Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

Abstract

Fungal maleidrides are an important family of bioactive secondary metabolites that consist of 7, 8, or 9‐membered carbocycles with one or two fused maleic anhydride moieties. The biosynthesis of byssochlamic acid (a nonadride) and agnestadride A (a heptadride) was investigated through gene disruption and heterologous expression experiments. The results reveal that the precursors for cyclization are formed by an iterative highly reducing fungal polyketide synthase supported by a hydrolase, together with two citrate‐processing enzymes. The enigmatic ring formation is catalyzed by two proteins with homology to ketosteroid isomerases, and assisted by two proteins with homology to phosphatidylethanolamine‐binding proteins.

A Cascade of Redox Reactions Generates Complexity in the Biosynthesis of the Protein Phosphatase‐2 Inhibitor Rubratoxin A

“…Bioinformatics analysis led to the identification of 11 gene clusters that encode ah ighly reducing polyketide synthase (HR-PKS). [9,12] Ther ole of the rbt gene cluster in the biosynthesis of 1 and 2 was confirmed through inactivation of the PKS,s ince in DrbtJ strain the production of all related compounds was completely abolished ( Figure 2b,t race ii and Figure S12). [9,12] Ther ole of the rbt gene cluster in the biosynthesis of 1 and 2 was confirmed through inactivation of the PKS,s ince in DrbtJ strain the production of all related compounds was completely abolished ( Figure 2b,t race ii and Figure S12).…”

“…[1] Many of the redox transformations are unprecedented in primary metabolism and can lead to significant morphing of the polyketide product, as illustrated in the biosynthesis of aflatoxin. [6] Compared to other nonadrides such as phomoidride A, [7] zopfiellin, [8] and byssochlamic aicd, [9] rubratoxins are structurally the most complex, with the highest degree of redox modifications, including am ultitude of hydroxylations at sp 3 carbons,a n unsaturated lactone,a nd a g-hydroxybutenolide in 1.C ollectively,these redox modifications make 1 ahighly specific and potent inhibitor of protein phosphatase 2( PP2A), and thus apotential lead compound for the development of anticancer drugs. Structurally,r ubratoxins belong to the family of nonadrides,agroup of fungal natural products with ac yclononane ring and two fused maleic anhydrides to form the 5/9/5 core ring system.…”

“…Structurally,r ubratoxins belong to the family of nonadrides,agroup of fungal natural products with ac yclononane ring and two fused maleic anhydrides to form the 5/9/5 core ring system. [6] Compared to other nonadrides such as phomoidride A, [7] zopfiellin, [8] and byssochlamic aicd, [9] rubratoxins are structurally the most complex, with the highest degree of redox modifications, including am ultitude of hydroxylations at sp 3 carbons,a n unsaturated lactone,a nd a g-hydroxybutenolide in 1.C ollectively,these redox modifications make 1 ahighly specific and potent inhibitor of protein phosphatase 2( PP2A), and thus apotential lead compound for the development of anticancer drugs. [10] Understanding the biosynthesis of 1 and 2 could therefore provide significant new insight into how nature builds structural complexity and unusual biosynthetic features starting from relatively simple precursors.…”

“…While the biosynthesis of the nonadride core in byssochlamic acid was recently reported, [9] including the polyketide synthase (PKS) product and the dimerization strategy,t he enzymatic basis for the post-dimerization redox modifications in this family of natural products have remained unexplored. Ap articularly interesting feature in 1 is the partial and regioselective reduction of one maleic anhydride moiety to a g-hydroxybutenolide.Strikingly,the g-hydroxybutenolide is essential for the binding of 1 to PP2A, since 1 displays an approximately 100-fold stronger inhibition of PP2A (K i = 28.7 nm)t han that of 2 (K i = 3.1 mm).…”

“…Using reverse transcriptase (RT)-PCR to aid in defining cluster boundaries, we arrived at at entative rbt gene cluster that contains 23 genes ( Figure S2). [9] Most relevant for this study,av ariety of redox enzymes,i ncluding aP 450 monooxygenase (RbtI), af lavin-dependent monooxygenase (FMO,R btA), a-ketoglutarate-dependent dioxygenases (a-KGDs;RbtB,RbtG,RbtE, and RbtU), and aferric reductase (RbtH) are also present and are likely involved in the postdimerization tailoring of the nonadride core into 1 and 2. [9] Most relevant for this study,av ariety of redox enzymes,i ncluding aP 450 monooxygenase (RbtI), af lavin-dependent monooxygenase (FMO,R btA), a-ketoglutarate-dependent dioxygenases (a-KGDs;RbtB,RbtG,RbtE, and RbtU), and aferric reductase (RbtH) are also present and are likely involved in the postdimerization tailoring of the nonadride core into 1 and 2.…”