Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

Abstract

Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.secondary metabolites | fungal genetics

The Combinatorial Biosynthesis of “Unnatural” Products with Polyketides

“…The biosynthesis of benzenediol lactone (BDL) polyketide, an important pharmacophore, is based on a pair of sequentially acting iterative polyketide synthases (iPKSs). Xu et al [48] also used a plug-and-play combinational approach to express random pairs of iPKS subunits from four BDL model systems in yeast and created a diverse library of BDL congeners, including radilarin with an unnatural DAL 14 skeleton and heat-shock response-inducing activity.…”

Combinatorial Synthesis of Flavonoids and 4-Hydroxy-δ-lactones by Plant-Originated Enzymes

“…成方面也取得突破, 合成了一系列结构新颖的类似 物 [13,14] . 另一方面, 随着蛋白质研究技术的发展, 很多 与次生代谢产物生物合成相关的调节酶被克隆表达并 鉴定, 其晶体结构被解析, 结构和功能的关系得以深入 研究.…”

Fungal polyketide engineering comes of age

“…Engineering of natural products via coexpression of heterologous gene combinations has been realized for bacterial polyketide antibiotics (6, 7). In PNAS, Xu et al (8) extend this paradigm for a class of fungal polyketide natural products called the benzenediol lactones (BDLs) and generate novel "nonnatural" natural products while also providing means to generate previously described minor analogs in much improved yields.…”

“…1). As more and more BDL structures and their associated biosynthetic gene clusters are revealed (11), a straightforward bioengineering approach to expand the chemical space naturally explored by BDL structures was envisaged by Xu et al (8) to recombine unnatural pairs of BDL hrPKS and nrPKS elements. A major knowledge gap prior to this study by Xu et …”

“…Tremendous insight has been achieved with high-resolution structures of the complete fungal fatty acid synthase (19) and recently for bacterial PKS modules (20,21) that begin to resolve the mechanistic decision-making process of these multifunctional enzymes. Such structural efforts in combination with prior (12,13) and current (8) genetic reprogramming studies, such as those by Xu et al, are together contributing toward a clearer mechanistic understanding of PKS design rules that will undoubtedly facilitate how they can be tweaked to generate even greater chemical diversity.…”