Construction method for efficient biosynthesis of streptomycete drugs

A biosynthesis and construction method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low fermentation yield and difficult industrial production, and achieve overcoming side effects, convenient operation, and overcoming difficulties in preparation or The effect of purchase

Active Publication Date: 2015-11-04
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

The bioeconomics principle of microorganisms always limits the excessive synthesis of each secondary metabolite, especially the PKS pathway of wild bact

Method used

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  • Construction method for efficient biosynthesis of streptomycete drugs
  • Construction method for efficient biosynthesis of streptomycete drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of Gradient Induced Expression Plasmids

[0043] The three DNA sequences of SEQ ID NO: 1, 2 and 4 were amplified by polymerase chain reaction (PCR), and the amplified DNA fragments of NO 1 were digested with DNA restriction endonucleases and inserted into plasmid pIJ8630 , forming pIJ8630- O R O lac , plus the plasmid pIJ8630 carried by itself egfp , which just constitutes pIJ8630- O R O lac - egfp . The amplified DNA fragment of SEQ ID NO: 4 was digested with DNA restriction endonuclease and inserted into plasmid pIJ8630- O R O lac - egfp Plasmid pIJ8630- O R O lac -egfp - P L tetO1 - lacI .

[0044] The DNA fragment of NO 2 amplified by PCR was digested with DNA restriction endonuclease and inserted into plasmid pIJ8630 to form pIJ8630- ermEp* , plus the plasmid pIJ8630 carried by itself egfp , which just constitutes pIJ8630- ermEp* - egfp .

[0045] The four DNA sequences of SEQ ID NO: 5-8 were respectivel...

Embodiment 2

[0047] Example 2 Obtaining of Streptomyces chattanooga Recombinant Strain

[0048] Inoculate Escherichia coli ET12567 (already containing plasmid pUZ8002) containing the above recombinant plasmids into 5 ml LB liquid medium (containing corresponding antibiotics), and culture at 37°C until OD 600 0.6, collect the bacteria by centrifugation, wash once with 10 ml LB liquid medium, and resuspend with 0.5 ml 2×YT liquid medium; add 20 μl spores of Streptomyces chattanooga to 0.5 ml 2×YT liquid Resuspend in culture medium, heat shock at 45 °C for 10 min, then cool in ice bath for 2 min; mix the spores of E. μg / ml of nalidixic acid and corresponding antibiotics, continue to cultivate at 30 °C for 4-5 days; culture the Streptomyces transformants obtained in the appeal for 4-5 days on the slant medium containing corresponding antibiotics, collect spores, and use PCR method verify.

Embodiment 3

[0049] Example 3 Induced expression of target genes

[0050] (1) Under sterile conditions, inoculate the inducible strain of Streptomyces chattanooga in the slant medium, and place it in an incubator for cultivation;

[0051] (2) After cultivating on the slant, insert the cultivated spore mass into a triangular flask filled with shaker flask seed culture medium, and cultivate on a shaker for 18-22 hours;

[0052] (3) Transfer the seed solution obtained in step (3) to 4% YEME medium. For the inducible mutant strain, add the inducer IPTG after 12 hours, and continue to shake the flask for 48 hours. The 4% YEME medium is 3.0 g / L of yeast extract, 3.0 g / L of malt extract, 5.0 g / L of peptone, 40 g / L of glucose, the rest is water, and the pH is natural;

[0053] (4) Absorb the Streptomyces bacterium liquid cultivated by an appropriate amount of liquid, collect the thalline by centrifugation, and wash the thalline once with PBS buffer solution;

[0054] (5) Analyze the expression c...

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Abstract

The present invention provides a construction method for efficient biosynthesis of streptomycete drugs. The construction method comprises: based on an inducible promoter O[R]O[lac] and a strong promoter ermEp<*>, firstly constructing a gradient induction expression system in streptomycete; controlling the expression level of target gene through the gradient induction, and carrying out coupled analysis on the relationship between the expression level of the target gene and the yield of the target drug so as to reveal the rate-limiting reaction in the drug biosynthesis path and the suitability of the rate-limiting enzyme; and through a constitutive promoter ermEp<*>, rationally carrying out series coupling on the encoding genes highly expressing the relevant rate-limiting enzyme so as to obtain a strain of the high-yield S Chattanoogensis producing strain L12, and transporting the strain to the China General Microbiological Culture Collection Center to preserve, wherein the preservation number is CGMCC No.10157. According to the present invention, the shake flask fermentation results show that the Natamycin yield in the S Chattanoogensis L12 is 3.3 times the yield in the S Chattanoogensis L10, and the 50 L fermentation tank small test results show that the highest yield achieves 15.7 g/L; and the method of the present invention has characteristics of efficiency, accuracy and simple operation, and is generally suitable for other streptomycetes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a construction method for efficient biosynthesis of Streptomyces drugs, in particular to the establishment of a Streptomyces gradient-induced expression system, which reveals the rate-limiting reactions and rate-limiting enzymes in Streptomyces drug biosynthesis pathways. Compatibility, improving the biosynthesis efficiency of Streptomyces medicaments, so as to increase the yield of medicaments, belongs to the technical field of industrial microorganisms. Background technique [0002] Natamycin (Natamycin), also known as Pimaricin (Pimaricin), natamycin, is a polyene macrolide antibiotic. Streptomyces chattanooga S. chattanogenesis Streptomyces natalis S. natalensis Streptomyces chrysospora S. gilvosporeus Streptomyces lydides S. lydicus and other Streptomyces produced. Natamycin has the molecular formula C 33 h 47 o 13 , with a molecular weight of 665.53, a lactone ring wit...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N15/52C12P19/62C12R1/465
Inventor 李永泉刘水平卜庆廷江辉
Owner ZHEJIANG UNIV
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