No abstract

On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.The aromatic polyketide actinorhodin (Act) (Fig. 1) is a secondary metabolite produced by Streptomyces coelicolor A3(2), genetically the most studied member of the genus. A C 16 carbon chain forms the backbone of each half-molecule of Act and is synthesized from one acetyl coenzyme A (acetyl CoA) starter and seven malonyl CoA extender units. These units are condensed together (with loss of CO 2 ) in a sequential fashion by the Act polyketide synthase (PKS) (14), a process which shows clear similarities to the mechanism of fatty acid biosynthesis (21, 33). The linear intermediate then undergoes cyclizations and further, ''tailoring'' modifications to yield the final molecule. A set of genes (act) involved in the synthesis of actinorhodin has been identified and located on one continuous (22-kb) section of the S. coelicolor chromosome (28). The act cluster has been completely sequenced (4,(10)(11)(12)16); it contains a set of some 22 genes that include the structural genes for the Act PKS, genes which encode the enzymes involved in the subsequent modification of the PKS product, and genes for activation of expression of the act cluster and for actinorhodin export.The act PKS itself consists of a series of discrete open reading frames (ORFs) encoding several proteins that share considerable amino acid sequence similarity with the components of Escherichia coli fatty acid synthase (FAS) (11). These are (i) a ketosynthase (KS), which catalyzes the condensation of the acyl building units; (ii) an acyl carrier protein (ACP), which serves a dual purpose as the recipient for the ...