Top The Cephalosporin Mold Of The Preliminary Study Of The Genetics And Breeding Methods And The Cephalosporin C Fermentation Behavior Of Mutation Screening

This thesis mainly studied methods of breeding A.chrysogenum, and further improved its ability to produce cephalosporin C. The study consisted of three parts: firstly, three strains with improved phenotypes were obtained by using classical strain improvement(CSI) for different pressure. Secondly, all these mutant strains were then recursive protoplast fused using Genone Shuffling technique and further screened and finally one fusion strain was obtained with all the mutants' characteristics. Lastly, the obtained fusion strain was subjected to molecular genetic manipulation. Substrate competition was released and recombinant protein-VHb was expressed by constructing plasmids and transformed into A.chrysogenum, so that cephalosporin C production and oxygen absorption ability under low dissolved oxygen conditions were improved.In the first part of the study, we obtained eighty mutant strains by recursive mutation and screening, among which three strains had obvious better characteristics than the original strain determined by fermentation and characteristic analysis. The results is that cephalosporin C production increased by 33%, time for max production was shortened by 20 hours and the ability to produce conidiospore improved by 50%.In the second part of the study, method of parents inactivated protoplast fusion was determined by optimizing the condition of production and regeneration of protoplast. Then four series of fusion were applied to the three mutant strains using Genome Shuffling Technique, with each series screening 100 strains of high malonate concentration resistant fusions, among which fusions with early differentiation were picked and production of cephalosporin C was analyzed, and the results showed that production in eleven strains were significantly increased. Further analysis showed that one of the fusions poccesses all characteristic of its parents.In the third part of the study, expression vector pANCVHb with hygromycin B-resistant gene was constructed, and then another vector pANCVHb-lys was obtained by insertin a fragment for part of the gene lys in A. chrysogenum. In the eleven transformants, production in three of them increased by 10%. Under low soluble oxygen conditions, biomass increased by 10%-15% in 70% of the transformants, indicating that feedback inhibition by substrates was released and functional VHb was expressed.