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Method for improving yield of avermectin and producing strain

A technology of abamectin and strains, applied in the field of microbial genetic engineering and fermentation engineering, can solve the problems of low fermentation unit and high production cost, and achieve the effects of increasing fermentation unit, increasing yield and reducing production cost

Active Publication Date: 2018-11-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the production strains of avermectin in my country still have problems such as low fermentation unit and high production cost.

Method used

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  • Method for improving yield of avermectin and producing strain
  • Method for improving yield of avermectin and producing strain
  • Method for improving yield of avermectin and producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The construction of embodiment 1mce gene overexpression vector

[0027] 1. Cloning of Streptomyces avermitilis mce gene

[0028] According to the mce gene [NC_003155.5 (3499939..3500430)] in the Streptomyces avermitilis wild-type strain ATCC 31267 that Genbank announces, the sequence design PCR primer of erythromycin resistance gene ermE*p promoter and ORF13p promoter:

[0029] Primer mce_1: 5′-gctctagagtgatcgcgcgcacgttg-3′

[0030] Primer mce_2: 5′-cgggatcccaaccctaccggcccata-3′

[0031] Primer erme*p_1: 5′-cggaattcgtgtccgttcgagtggcg-3′

[0032] Primer erme*p_2: 5′-gctctagagctcaccgctggatcctac-3′

[0033] Primer orf13p_1: 5′-cggaattctgtacggcgctgattact-3′

[0034] Primer orf13p_2: 5′-gctctagaggtgacagacatggaggtctc-3′

[0035]Both ends of primers mce_1 and mce_2 were respectively introduced with XbaI / BamHI restriction sites (underlined part), and both ends of ermE*p_1, ermE*p_2 and ORF13p_1 and ORF13p_2 were respectively introduced with EcoRI / XbaI restriction sites (un...

Embodiment 2

[0038] Transformation of embodiment 2 recombinant plasmids

[0039] The mce overexpression plasmids pKC-ermE-mce and pKC-ORF13-mce were constructed according to Example 1, and the original plasmid used as a control was pKC1139.

[0040] In this embodiment, the wild-type strain of Streptomyces avermitilis ATCC31267 and the high-yielding strain of abamectin 76-02-e were respectively selected as starting strains. The protoplasts of these two S. avermitilis strains were prepared, and the mce overexpression recombinant plasmids pKC-ermE-mce and pKC-ORF13-mce extracted from Escherichia coli were transformed into the wild-type strain and the avermectin high-yielding strain respectively76 The protoplasts of -02-e were spread on the protoplast regeneration medium RM14 plate respectively, and cultured at 28°C for 16-20h. When fog appeared on the plate, apply 1mL aqueous solution containing 1000μg apramycin on the plate. , continue culturing at 28°C for 7-10 days, and the grown colonies...

Embodiment 3

[0042] Embodiment 3 In Streptomyces avermitilis wild-type bacterial strain ATCC31267, the impact of overexpressing mce on the synthesis of abamectin

[0043] 1. Shake flask fermentation of Streptomyces avermitilis

[0044] Seed medium: 30g soluble starch, 2g malt extract, 2g soybean peptone, CoCl 2 ·6H 2 O 5mg, add distilled water to 1L, adjust pH to 7.0-7.2.

[0045] Fermentation medium: 50g soluble starch, 12g yeast powder, MgSO 4 ·7H 2 O 0.5g, K 2 HPO 4 ·3H 2 O 0.5g, KCl 4g, CaCO 3 2g, CoCl 2 ·6H 2 O 5mg, add distilled water to 1L, adjust pH to 7.0-7.2.

[0046] 2. HPLC analysis of fermentation products

[0047] Abamectin extraction: take 1.0mL fermentation broth, add 4.0mL methanol, soak for more than 30 minutes, oscillate once every 10 minutes, centrifuge at 12000rpm for 5 minutes, centrifuge twice, take supernatant and inject for analysis.

[0048] HPLC analysis conditions: C 18 Reversed-phase column, column length 150mm, column inner diameter 4.6mm, column...

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PUM

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Abstract

The invention provides a method for improving the yield of avermectin and a producing strain. The method is to construct an overexpressed genetically engineered strain of a gene mce (SAV2857) encodedby a methylmalonyl-CoA epimerase in streptomyces avermitilis. The genetically engineered bacterium obtained by the invention can be directly used for the fermentation production of the avermectin; thefermentation unit of the avermectin is improved, the yield of avermectin B1a is increased by about 25% compared with that of an original strain, and the production cost is lowered; and the genetically engineered bacterium has important industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering and fermentation engineering, and in particular relates to a recombinant bacterial strain constructed by a genetic engineering method and the application of the bacterial strain in improving the production of abamectin. Background technique [0002] Avermectins are a class of sixteen-membered ring macrolide antibiotics produced by Streptomyces avermitilis, which can be used to kill arthropod parasites and nematodes, with high efficiency and low toxicity , No residue and other advantages, it is widely used in agriculture, medicine and animal husbandry and other fields. Abamectin is composed of eight components (A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b), among which B1 component has the strongest insecticidal activity, and commercialized abamectin is B1 component . Ivermectin (ivermectin) is made from avermectin B1 by hydrogenation reduction at C22-C23 position. It has the ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P19/62C12R1/465
CPCC12N9/90C12N15/76C12P19/623C12Y504/99002
Inventor 陈芝刘兰杰文莹李季伦
Owner CHINA AGRI UNIV
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