Streptomyces thioluteus antibiotic regulation gene and method for increasing yield of streptomyces antibiotic

A technology for regulating genes of Streptomyces thioluteus luteus, applied in the field of genetic engineering, can solve problems such as lack of research reports on functions and mechanisms of action, and achieve the effect of broad application prospects

Inactive Publication Date: 2013-09-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bioinformatics comparative analysis shows that ORF4 is a "possible regulatory gene", and there is no research report on its function and mechanism of action in the existing data, and it is a brand new gene

Method used

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  • Streptomyces thioluteus antibiotic regulation gene and method for increasing yield of streptomyces antibiotic
  • Streptomyces thioluteus antibiotic regulation gene and method for increasing yield of streptomyces antibiotic
  • Streptomyces thioluteus antibiotic regulation gene and method for increasing yield of streptomyces antibiotic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Method of using ORF4 gene to increase the production of actinomyces purpurin and undecyl prodigiosin in Streptomyces lividans

[0026] 1) Construction of recombinant plasmid pZMY9-3 containing ORF4 gene

[0027] Streptomyces sulpholigue can produce a variety of natural active substances. Our laboratory has conducted in-depth research on this strain, performed genome scanning and constructed a genome library, from which cosmid9-3 is derived. Sau3AI was used to partially digest cosmid9-3, and a fragment of about 4kb was recovered and connected to pJTU2554 (Li L. The mildiomycin biosynthesis: initial st eps for sequential generation of 5-hydroxymethylcytidine 5'-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119.2008 , Chembiochem, 9:1286-1294), by transforming into Streptomyces lividans ZX1, selecting a recombinant plasmid that can make Streptomyces lividans ZX1 actinorubicin high-yield and sequencing, found that there is an unknown...

Embodiment 2

[0077] Example 2: Method of using ORF4 gene to increase the production of olive leaf element in Streptomyces hygroscopicus

[0078] 1) Construction of recombinant plasmid pZMY9-3 containing ORF4 gene (same as Example 1)

[0079] 2) The recombinant plasmid pZMY9-3 was introduced into Streptomyces hygroscopicus ATCC29253 by conjugative transfer (same as Example 1)

[0080] Obtained Streptomyces hygroscopicus ZMY2 containing ORF4, and at the same time introduced unloaded pMSB-WS052 named ATCC29253-CK as a control.

[0081] 3) Determination of antibiotics in ZMY2

[0082] ZMY2 and the control ATCC29253 were cultured for 7 days with olive leaf element fermentation medium, 250 rpm, 30°C. After centrifugation, the supernatant was removed, the mycelium was extracted with acetone, evaporated to dryness under reduced pressure and dissolved in methanol, and the biological activity was determined using Bacillus subtilis (see below for the method). Compared with the control, the antibacterial acti...

Embodiment 3

[0089] Example 3: Using ORF4 gene to increase the production of cephanomycin C in Streptomyces coryneformis

[0090] 1) Construction of recombinant plasmid pZMY9-3 containing ORF4 gene (same as Example 1)

[0091] 2) The recombinant plasmid pZMY9-3 was introduced into Streptomyces coryneformis NRRL3585 by conjugative transfer (same as Example 1)

[0092] Obtained Streptomyces hygroscopicus ZMY3 containing ORF4, and at the same time introduced unloaded pMSB-WS052 named NRRL3585-CK as a control. .

[0093] 3) Determination of antibiotics in ZMY3

[0094] ZMY3 and the control wild-type strain NRRL3585 were cultured using cephalosporin C fermentation medium, and Bacillus subtilis was used for biological activity determination (see the method below). Compared with the control wild-type strain NRRL3585, ZMY3 has significantly improved antibacterial activity ( Picture 11 ). At the same time, the fermentation broth was analyzed by HPLC, and a peak with a maximum absorption wavelength of 263...

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Abstract

The invention discloses a streptomyces thioluteus antibiotic regulation gene ORF4, wherein the nucleotide sequence is shown by SEQ ID No:1, or the nucleotide sequence codes the same protein with the nucleotide shown by the SEQ ID No:1. The invention also discloses an application of the gene in preparing an antibiotic high-yield streptomyces gene mutant strain and a method for increasing the yield of the streptomyces antibiotic. According to the gene and method disclosed by the invention, by adopting the streptomyces thioluteus antibiotic regulation gene ORF4, the problems of high workload and high blindness of traditional streptomyces breeding are solved, the function is realized in multiple antibiotic producing bacteria, the antibiotic high-yield strain bred by genetic engineering is obtained, and the application prospect is broad.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a Streptomyces sulphureus antibiotic regulation gene, its use and a method for improving the production of Streptomyces antibiotics. Background technique [0002] Streptomyces is a highly commercially valuable actinomycete. Among the discovered microbial-derived antibiotics, about 2 / 3 are produced by actinomycetes, of which about 80% are produced by streptomyces (Kieser et al., 2000, Practical Streptomyces Genetics. The John Innes Foundation, Norwich) , They play an important role in anti-bacterial, anti-insect, anti-tumor, anti-fungal and other aspects. [0003] Because of the commercial value of streptomyces, methods to increase the production of streptomyces antibiotics are very important. These methods include two types: one is to mutagenize streptomyces by physical and chemical methods and to select high-yielding strains from mutant strains; the other is thr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12N15/63C12R1/465C12R1/55
Inventor 何璟朱梦奕赵瑞芳王利娟
Owner HUAZHONG AGRI UNIV
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