Abstract
Three examples are presented describing the application of genes cloned from biosynthetic pathways of penicillins and cephalosporins produced by Penicillium chrysogenum, Cephalosporium acremonium, Streptomyces clavuligerus, and Streptomyces lipmanii. In the first example, ring expansion of penicillin N was determined to be the rate limiting step in synthesis of cephalosporin C in a production strain of C. acremonium by assay of metabolites in production fermenters. Penicillin N expandase/deacetoxycephalosporin C [DAOC] 3’-hydroxylase was purified to homogeneity and reverse genetics was used to clone the corresponding gene, cefEF. A system for genetic transformation for C. acremonium was developed and used to insert an extra copy of the C. acremonium cefEF gene back into the production strain. The transformant produced twice as much expandase/hydroxylase as the production strain recipient. The production strain accumulated penicillin N in stirred fermenters and the transformant did not. The transformant produced about 10% more cephalosporin C than the production strain recipient. The second example illustrates concepts of pathway engineering. Using promoters and terminators from the P chrysogenum penDE and pcbC gene and open reading frames from the S. lipmanii cefD and S. clavuligerus cefE gene, a vector suitable for expressing isopenicillin N epimerase and penicillin N expandase was constructed and used to produce deacetoxycephalosporin C [DAOC] in Penicillium chrysogenum. Production of DAOC by fermentation and conversion to 7-amino-deacetoxycephalosporanic acid [7-ADCA] by enzymatic hydrolysis would provide a substantially aqueous process for making 7-ADCA, an important industrial intermediate used in the manufacture of oral cephalosporin antibiotics. In the third example, high level expression of various pcbC genes in E. coli makes large quantities of isopenicillin N synthetase available for the conversion of synthetic substrate analogs to a variety of penicillins and novel β-lactam compounds that can be screened for antibiotic activity, inhibition of β-lactamases, and other pharmacological activities.
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Queener, S.W. (1992). Molecular Biology of Penicillin and Cephalosporin Biosynthesis: Application of Cloned Genes. In: Vardar-Sukan, F., Sukan, Ş.S. (eds) Recent Advances in Biotechnology. NATO ASI Series, vol 210. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-2468-3_10
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