Breeding Of Streptomyces Avermitilis Based On ARTP Mutagenesis And Optimization Of Avermectins Fermentation Processes

Avermectin is a sixteen-membered macrolide antibiotic with remarkable beneficial properties.It has been widely utilized in agriculture and animal husbandry as a broad-spectrum insecticide.At present,China is the only and the largest Avermectins producing country in the world.However,the titer of Avermectin B1a is still lower as compared with other antibiotics of the similar class.Therefore,this work is mainly focused on the breeding of Streptomyces avermetilis followed by the optimization of fermentation process.This study has achieved remarkable results.The obtained results from this study have been briefed as below:1.Establishment of strain mutation and high-throughput screening method based on ARTP mutation.Based on the combination of ARTP mutagenesis with L-isoleucine to breed a high component B1a yield strains.The high-throughput culture of 24-deep microtiter plates(MTPs)and high-throughput detection method by 96 well microtiter plate under 245 nm were used.The spore suspension of Streptomyces avermitilis was treated with ARTP for 40 s and combined with 0.1%L-isoleucine,a mutant strain named 1-I/B-4 was successfully obtained,which results in a higher B1a titer.As compared with the starting strain,the B1a titer was increased by 28.22%.After four generations of passage,its Bla concentration was 14.1%higher than the starting strain,reaching 5710 mg/L.2.Optimization of shaking flask fermentation medium and culture conditions of mutant strains.(1)The response surface method(RSM)was used for the optimization of shake flask fermentation medium of the mutant strain.PB test,steepest climbing experiment and central response surface experiment were used to optimize the fermentation medium of mutant strains.The Bla production of the optimized medium was 17.44%higher than that of the original medium.(2)Optimization of shake flask culture conditions for increasing the avermectin titer and its components of the mutant strain.At the shake flask level,the effects of dissolved oxygen,phosphate,nitrogen sources,L-isoleucine and soybean oil on B1a and B1a/B1 were investigated.Adding 0.02-0.06%KH2PO4 to the initial medium is beneficial for mycelial growth,but it can prolongs the time of primary metabolism and inhibits product synthesis.Yeast powder is the best nitrogen source for avermectin fermentation.The analysis of various metabolic parameters found that the nitrogen source maintained a significant effect on the synthesis of avermectin in the middle and late stages of fermentation.Adding 1.0g/L,1.5g/L,2.0g/L L-isoleucine has no promoting effect on avermectin synthesis,and has no significant effect on B1a/B1,but promotes sugar consumption.Moreover,adding soybean oil in secondary metabolism stage promote Bla synthesis and increased with increasing amounts in the range of 0.5-1.5%,but the addition of primary metabolism may inhibit B1a synthesis.3.Optimization of fermentation processes for the production of mutant and control strains.Analysis of the parameters related to metabolic characteristics was performed(by online and offline).The seed transfer time was quickly determined based on online CER,pH and off-line mycelial morphology.The C/N ratio and nutrient structure of seed medium were also optimized;Conclusively,the current study developed a feeding strategy based on CER supplemented yeast powder,which successfully increased the B1a titer by 26.9%,reaching 8697 mg/L.