Chemical space expansion for fungal polyketides by reaction flux derailing strategy

Natural products (NPs) remain a resource of pharmaceuticals and agrochemicals, but NPs that follow from the native biosynthetic logics have been intensely investigated, thereby leading to the frequent re-isolation of known compounds. Here we present an innovative, or RFD (reaction flux derailing)-based, approach characterized by rendering a native biosynthetic machine to produce unnatural molecules outside the boundary of NPs' chemical space. The prowess of the strategy was exemplified by the generation of architecturally undescribed naphthoquinone-chromane hybrids, trivially named daleslawsone A and B, in the mutant (∆pksTL) culture of Daldinia eschscholzii IFB-TL01 supplemented with lawsone, a pharmacophore-bearing xenobiotic predicted and expected to substitute for the native polyketide intermediate-1,3,6,8-tetrahydroxynaphthalene and its 2-acetyl analogue. Through a combination of spectroscopic and X-ray crystallographic analyses, daleslawsones A and B were elucidated to share an unpredicted 6/6/5/6/6 pentacyclic molecular framework. Such a unique carbon skeleton was demonstrated to form from the covalent coupling of lawsone with 1-(2,6-dihydroxyphenyl)but-2-en-2-one, a native fungal biosynthetic intermediate, and rationalized or found to be mediated via a bicyclo[3.2.1]octane-2,8-dione substructure. The cyclopentanone (but not cyclohexanone) moiety in the dione system was tailored both by a spontaneous ketalization to form daleslawsone B, and by an enzymatic reduction to yield the cyclopentanol nucleus of daleslawsone A. Such a ketone reduction was shown to be promoted by two kinds of ketoreductases, DeAKR (an aldo-keto reductase (AKR)) and De3HNR (a trihydroxynaphthalene reductase), which were identified through our BLAST analysis, heterologous overexpression of potential enzymes, and pure protein-based transformation experiment. In a chirality-dependent manner, daleslawsone A and B displayed the anti-inflammatory action in lipopolysaccharide-induced RAW264.7 cells, with (-)-daleslawsone A being more active than other stereoisomers. Collectively, the RFD-based strategy for generating new molecules is established to enable the production of new-to-nature and difficult-to-synthesize compounds that may be helpful for the drug or pesticide discovery field.