Antibiotic Overproduction Of Streptomyces Diastatochromogenes 1628 By Ribosome Engineering

Streptomyces diastatochromogenes 1628,isolated from Tianmu Mountain in Zhejiang Province,has a high antibacterial activity against various plant pathogenic fungi,and can biosynthesize at least 4 kinds of antibiotics,including toyocamycin,tetramycin A,tetrin B and tetramycin P.However,the yield of wild-type strain is very low.In this study,the ribosome engineering method was applied to increase the yield of S.diastatochromogenes 1628.The results are as follows:1.A triple mutant strain T3-145,obtained by screening for resistance to streptomycin and paromomycin,can produce large amounts(1013 mg/L)of the toyocamycin.The productivity of toyocamycin is 17-fold higher than that of the wild type.The results of gene sequencing showed that the rsmG gene in T3-145 has a nonsense mutation,the 48th codon encoding the cysteine becomes the stop codon.But the mutation on rpsL gene was not detected.2.A pentuple mutant strain G5-59,obtained by screening for resistance to streptomycin,rifampicin and paromomycin,can produce huge amounts of tetramycin P,tetrin B and tetramycin A(3131,1033,768.9 mg/L,respectively),which were 40-,29-,13-fold higher than the level produced by the wild type,respectively.The results of gene sequencing showed that the rpsL gene in G5-59 has a point mutation,the 42th codon encodes an amino acid from proline to serine;the rsmG gene undergoes a point mutation too,and the 437 codon-encoded amino acid changes from alanine to valine;the rpoB gene also undergoes a point mutation,the 424 codon encodes the amino acid from glutamic acid to leucine.3.To screen out the most stably expressed endogenous reference genes in different drug resistance overproduction mutants of S.diastatochromogenes 1628,six candidate reference genes were selected for real-time quantitative PCR,and two kinds of software were adopted to analyze the stability.The results revealed that the optimum reference gene analyzed by different software was slightly different,and rpoA is the most suitable reference gene in all tested strains.4.The growth characteristics of the high-yielding mutants were studied in a 5L fermentor.The results showed that the mutants grow slowly.Under the condition of fermentation tank,the yield of secondary metabolites of each mutant is changed obviously.The yield of tetramycin P is decreased,while the yield of tetrin B and tetramycin A are improved,compared to the flask-fermentation.In the mutant strains,the expression levels of toyocamycin biosynthesis regulatory gene toyA and tetramycin A biosynthesis gene tetRI impro-ve significantly than those of the wild type strains,but the difference among the mutant strains is not significant,suggesting that there are other mechanisms that affect the yield of mutants.The results of this study provided new idea and method for the breeding of S.diastatochromogenes 1628.Introduction of combined drug resistance to 1628 by ribosome engineering can effectively enhance its productivity of secondary metabolites,and the method is more economical and fast comparing to traditional breeding methods.Furthermore,the results of mutant points mutations and expression of high-yield mutant genes also provide a theoretical basis for the study of mechanism about ribosome engineering.