The Study Of Heavy Ion Beam Breeding And High-throughput Screening Of Pleuromutilin-high-production Strains

Pleuromutilin is a kind of double terpenoid antibiotic from fungal, Clitopiluspasseckerianus. It is an active substance against penicillin-streptomycin-resistantstaphylococci. Pleuromutilin class antibiotics have excellent antibacterial activity and theyare known as a powerful weapon to fight against drug-resistant. The benefits of thepleuromutilin development are obvious; there are many antibiotics of pleuromutilin classlisted in the all study stages. So the demand for the pleuromutilin antibiotics will increase.And the production of pleuromutilin slowed the development speed of pleuromutilin classantibiotics. In this study, pleuromutilin-product strain is improved.The matters must be considered in the breeding of high-production strains are themethod of mutagenicity, the optimization of fermentation, the method of screening. Previousused a variety of mutagenesis methods to breed high-yield strains, but Clitopiluspasseckerianus is a kind of fungi and its ability to repair itself is good, general mutagen is notsuitable for its breeding. To optimize the pleuromutilin fermentation conditions, previousused single-factor test or orthogonal test. The number of trials is large, and the results wereunable to find the best combination of factor levels. The breeding of pleuromutilinhigh-production strain is time-consuming and heavy workload, which limits the developmentof pleuromutilin. The method of quick screening is in urgent need. Then, the methods of thisstudy are:1. Heavy Ion Irradiation breeding pleuromutilin-production strain.Because of the high energy, strong mutagenic effects, and the mutation cannot berepaired, the heavy ion beam generated by HIRFL in Institute of Modern Physics, ChineseAcademy of Sciences energy, is used in the microbial mutagenesis more and more, and theresults are good. In this study, heavy ion irradiation breeding methods is used to breedClitopilus passeckerianus for high-production strains. The optimum dose of ion implantationirradiation is40Gy. And a high-production strain K40-3is obtained. The production of K40-3is16120μg/mL, increased by25.3%.2. Response Surface Method optimization of fermentation conditions of pleuromutilin production strains.The experiment was used response surface method to optimize Clitopiluspasseckerianus fermentation conditions. According to Response Surface Methodology, theoptimal culture conditions is glucose5.0%, soybean oil0.33%, yeast extract0.6%, corn steepliquor3.0%, MgSO₄0.035%CaCO₃0.05%, initial pH6.9, loaded amount35mL/500mLâ–³.The highest production was24750μg/mL. It is increased by53.7%than the original medium.3. The establishment of High-throughput screening method of pleuromutilin highproduction strain.Because of its miniaturization, high efficiency, low-cost, the high-throughput methoddeveloped in recent is very suitable for the screening of high-production strains. This trendwill shorten the breeding time. In this study, a new method of high-throughput screening forthe high-yield pleuromutilin strains was established. A number of pleuromutilin strains to bescreened can be quickly determined with this method, greatly shortens the time needed in thestrain improving process.