ISOLATION AND CHARACTERIZATION OF GENES FROM BETA-LACTAM ANTIBIOTIC PRODUCING ORGANISMS (PENICILLIUM CHRYSOGENUM, RIBOSOMAL-RNA, CEPHALOSPORIUM ACREMONIUM, ISOPENICILLIN N SYNTHETASE)

The potential exists to increase the production of existing beta-lactam antibiotics and to invent new antibiotics by genetic engineering of Penicillium chrysogenum and Cephalosporium acremonium. The purpose of this study was to further characterize the genetics of these fungi and to isolate and characterize genes so that they could be manipulated to increase beta-lactam production and create new antibiotics.; The isopenicillin N synthetase gene from P. chrysogenum and the rRNA genes from C. acremonium were isolated from recombinant lambda bacteriophage libraries using heterologous probes. The protein coding region of the P. chrysogenum isopenicillin N synthetase gene was about 74% homologous to the C. acremonium isopenicillin N synthetase gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. E. coli cells with the P. chrysogenum isopenicillin N synthetase gene contained isopenicillin N synthetase activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti - C. acremonium isopenicillin N synthetase antiserum.; The rRNA genes of C. acremonium were located on a 9.2 kb PstI restriction fragment in genomic DNA digests. A partial restriction map was generated of the 9.2 kb rDNA repeat and hybridization analysis revealed the order of the genes to be 18S, 5.8S and 28S. Ribosomal DNA sequences were incorporated into C. acremonium transformation vectors to increase the transformation frequency and thus facilitate identifying a high producing strain.