Functional Dissection of a Multimodular Polypeptide of the Pikromycin Polyketide Synthase into Monomodules by Using a Matched Pair of Heterologous Docking Domains

Abstract

The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and 6 extension modules which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of an S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a 10-fold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.

Protein–protein interactions in “cis-AT” polyketide synthases

“…This was illustrated by genetic manipulation of the producing strains for pikromycin and erythromycin. Separation of the naturally fused modules 1 and 2 reduced pikromycin production more than 1000-fold 82 . Conversely, fusion of the naturally in trans polypeptides DEBS1 and DEBS2, or DEBS2 and DEBS3, maintained or even enhanced erythromycin production relative to the wild type 83 .…”

Structural and Functional Trends in Dehydrating Bimodules from trans-Acyltransferase Polyketide Synthases

“…Trans -AT PKSs lack the N- and C-terminal docking domains (NDD and CDD regions) that noncovalently connect ACP and KS domains in cis -AT systems (Weissman 2006; Yan, 2009; Whicher, 2013). The split nature of dehydrating bimodules is representative of many uncharacterized disconnections between trans -AT polypeptides.…”

Cyanobacterial Polyketide Synthase Docking Domains: A Tool for Engineering Natural Product Biosynthesis

“…When successive modules operate from independent proteins, non-covalent association of C- and N-terminal docking domains promote protein-protein interaction of the upstream ACP and downstream KS (Gokhale et al, 1999) (Figure 1A). Docking domains, ACP dd at the ACP C-terminus of the upstream module and dd KS at the KS N-terminus of the downstream module, are essential to ensure correct transfer of polyketide chain elongation intermediates (Gokhale et al, 1999; Kittendorf et al, 2007; Kumar et al, 2003; Tsuji et al, 2001; Weissman, 2006a, b; Wu et al, 2002; Wu et al, 2001), and thus are essential structural elements for engineering these pathways to generate novel small molecules by rearrangement or recombination of PKS modules (Menzella et al, 2007; Menzella et al, 2005; Reeves et al, 2004; Yan et al, 2009). Although early studies demonstrated that cognate docking domains can facilitate intermediate transfer between modules that do not naturally associate (Menzella et al, 2007; Menzella et al, 2005; Reeves et al, 2004; Wu et al, 2002; Yan et al, 2009), none of the systems explored docking domain structure and function across broad phylogenetic groups.…”