Optimization Of The Transformation Platform Of Acremonium Chrysogenum And The Construction Of The Ku70 Homologous Gene Knock-out Plasmid

Semisynthetic cephalosporin is one of the anti-infective drugs used in clinic all over the world. The sales account for 65% shares of the world market for antibiotics. And this ratio is growing steadily in the past several years.But the genetic modification of filamentous fungi Acremonium chrysogenum is very difficult for the two reasons. First, the transformation frequency of Acremonium chrysogenum is very low; Second, the exogenous genes transformed into fungi cell tend to insert into the chromosomes randomly. That means the directed manupilation by homologous recombination is extremely hard in Acremonium chrysogenum. The strategy of Ku70 knock-out to improve the gene targeting efficiency has been used in many kinds of filamentous fungi. So the improvement of the transformation platform and the construction of ?Ku70 gene mutant of Acremonium chrysogenum could be very helpful for the genetic modification of the strain.In this study, we optimized the preparation and regeneration conditions of Acremonium chrysogenum protoplast. The influences of cell wall degrading enzymes, digesting time, enzymes adding amounts, morphology of hyphae, culture media composition and spreading ways on protoplast preparation and regeneration of Acremonium chrysogenum were systematically examined. The final optimum conditions of protoplast preparation were as follow: moderately swollen hyphae of Acremonium chrysogenum was digested by 1% lysing enzyme for 2h at 30?. The number of protoplast reached 2×108 per milliliter. Double-plated the protoplast preparation on HCM medium, and the regeneration ratio reached as high as 20%? And then, we tested the transformation of the protoplast by plasmid pYG233. The transformation efficiency could reach 10 transformants/?g plasmid. The positive transformation had the potential to convert CPC to 7-ACA.In addition, we try to construct the plasmid to knock out Ku70 gene in Acremonium chrysogenum. This plasmid contained the upstream and downstream fragments of the Ku70 homologous gene of Acremonium chrysogenum, phleomycin resistance marker and pcbAB promoter cloning from Acremonium chrysogenum.These research results made good basis for metabolic engineering of Acremonium chrysogenum.