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Recombinant streptomyces ansochromogenes, preparation method thereof and use thereof

A technology of Streptomyces chromogenes and recombinant plasmids, applied in biochemical equipment and methods, recombinant DNA technology, microbial-based methods, etc., can solve problems such as difficult to obtain full-length gene clusters, and achieve increased yield and increased yield Effect

Inactive Publication Date: 2011-02-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene cluster of nikkomycin biosynthesis is larger than 35kb, and it is difficult to obtain the full-length gene cluster by general molecular biology methods

Method used

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  • Recombinant streptomyces ansochromogenes, preparation method thereof and use thereof
  • Recombinant streptomyces ansochromogenes, preparation method thereof and use thereof
  • Recombinant streptomyces ansochromogenes, preparation method thereof and use thereof

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Embodiment 1

[0031] Embodiment 1, the construction of the recombinant plasmid pNIK containing nikkomycin biosynthesis gene cluster

[0032] 1. Preparation of recombinant cosmids containing nikkomycin biosynthesis gene cluster fragments

[0033] Streptomyces chromogenes 7100CGMCC 4.321 (purchased from the General Microorganism Center of China Microbiological Culture Collection Management Committee), its spores are in the shape of silk hooks, or coils, and rarely coils; the spores are spherical or oval.

[0034] Genomic DNA was extracted from Streptomyces chromogenes 7100CGMCC 4.321, digested with Sau3AI, and separated by pulse electrophoresis to recover a 40-50kb DNA fragment. Phosphorylated and recovered DNA fragments with T 4 DNA ligase linking, transforming Escherichia coli XL 1-blue MR (Stratagene) to obtain a recombinant cosmid library containing a fragment of the nikkomycin biosynthetic gene cluster, using a partial fragment of the sanF gene (500bp, sequence 1) to digoxin Positive c...

Embodiment 2

[0043] Embodiment 2, the construction of nikkomycin engineering bacteria

[0044] 1. The acquisition of the recombinant Streptomyces ansochromogenes (Streptomyces ansochromogenes) transferred to the recombinant plasmid pNIK

[0045] The recombinant plasmid pNIK containing the entire nikkomycin biosynthetic gene cluster can be used to increase the copy number of the nikkomycin biosynthetic gene cluster on the Streptomyces chromogenes genome, since the recombinant plasmid pNIK can be integrated in the chromogenic The Streptomyces genome replicates along with the chromosome, so the nikkomycin high-yielding engineered bacteria constructed with this plasmid are genetically stable. The specific method is as follows:

[0046] The recombinant plasmid pNIK was transferred into E.coli ET12567 (John Innes Institute, Norwich, UK) to remove the restriction modification, and apramycin was used as a selection marker, and the recombinant plasmid pNIK was transferred into the Streptomyces chr...

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Abstract

The invention discloses recombinant streptomyces ansochromogenes, a preparation method thereof and use thereof. The recombinant streptomyces ansochromogenes are recombinant bacteria obtained by introducing nikkomycin biologic synthesis gene clusters into the streptomyces ansochromogenes, wherein the nikkomycin biologic synthesis gene clusters can be introduced into the streptomyces ansochromogenes by recombinant plasmids pNIK. Compared with the streptomyces ansochromogene strain 7100 CGMCC4.321, the recombinant streptomyces ansochromogenes of the invention can improve the nikkomycin Z yield by about 1.8 times and the nikkomycin X yield by about 3 times. The recombinant streptomyces ansochromogenes obviously improve the yield of the nikkomycins and provides necessary technical preparation for the large-scale production of the nikkomycins.

Description

technical field [0001] The invention relates to a recombinant Streptomyces chromogenes and its preparation method and application. Background technique [0002] Nikkomycin belongs to nucleoside peptide antibiotics, which is similar in structure to UDP-N-acetylglucosamine, the natural substrate of chitin synthase, and is a strong competitive inhibitor of chitin synthase. Since chitin is an important part of fungal cell walls, insects and other invertebrate exoskeletons, nikkomycin can effectively inhibit fungi, insects and mites, and is non-toxic or extremely toxic to mammals, bees and plants Low, and it is easy to degrade in nature, so it is an ideal agricultural antibiotic. Field experiments have proved that nikkomycin has a good control effect on many plant diseases, such as wheat powdery mildew, tobacco red spot, cucumber sclerotinia, tomato leaf mold, apple ring spot and so on. At the same time, studies have shown that nikkomycin can effectively inhibit the deep infect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/76C12P21/02C12R1/465
Inventor 谭华荣廖国建李金娥
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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