Construction And Identification Of Penicillium Chrysogenum Mutant Library

Penicillin is one of the important strategic antibiotics.Penicillium chrysogenum,penicillin producing strain,is an important industrial microorganism which has great industrial value.Current industrial strains are derived from a single natural isolate of P.chrysogenum NRRL 1951.The pennicillin production increased by more than 1000 times.So it is difficult to improve pennicillin production by traditional breeding and fermentaion technology.It is necessary to obtain a pennicillin high-producing strain by modern molecular biology technology.This study is intended to improve the industrial P.chrysogenum strain by genetic engineering technology to increase the yield of penicillin.The wildtype strain in our research is industrial P.chrysogenum.The exogenous bleomycin resistant gene,linked with T-DNA which was synthesized by PCR,was transformed into P.chrysogenum protoplast mediated by PEG.A large number of transformants were screened by antibiotic.The mutants obtained in this way laid the foundation for screening penicillin high-producing mutant strains.To identify if the exogenous gene was transfomed into P.chrysogenum,a simple and high throughput genomic DNA extraction method was developed.The method is based on enzyme lysis combining with heating and ultrasonic treatment.The genomic DNA isolated by the method can be used as PCR template.Two enzymes treatment,6 mg/mL snailase and 4000 U/mL cellulase,is the best way to lyze the mycelia.The mycelia is treated by the following steps,ultrasonic treatment 10-15 min,lyses at 28 for 14 h,heating at 90?-98 ?for 10-15 min,then add 50-100 ?LddH2O,mix until the layers separated,The supernatant could be used as the PCR template directlyand the PCR bands are clear and accurate.The mutants were cultivated with 24-well plates screen the pennicillin production based the high-throughput detection techniques.The results showed that 324 transforments were identified as mutant strains,including 12 penicillin high-producing strains,134 penicillin low-producing strains and 9 spore earlier-forming strains.The high-throughput genome extraction method established by our research can not only satisfy the subsequent molecular identification of P.chrysogenum mutants but also provide a reference for other filamentous fungi.The detection process will be finished within short time for the efficient transformation system combing with high-throughput screening platform.The effiency was improved.