Allophane increases the protein levels of several cephamycin biosynthetic enzymes in Nocardia lactamdurans

Abstract

Addition of allophane, a phosphate-trapping agent, to two different strains of Nocardia lactamdurans exerted a large stimulatory effect on cephamycin biosynthesis. The biosynthesis of cephamycin is inhibited by inorganic phosphate at concentrations above 5 mM and allophane reversed this inhibitory effect. Allophane-supplemented cultures showed increased activities and/or an extended life in the cell of four cepharnycin biosynthetic enzymes : isopenicillin N synthase, the two-protein-component 7a-cephem methoxylase (7a-cephem hydroxylase and 7-hydroxycephem methyltransferase) and 3'-hydroxymethylcephem O-carbamoyltransferase. However, the first enzyme of the pathway, lysine 6-aminotransferase, was not stimulated by allophane. Allophane-supplemented cultures showed increased protein levels of (i) a-aminoadipyl-cysteinyl-valine synthetase (the condensing multienzyme that forms the tripeptide intermediate), and (ii) the two proteins involved in the 7a-cephem methoxylase, as shown by immunoblotting with antibodies against each of these proteins. Phosphate repressed the de novo synthesis of these proteins but did not increase their degradation. These results indicated that allophane stimulates expression of the cluster of genes extending from the pcbAB gene (encoding a-aminoadipyl-cysteinyl-valine synthetase) to cmcl-cmcl (encoding the two-protein methoxylase) and cmcH (encoding the 0-carbamoyltransferase).

Inducing Effect of Diamines on Transcription of the Cephamycin C Genes from the lat and pcbAB Promoters in Nocardia lactamdurans

“…Growth was estimated as cell DNA content, using the diphenylamine reagent (2) and salmon sperm DNA standard as described previously (18).…”

Preliminary Studies for Cephamycin C Purification Technique

“…Cephamycin C (CepC) is a β-lactam compound first isolated from Streptomyces clavuligerus as an inhibitor of the synthesis of the Gram-negative cell wall [1][2][3]. According to Baggaley et al [4], cephamycin C, a cephem antibiotic produced by actinomycete bacteria, is more stable against inactivation by β-lactamase than the cephem produced by fungi, cephalosporin C. Cephamycin C is used as raw material in the production of several semi-synthetic antibiotics, such as cefoxitin and cefotetan.…”