Top Cephalosporin Mold The Transformation System And Application

The industrial production of p-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples for the application of biotechnology in fermentation industry. Today, the P-lactam antibiotics, particularly penicillins and cephalosporins, represent the major products of antibiotics with worldwide sales for approximately 15 billion dollars and account for approximately 65% shares of the total world market for antibiotics. Traditional natural isolation, mutation and selection, rational screening and protoplast fusion have made a great progress in the improvement of both Penicillium chrysogenum and Cephalosporium acremonium, two main industrial strains for production of P-lactam antibiotics. Rapid developments in molecular biology have become another powerful tool for strain breeding.Establishment of genetic transformation system is an important prerequisite for application of molecular genetics to extensive study of C. acremonium. However, low frequency of integrative genetic transformation is a major obstacle to gene cloning in Cephalosporium acremonium, for example, screening and identification of genes from a DNA bank by complementation of blocked mutants, knock out or replacement of genes. An effective vector for transformation will to some extent depend on the promoter function located in the upstream of selective marker. It is necessary to clone an appropriate promoter when a vector for transformation is constructed. Trp1 gene in an E. coli.-yeast shuttling plasmid pGBT9 complements the synthesis of tryptophan in an auxotroph of Saccharomyces cerevisiae Y153. A promoter-trapping vector pGBT14 containing two Trpl genes linked by head to head but without promoter region was constructed in order to select DNA segments with promoter activity. Saccharomyces cerevisiae Y153 containing pGBTH could not grow up on a complete minimal dropout medium without tryptophan. Using this vector, a 0.5-2.0Kb chromosomal DNA library of C. acremonium was constructed, and twenty four DNA fragments exhibiting promoter activity in Saccharomyces cerevisiae Y153 were selected by expressing the DNA library. It was demonstrated that the promoter activity for those DNA fragments was different in Saccharomyces cerevisiae Y153. A pair of primers was designed based on the published pcbAB-pcbC promoter sequence. 1.3Kb DNA fragment containing the full-length bidirectional pcbAB-pcbC promoter region was amplified by PCR method from chromosomal DNA isolated from filamentous fungus C. acremonium. DNA sequencing for the amplified fragment was determined and sequence alignment between amplified and known fragments was analyzed by Vector NTI software. Two plasmid vectors designated as pYG8 and pYG9 were constructed, which contain a bleomycin resistant gene downstream pcbAB promoter or pcbC promoters respectively. It was found thatthe activity of pcbC was much stronger than that of pcbAB depending on the resistance of their separate transformants to bleomycin. A plasmid vector pYG13 was constructed by inserting a VHb gene downstream pcbC promoter. It was demonstrated that the frequency of transformation was significantly increased as compared with plasmid pYG715/Vgb containing TrpC gene promoter from Aspergillus nidulans and could reach 9.8 transformants per ug DNA when Cephalosporium acremonium was transformed with pYG13. Southern blotting and Carbon monoxide-binding analysis reveal that vgb gene was integrated into the genomic DNA of C. acremonium and functional vgb gene was expressed in C. acremonium.CefEF gene encoding DAOC synthetase/hydroxylase was cloned with PCR technique. The alignment of sequences revealed that three base mutation exist in the amplified cefEF gene, that is T77→C77; G451→A451; G803→C803, respectively, which resulted in the changes of three codons: GTC→GCC; GAT→AAT and CGG→CCG. The first one is silence mutation, the others are Asp→Asn, Arg→Pro. It is unknown whether such changes were caused by PCR error or real mutation resulting from the processes of breeding performed in thi...