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.

Keywords: fungal genetics; secondary metabolites.

Fig. 1.

Biosynthesis of natural benzenediol lactones. Biosynthetic assembly of monocillin II, trans-resorcylide, desmethyl-lasiodiplodin, and 10,11-dehydrocurvularin (the “on-program” main metabolites) in recombinant S. cerevisiae BJ5464-NpgA (24, 40) strains by native BDLSs (12, 14, 25). R indicates the hrPKS-generated priming unit that is elaborated by the nrPKS. Numerals in the colored spheres indicate the number of malonate-derived C₂ units (C−C bonds shown in bold) incorporated into the polyketide chain by the hrPKS vs. the nrPKS (“division of labor” or “split” by the BDLS: e.g., 5+4 indicates a pentaketide extended by four more malonate units). Stutter products are minor metabolites produced by extra extension cycles resulting in irregular “splits” (25).

Fig. 2.

Combinatorial biosynthesis using AzResS2. Biosynthesis of on-program and stutter products by S. cerevisiae BJ5464-NpgA (24, 40) coexpressing AzResS2 with heterologous BDL hrPKSs (12, 14, 25). Product structures are colored to indicate the origin of the assembled biosynthons. Stars over peaks in product profiles (HPLC traces recorded at 300 nm) indicate host-derived metabolites. See Fig. 1 for additional explanations.

Fig. 3.

Combinatorial biosynthesis using CcRadS2. Biosynthesis of on-program and stutter products by S. cerevisiae BJ5464-NpgA (24, 40) coexpressing CcRadS2 with heterologous BDL hrPKSs (12, 14, 25). CcRadS2* and the split-color box indicates that SAT_CcRadS2 was replaced by SAT_AtCurS2 to facilitate coupling with AtCurS1. See Fig. 2 for additional explanations.

Fig. 4.

Combinatorial biosynthesis using LtLasS2. Biosynthesis of on-program and stutter products by S. cerevisiae BJ5464-NpgA (24, 40) coexpressing LtLasS2 with heterologous BDL hrPKSs (12, 14, 25). LtLasS2* in a split-color box indicates replacement of SAT_LtLasS2 with the SAT cognate for the heterologous hrPKS (e.g., SAT_LtLasS2 replaced by SAT_CcRadS2 to facilitate collaboration of LtLasS2 with CcRadS1). See Fig. 2 for additional explanations.

Fig. 5.

Combinatorial biosynthesis using AtCurS2. Biosynthesis of on-program and stutter products by S. cerevisiae BJ5464-NpgA (24, 40) coexpressing AtCurS2 with heterologous BDL hrPKSs (12, 14, 25). AtCurS2* in a split-color box indicates replacement of SAT_AtCurS2 with SAT_CcRadS2. See Fig. 2 for additional explanations.