Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model

Abstract

A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes͞molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.

A phosphopantetheinylating polyketide synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis

“…Subjecting NcsE to the identical MS analyses that were performed with SgcE revealed S1007, the NcsE equivalent of S974, was also postranslationally modified with the 4Ј-PP cofactor (SI Fig. 7), thereby strengthening the conclusions deduced from active site mapping of SgcE and consistent with our prior prediction that all PKSE have identical domain functionality (12).…”

“…2A). The remaining regions, one located between the AT and KR of Ϸ220 aa and the other residing at the C terminus (Ϸ350 aa), are shared among the PKSE family (minimum of 20% identity) but have no apparent sequence homology to other proteins, as revealed by using bioinformatics analysis (12). However, the head-to-tail domain organization of PKSE is very similar to that found in polyunsaturated fatty acid (PUFA) synthases involved in eicosapentaenoic acid and docosahexaenoic acid biosynthesis (33), and the KS and AT domains of PUFA synthases and PKSE show significant sequence homology [supporting information (SI) Fig.…”

“…1) or a 10-membered ring such as CAL and dynemicin (4)(5)(6). To date, there are 11 enediynes that have been structurally elucidated (4, 7) with several more potential enediynes in existence, including two marine-derived compounds hypothesized to originate as enediynes and to undergo aromatization upon isolation (8,9) and several structurally uncharacterized enediynes that have been predicted based on conserved and apparently cryptic genetic information (10)(11)(12).…”

“…The biosynthetic gene clusters for C-1027 (13), NCS (14), MDP (15), and CAL (16) have been cloned and sequenced, and the first biosynthetic step leading to the enediyne core was speculated to be catalyzed by a new type of iterative polyketide synthase (PKS) that is shared among the entire enediyne family (12)(13)(14)(15)(16). PKSs catalyze decarboxylative condensation similar to fatty acid synthases (FASs) and likewise share homologous ketosynthase (KS) domains, responsible for decarboxylation and condensation, and acyltransferase (AT) domains, responsible for selection and loading of an acyl CoA (CoA)-substrate to a free thiol of an acyl carrier protein (ACP).…”

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“…34,35 In Streptomyces strains, the PKS-I gene in isolates from plant roots (38%) was found far less than in isolates from soils (92%), while there was little difference in the percentage of NRPS and PKSE genes detected. In rare actinomycetes, PKSE genes from plant root isolates (55%) were more than those detected in soil isolates (21%; Table 2).…”