Study On Calcimycin (A23187) Biosynthesis

Calcimycin (A23187) is an important natural ionophore antibiotic. Calcimycin (A23187) inhibits the growth of Gram-positive bacteria, and also inhibits the activity of ATPase and uncouples oxidative phosphorylation of mammalian cells, and even induces apoptosis of cultured cells. Calcimycin (A23187) which is specifically transport divalent cations such as calcium and magnesium has been widely used to study calcium ion transport across membrane in vivo and in vitro and as a probe fro calcium-potential biology processes. Calcimycin (A23187) is consisted of three structure units:α-ketopyrrole, a spiroketal ring and an aminomethyl substituted benzoxazole. Feeding experiments showed that theα-ketopyrrole moiety could arise from proline while the spiroketal ring could be formed by 2 molecules acetate and 4 molecules propionate. The benzoxazole moiety could be derived from 3-hydroxy-anthrannilic acid via a chorismate intermediate. Considering the special structure and action mechanism, scientists exhibited great research interest in calcimycin (A23187) for a long time. More than16,000 publications have been reported since its discovery in 1972. However, the mechanisms for the biosynthesis of calcimycin (A23187) have not been elucidated up to now, and this study aims to clarify the unique mechanism for calcimycin (A23187) biosynthesis at molecular level so as to set the theoretical basis for creating new drugs via combinatorial biosynthesis method.Degenerate primers were designed based on the conserved domains of bacterial proline adenytransferase genes, the proline adenytransferase gene was cloned and then the calcimycin (A23187) biosynthetic gene cluster was cloned from the S. chareusis genomic library by PCR amplification. Three overlapping positive cosmids, p14F11, p16F9 and p6F5, were selected for sequencing. Bioinformatics analysis showed that the sequenced 101 kb was revealed to contain 58 ORFs. 27 of the 58 ORFs are responsible for the biosynthesis of calcimycin (A23187) and spanning 64 kb DNA region. And the 27 ORFs included three for the biosynthesis of theα-ketopyrrole moiety (calN1-N3), five genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring (calA1-A5), four genes for the biosynthesis of 3-hydroxy anthranilic acid (calB1-B4), a N-methyltransferase tailoring gene (calM), a resistance gene (calT), a type II thioesterase gene (calG), three regulatory genes (calR1-R2), four other function genes (calCDFH), and five unknown genes (calU1-U5). Four genes (calN1N2B2M) were knocked out by in-frame deletion and identified to be responsible for the biosynthesis of calcimycin (A23187). Enzymatic activity in vitro showed that CalM catalyzed the last N-methylation tailoring step. Based on the experimental data, bioinformatics analysis and previous feeding experiments, the calcimycin (A23187) biosynthetic pathway was successfully proposed.CalN1 is a prolyl dehydrogenase which oxidizes the prolyl ring to createα-ketopyrrole. CalN2 is a proline adenyltransferase which specially recognizes and activates proline. CalB2 is an isochorismatase which catalyzes 2-amino-2-deoxyisochorismate (ADIC) to produce trans-2, 3-dihydro-3-hydroxyanthranilic acid (DHHA). CalM is an N-methyltransferase which catalyzes N-demethyl calcimycin (A23187) to get calcimycin (A23187). The reaction catalyzed by CalM is the last modification step in the calcimycin (A23187) biosynthesis. In vivo experiments showed that calN1N2B2M are directly relevant to calcimycin (A23187) biosynthesis; the calN1N2B2 respectively defective mutants not only lost the production of calcimycin (A23187), but also the production of the two intermediates cezomycin and N-methyl calcimycin (A23187); the calM defective mutant lost the production of calcimycin (A23187), but still accumulated the production of the two intermediates cezomycin and N-methyl calcimycin (A23187); and both calN2M respective complementation strains restored the production of the three antibiotics. Further in vitro enzymatic activity proved that CalM catalyzed the formation of calcimycin (A23187) from N-demethyl calcimycin (A23187) in the presence of S-adenosyl-methionine (SAM), conforming to in vivo inactivation and complementation experiments.The biosynthesis of calcimycin (A23187) is derived form proline, and then to produceα-ketopyrrole ring by adenylation and dehydrogenation. After 6 rounds of decarboxylative condensation usingα-ketopyrrole as starter unit and two malonyl CoA and four methylmalonyl CoA as extender units, the full length polyketide chain are biosynthesized. It is proposed that CalG may cleave the mature linear polyketide or catalyse directly the condensation of 3-hydroxy anthranilic acid and the polyketide chain to form the benzoxazole moiety. Hydroxylation, amination and N-methylation finally generate calcimycin (A23187).