Method for high-yield bacterial strains by high-flux screening of gentamycin based on ARTP composite mutagenesis technique

Abstract

The invention relates to a method for high-yield bacterial strains by high-flux screening of gentamycin based on an ARTP composite mutagenesis technique. The method comprises the steps of preparing aspore suspension, performing ARTP mutagenesis composite mutagenesis, performing quick detection on products, prescreening high-yield bacterial strains with gentamycin and performing screening once again to obtain high-yield bacterial strains and performing stability inspection on the high-yield bacterial strains. Compared with the prior art, the method has the advantages that the principle that OPA and the gentamycin are subjected to a reaction to form characteristic absorption peaks of about 330nm is adopted, through a microplate reader, quick determination of an OD value is realized, so thatfast detection of the gentamycin can be successfully realized. The interference problems that poor specificity of a phosphotungstic acid method and a method for forming precipitate with the gentamycin to cause reduction of the OD value, fermentation liquid color and the like, are solved. The method has the characteristics of being quick, high-flux, convenient to operate, accurate and the like, and the mutation breeding and screening efficiency of the gentamycin is improved to a great extent, so that the probability for obtaining the high-yield bacterial strain with the gentamycin is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of microorganism culture, and in particular relates to a method for high-throughput screening of high-yielding gentamicin strains based on ARTP compound mutagenesis technology. Background technique [0002] Gentamicin is an aminoglycoside antibiotic produced by fermentation and extraction of micromonas (micromonas crimson or micromonas aculeatus), which can inhibit the biosynthesis of bacterial proteins, thereby inhibiting Bactericidal effect, has a strong bactericidal effect on Gram-negative bacteria and some Gram-positive bacteria. Gentamicin is the main aminoglycoside antibiotic, and its market accounts for 50% of aminoglycoside antibiotics. Therefore, it is of great significance for practical application to develop high-throughput screening mutagenesis for gentamicin industrial production strains to obtain high-yielding strains. [0003] Most of the strains of Micromonas crimson rubrum or Micromonas ac...

AI Technical Summary

Problems solved by technology

[0003] Most of the strains of Micromonas crimson rubrum or Micromonas aculeatus that produce gentamicin in actual enterprises are obtained by traditional screening methods, such as shaking flask culture, liquid phase or biological method to detect titer, which has high investment and long screening period. The screening throughput is small, the screening method is single, and the screening efficiency is low, so the increase in the production capacity of gentamicin is relatively low

There is no complete set of methods for large-scale high-throughput mutagenesis screening of gentamicin-producing strains in the prior art

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