the fermentation of obtained recombinant bacteria to produce abamectin, which solves the problem of low yield of existing abamectin production strains

Method used

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  • A kind of recombinant bacterium, its construction method and the method for improving avermectin output
  • A kind of recombinant bacterium, its construction method and the method for improving avermectin output
  • A kind of recombinant bacterium, its construction method and the method for improving avermectin output

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Embodiment 1

[0032] Embodiment 1, construction of sucD1 gene knockout vector

[0033] 1. Amplification of sucD1 gene upstream and downstream fragment sequences sucD1 L-arm and sucD1 R-arm

[0034] Design primers for amplifying the sucD1 gene upstream fragment (as shown in the sequence table SEQ ID NO: 1) and the downstream fragment (as shown in the sequence table SEQ ID NO: 2) located on the chromosome of Streptomyces avermitilis MA4680 (ATCC31267) sucD1 L-arm upstream primer sucD1 L-F (5'-AGGTCGACGGTATCGATAAGCTTGCGTCCCAGTCGGTGTAGTTC-3'), downstream primer sucD1 L-R (5'-GTTCCTATTCCGAAGTTCCTATTCGCGTCATGCCGTGTCACTACCTTC-3'), the amplified product should be 941bp.

[0035] sucD1 R-arm upstream primer sucD 1 R-F (5'-ACTTCGAAGTTCCTATAACTTGTCGGGCTCCTTCAGCCGGC-3'), downstream primer sucD1-R-R (5'-TCCCCCGGGCTGCAGGAATTCTGTACGAACACCAGGCAAGGGAA-3'), the amplified product should be 1135bp sequencing verification.

[0036] Using the total DNA of Streptomyces avermitilis MA4680 (ATCC31267), a high-yiel...

Embodiment 2

[0042] Embodiment 2, transformation of recombinant plasmid

[0043] Due to the strong restriction modification in Streptomyces avermitilis, the transformation efficiency is extremely low when E.coli DH5α is directly used for conjugative transfer with Streptomyces avermitilis, sometimes even no transformant can be obtained. However, using E.coliET12567 (PUZ8002) without restriction modification for conjugative transfer, the transformation efficiency was significantly improved. Therefore, the constructed recombinant plasmid was transformed into E.coliET12567 (pUZ8002) (Kieser T, Bibb M J, Buttner M J, et al. Practical Streptomyces Genetics, 2000, Norwich: The John Innes Foundation.) (within twenty years from the date of application The public can obtain unmethylated DNA from the Institute of Microbiology, Chinese Academy of Sciences, and then carry out conjugative transfer.

[0044] In this example, select Streptomyces avermitilis MA4680 (ATCC31267), (the public can obtain from...

Embodiment 3

[0047] Embodiment 3, the fermentation research of transformant

[0048] 1. Shake flask fermentation of Streptomyces avermitilis

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Abstract

The invention relates to recombinant strain, a construction method thereof, and a method for improving yield of abamectin. The recombinant strain is obtained by knocking off alpha-subunit sucD1 genes from succinyl-coa synthase in streptomyces avermitilis. By knocking off the alpha-subunit sucD1 genes from succinyl-coa synthase in streptomyces avermitilis, succinyl-coa synthase in streptomyces avermitilis is lost, then the metabolic pathway of tricarboxylic acid is weakened, abundant acetyl CoA flows to the synthetic route of the abamectin precursor, then the flow of the pentose phosphate of the pathway metabolic pathway is indirectly increased, the reducing power supply is improved, the synthesis of the abamectin precursor is increased, and the yield of abamectin is increased.

Description

technical field [0001] The present invention generally relates to the field of biotechnology, in particular to a recombinant bacterium, its construction method and a method for improving the production of abamectin. Background technique [0002] Avermectin is a class of macrolide antibiotics fermented by Streptomyces avermitilis, which has a wide range of anthelmintic and insecticidal activities. Streptomyces avermitilis fermented liquid usually contains 8 kinds of abamectin components A1a, A1b, A2a, A2b, B1a, B1b, B2a, B2b, among them, the insecticidal effect is best with B1 component, especially B1a component . Abamectin has a unique mechanism of action, which is different from conventional chemical pesticides. The insecticidal effect is dozens of times that of general chemical pesticides, and it is not easy to make pests develop resistance. The target is the neurotransmitter γ-aminobutyric acid of nematodes and arthropod parasites. It has low toxicity to mammals, high s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/76C12P19/62C12R1/465
Inventor 张立新帅辉刘梅张庆杨军强高鹤永
Owner 内蒙古新威远生物化工有限公司
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