Solanapyrone Synthase, a Possible Diels–Alderase and Iterative Type I Polyketide Synthase Encoded in a Biosynthetic Gene Cluster from Alternaria solani

Abstract

The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes-sol1-6-coding for a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels-Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin-dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (-)-solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels-Alder enzyme.

A Novel Type Pathway-Specific Regulator and Dynamic Genome Environments of a Solanapyrone Biosynthesis Gene Cluster in the Fungus Ascochyta rabiei

“…Recently, the solanapyrone biosynthesis gene cluster was identified in Al. solani (22). However, a lack of genomic information on the solanapyrone gene cluster in other fungi has hindered further examination of the HGT hypothesis.…”

Effective Use of Heterologous Hosts for Characterization of Biosynthetic Enzymes Allows Production of Natural Products and Promotes New Natural Product Discovery

“…[17][18][19][20] However, only five examples of biotransformation and their reaction mechanisms have been examined in detail using purified natural enzymes that catalyze a pericyclic reaction. Those five enzymes, SpnF in spinosyn biosynthesis, 21) solanapyrone synthase (Sol5), 22) LovB from lovastatin biosynthesis, 23,24) macrophomate synthase (MPS), 22,25) and riboflavin synthase (RibC) 26,27) were reported previously as enzymes that catalyze a Diels-Alder reaction based on their observed ability to promote a cycloaddition reaction (Chart 1). SpnF has been described as the first enzyme for which specific acceleration of a [4+2] cycloaddition reaction has been verified experimentally as its only observable function.…”

“…23) Macrophomate synthase has been studied extensively by this author and his colleagues as the first Diels-Alder-catalyzing enzyme. 28) However, recent studies have suggested that the tandem Michael-Aldol reaction is a more plausible reaction pathway for this transformation, 29,30) Other multifunctional enzymes, Sol5 and RibC, have been proposed to participate in hydroxyl oxidation 22) and hydride transfer, 27) respectively, in addition to the [4+2] cycloaddition reactions. However, the concertedness of how these enzymes catalyze the multiple reactions has yet to be elucidated.…”

Functional analysis of fungal polyketide biosynthesis genes

“…This compound was a demethylated form of prosolanapyrone I, which was the established biosynthetic precursor of solanapyrones by feeding experiments. 58,59 Thus, the PKS product was named desmethylprosolanapyrone I, and the Pks-AS2 coding PKS was prosolanapyrone synthase (PSS) 60 ( Figure 11).…”

“…These results unambiguously confirmed that the SPS encoded by sol5 catalyzes the oxidation and following exo-selective Diels-Alder cyclization to form (À)-solanapyrone A from prosolanapyrone II. 60 PKS-NONRIBOSOMAL PEPTIDE SYNTHASE HYBRID CPAA Gene cloning and sequencing were required for functional studies on target genes. In this context, we strived to clone PKS genes from fungi.…”