Synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus

Abstract

In vitro synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus was investigated using K-ketoglutarate, L-ascorbic acid, FeSO 4 , S-adenosyl-L-methionine, and 7K-demethoxycefminox as the substrates. The formation of cefminox was detected both by a biological assay with Proteus vulgaris GN 76/C-1 and by high performance liquid chromatography. Although the conversion rate of 7K-demethoxycefminox to cefminox was observed to be quite low, it still demonstrated the potential for an enzymatic process to replace the chemical steps which are currently in use for the production of cefminox. ß

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

“…Expression of the novel pathway was done in P. furianosis , which has optimal activity at 100°C; when the cell‐free reaction was run at 70°C, only the recombinantly expressed enzymes were active at the lower temperature. Crude extracts have demonstrated the ability to synthesize high‐value biologics and therapeutics; examples include single‐step conversion of diketides to chiral triketide lactones (polyketide building blocks) [75], lysine to ϵ‐poly‐L‐lysine (an antimicrobial) [76], and 7α‐demethoxycefminox to cefminox (an antibiotic) [77]. In another study, 14 C‐labeled substrates were used to elucidate possible biosynthetic pathways for manufacture of the antitumor agent Azinomycin B [78].…”

Improvement of 7α -methoxycephalosporins production by overexpression of cmcJ and cmcI controlled by promoter ermEp * in Streptomyces clavuligerus

“…The industrial production of 7a-methoxycephalosporins is carried out by chemical synthesis using 7a-methoxycephalosporin C (MCPC) as the main precursor (Kobayashi et al 1977;Elander 2003). Because the conventional process to obtain MCPC is the chemical conversion of the more abundant substrate cephalosporin C (CPC), which is inefficient and expensive, biotransformation of CPC to MCPC was considered as a potential alternative enzymatic method for the industrial production of 7a-methoxycephalosporins (O'Sullivan and Abraham 1980;Hood et al 1983;Kim et al 2000).…”

Establishing a cell-freeVibrio natriegensexpression system

“…Its rapid growth rate, which has been linked to high rates of protein synthesis 5 and metabolic efficiency 6,7 suggests that this host may be harnessed as a powerful cell-free expression system. Cell-free bioproduction of protein and chemicals has been extensively investigated in E. coli and recently expanded to several other bacteria [8][9][10][11][12][13] . Accordingly, we assessed V. natriegens crude cell extract productivity in small-scale batch reactions using super-folder GFP (sfGFP) controlled by a T7 promoter.…”