In Vivo and In Vitro Trans-Acylation by BryP, the Putative Bryostatin Pathway Acyltransferase Derived from an Uncultured Marine Symbiont

Abstract

Summary The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete ORF (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills

“…Additionally, phylogenetic analysis has shown that trtB falls within the clade of discrete FAS-like PKS malonyltransferases ( Fig. S8B) composed by discrete AT domains that hypothetically are involved or biochemically are proven to load malonyl-CoA units in a variety of trans-AT PKS systems (19).…”

Analysis of the Sorangicin Gene Cluster Reinforces the Utility of a Combined Phylogenetic/Retrobiosynthetic Analysis for Deciphering Natural Product Assembly by trans‐AT PKS

“…This mechanism requires that one of the two trans ATs present in the pathway exhibit specificity for hydroxymalonate instead of malonate. Alignment of the two Sor ATs against known trans ATs shows that AT b groups with malonatespecific domains, which are present in all clusters (e.g., those from the leinamycin, [29] bryostatin, [30] and bacillaene, [24] the specificities of which have been demonstrated directly in vitro; Supporting Information). On the other hand, AT a clades with a second group of trans ATs that are present only in some systems, consistent with the idea that it may exhibit alternative specificity.…”

The Combinatorial Biosynthesis of “Unnatural” Products with Polyketides

“…cis-acting ATs are coupled with the native ACP domains, whereas trans-acting ATs exist as stand-alone enzymes. The latter can transfer the extender unit onto one or more ACPs in a multi-modular PKS assembly line, such as the synthesis of disorazol [31] and bryostatin [32]. Compared to cis-AT, the trans-AT may have a wider substrate library, thus, it can be used to obtain "unnatural" analogs [33].…”