Using resting cells and extracts of Streptomyces clavuligerus NP1, we have been able to convert penicillin G (benzylpenicillin) to deacetoxycephalosporin G. Conversion was achieved by increasing by 45؋ the concentration of FeSO 4 (1.8 mM) and doubling the concentration of ␣-ketoglutarate (1.28 mM) as compared with standard conditions used for the normal cell-free conversion of penicillin N to deacetoxycephalosporin C. ATP, MgSO 4 , KCl, and DTT, important in cellfree expansion of penicillin N, did not play a significant role in the ring expansion of penicillin G by resting cells or cell-free extracts. When these conditions were used with 14 other penicillins, ring expansion was achieved in all cases. Biosynthesis of cephalosporins in Cephalosporium acremoniumand Streptomyces clavuligerus proceeds through a biosynthetic pathway that includes expansion of the five-membered thiazolidine ring of the intermediate penicillin N into the sixmembered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) (for reviews, see refs. 1 and 2). This step is catalyzed by the ␣-ketoglutarate-dependent dioxygenase, DAOC synthase (''expandase'') (3). In the fungus C. acremonium, the activity of expandase resides in a bifunctional enzyme (4) that catalyzes not only ring expansion but also the hydroxylation of the methyl group of DAOC to deacetylcephalosporin C whereas in the bacterium S. clavuligerus the two activities are associated with separate proteins (5, 6).We and others (3-10) have found that the enzyme, in cell-free extracts as well as after purification, has a very narrow substrate specificity and no detectable activity on readily available and inexpensive penicillins (such as penicillin G and V produced by Penicillium chrysogenum). Indeed, chemical ring expansion plus an enzymatic removal of the phenylacetyl side chain is used in industry to convert penicillin G into 7-aminodeacetoxycephalosporanic acid, an important intermediate for the manufacture of semisynthetic cephalosporins. However, this process requires several steps and is expensive and polluting. A simple biological route might replace the chemical process, requiring only two steps, i.e., ring expansion and enzymatic deacylation, thereby reducing costs and environmental problems. We now have identified the conditions that allow conversion of penicillin G to deacetoxycephalosporin G (DAOG; phenylacetyl-7-aminodeacetoxycephalosporanic acid). In this report, we describe the conversion of a number of penicillins, including penicillin G, into cephalosporins by using resting cells and cell-free extracts of the cephalosporin-deficient mutant S. clavuligerus NP1 (11). MATERIALS AND METHODSMicroorganism. S. clavuligerus NP1, a mutant producing only trace levels of cephalosporins (11), was used in this work. The absence of significant levels of cephalosporins in this strain facilitated detection of cephalosporins produced from added penicillins by ring expansion.Media and Culture Conditions. Mycelia were obtained by using 250-ml baffled flasks containing 40 ml of...

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