GENETIC-ENGINEERING APPROACH TO REDUCE UNDESIRABLE BY-PRODUCTS IN CEPHALOSPORIN-C FERMENTATION

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathwa y, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxyce phalosporanic acid (7-ADCA), when the cephalosporin C is converted che mically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In th e cephalosporin C biosynthetic pathway of A. chrysogenum, the bifuncti onal expandase/hydroxylase enzyme catalyzes the conversion of penicill in N to DAOC and subsequently deacetylcephalosporin C (DAC). By geneti cally engineering strains for increased copy number of the expandase/h ydroxylase gene, we were able to reduce the level of DAOC present in t he fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed th at one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amou nt of transcribed expandase/hydroxylase mRNA in one of the transforman ts is increased approximately two-fold over that in the untransformed host.