Identification and Characterization of the Carbapenem MM 4550 and its Gene Cluster in Streptomyces argenteolus ATCC 11009

Abstract

Nearly 50 naturally-occurring carbapenem β-lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C-2 and C-6 side chains as well as the stereochemistry at C-5/C-6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. While the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by S. argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that genes encoding the biosynthesis of the bicyclic core and the C-6 and C-2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22/23, encode a two-component response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.

Intraspecies comparison of Streptomyces pratensis genomes reveals high levels of recombination and gene conservation between strains of disparate geographic origin

“…In contrast, the beta-lactam gene cluster in S. clavuligerus ATCC 27064 produces cephamycin and is distantly related to the beta-lactam gene cluster found in S. pratentis . The S. pratensis beta-lactam gene cluster most closely resembles that reported for MM 4550 from S. argenteolus ATCC 11009 [ 40 ]. MM 4550 is within the carbapenem class of beta-lactams [ 41 ], but is distantly related to the cephamycin gene cluster (e.g.…”

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis

“… 156 Disruption of the ThnQ homolog involved in the biosynthesis of MM 4550 resulted in accumulation of an unhydroxylated product. 164 Some carbapenems have different C-6 side chains, such as an (8 S )-hydroxyl group or an (8 S )-sulfate group (as seen in MM 4550). 4 The epimerisation of the 8-hydroxyl group in MM 4550 is proposed to be catalysed by Cmm17, an enzyme homologous to the enoyl-CoA hydratases.…”

Consecutive radical S -adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis

“…By identifying favorable substrates for ThnK and elucidating its activity, we have gained additional clues about the unknown central steps in thienamycin biosynthesis as well. With formation of the ethyl side chain accounted for, ThnL and ThnP can provisionally be assigned nonmethyltransferase functions, potentially distinct chemistry for the RS-cobalamin family, given that both proteins are essential for carbapenem production (23,24). Because ThnK activity was observed only when a C2 side chain was present (compounds 10-12 vs. 2, 8, and 9), we deduce that C2-thioether formation, likely mediated by ThnL or ThnP, precedes C6-methylation in the pathway.…”