Anthracyclines are C-20 or C-21 aromatic polyketides produced by Streptomyces species and are clinically used in the treatment of various types of cancer. Anthracyclines are biosynthesized by a minimal polyketide synthase (minPKS) comprised of a ketoacyl synthase, chain length factor, acyl-carrier protein, and optionally, a type III acyltransferase. First ring aromatic polyketide biosynthesis then branches off into reducing PKS (i.e. C-9 ketoreduction) via an associated ketoreductase and aromatase enzymes or non-reducing PKS (i.e. 9-hydroxyl group) via aromatase/cyclase enzymes. Then, second-third ring cyclases install the tricyclic aromatic intermediate, which results in formation of an anthraquinone-like intermediate typified by nogalonic acid (C-20) or aklanonic acid (C-21). In this work, we engineered reducing-type and non-reducing type PKS cyclase constructs into Streptomyces coelicolor M1154 to reconstitute heterologous extended minPKS systems in vivo. Here, we developed a BioBricks toolkit of promoters, expression vectors, and engineered hosts for metabolic engineering of aromatases and cyclases. The activity of the different cyclase constructs was determined via complementation with a codon-optimized version of the nogalamycin minPKS (snoa123 (C-20 nogalamycin)), the aclacinomycin minPKS (aknBCDE2F, C-21 aclacinomycin), and the daunorubicin minPKS (dpsABCDG, C-21 daunorubicin). Our results indicate that the heterologous extended PKS systems exhibit significant enzymatic collaboration, which portends that aromatic PKS systems are amenable to new applications of combinatorial biosynthesis. The tools developed in this project will provide a basis for improved access to aklanonic acid and nogalonic acid analogues, which are useful for synthesis of pharmaceuticals and commodity chemicals.

Biopolymer-ARO/CYC: Biobricks polyketide metabolic engineering of aromatases and cyclases

ACS Fall 2021

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