Cross‐Module Enoylreduction in the Azalomycin F Polyketide Synthase

Abstract

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end‐product, has become a cornerstone of knowledge‐based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross‐modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly‐line catalysis.

New Glycosylated Polyene Macrolides: Refining the Ore from Genome Mining

“…A few polyketides are known for which biosynthesis of the polyketide chain begins with arginine-derived guanidinobutyryl or 4-aminobutyryl primers (Figure 5). These include antifungal linear polyene polyols ECO-02301 from Streptomyces aizunensis NRRL B-11277 [50], clethramycin from Streptomyces malaysiensis DSM4137, and mediomycin from Streptomyces mediocidicus [51][52][53] (Figure 5). Three enzymes convert L-arginine to guanidinobutyryl CoA: a decarboxylating arginine mono-oxygenase, an amide hydrolase, and a CoA ligase [51] (Figure 6).…”

“…Amycolatopsis saalfeldensis has a polyene BGC that includes homologues (57% identical and 71% similar) of the amine oxidase and acyl CoA synthetase that generate guanidinobutyryl CoA as a primer. The PKS assembly line starts with a loading ACP homologous to that for azalomycin PKS, which also uses a guanidinobutyryl primer [53] (Supplementary Materials Figures S1-S3). The predicted polyene structure is shown in Figure 7.…”

“…nobutyryl CoA as a primer. The PKS assembly line starts with a loading ACP homologous to that for azalomycin PKS, which also uses a guanidinobutyryl primer [53] (Supplementary Materials Figures S1-S3). The predicted polyene structure is shown in Figure 7.…”

Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition

“…Moreover, intermodule interactions also explain why the loss of function domain in a module tends to be accompanied by a downstream domain stuttering or action of a free-standing enzyme rather than domains in other modules in the assembly-line [14][15][16] . However, in our previous work [17][18][19] , we uncovered an unprecedented cross-module enoylreduction in which a switchable ER domain in module 1/2 acts in trans to catalyze the enoylreduction within the module 3 in azalomycin F (AZL) PKS assembly-line (Fig. 1).…”

“…1). Notably, we previously provided in vitro evidence showing that the ER1/2-KR1/2 didomain comprises the minimal cassette required for cross-module enoylreduction 19 . Therefore, we propose that the ER1/2 domain in modules 1/2 and 3 are arranged into a hybrid for full enoylreduction via previously unknown cross-module interactions.…”