In vitro conversion of penicillin G and ampicillin by recombinant Streptomyces clavuligerus NRRL 3585 deacetoxycephalosporin C synthase

The in vitro conversion of penicillin N analogues to cephalosporins was first demonstrated in resting cells and cell-free extracts of Streptomyces clavuligerus NP1. This study has optimized the conversion rate of a highly expressed soluble source of recombinant S. clavuligerus NRRL 3585 deacetoxycepalosporin C synthase (scDAOCS) through detailed characterization of its cofactor requirements. Inclusion of dithiothreitol (DTT) and ascorbate at various concentrations from 0.2–16 mM and 0.2–10 mM was found to be inhibitory to scDAOCS activity under specified reaction conditions examined. Simultaneous omission of these cofactors in a new modified reaction condition, comprising of 50 mM Tris HCl (pH7.4), 0.8 mM ATP, 1.28 mM α-ketoglutarate and 1.8 mM FeSO4, enhanced the conversion rate of two penicillin N analogues, penicillin G and ampicillin, through 20- and 35-fold respectively.