Molecular and Computational Bases for Spirofuranone Formation in Setosusin Biosynthesis

Abstract

The 3­(2H)-furanone unit is observed in many biologically active natural products, as represented by the antifungal medication griseofulvin. Setosusin (1) is a fungal meroditerpenoid featuring a unique spiro-fused 3­(2H)-furanone moiety; however, the biosynthetic basis for spirofuranone formation has not been investigated since its isolation. Therefore, in this study we identified the biosynthetic gene cluster of 1 in the fungus Aspergillus duricaulis CBS 481.65 and elucidated its biosynthetic pathway by heterologous reconstitution of related enzyme activities in Aspergillus oryzae. To understand the reaction mechanism to afford spirofuranone, we subsequently performed a series of in vivo and in vitro isotope-incorporation experiments and theoretical calculations. The results indicated that SetF, the cytochrome P450 enzyme that is critical for spirofuranone synthesis, not only performs the epoxidation of the polyketide portion of the substrate but also facilitates the protonation-initiated structural rearrangement to yield 1. Finally, a mutagenesis experiment using SetF identified Lys303 as one of the potential catalytic residues that are important for spirofuranone synthesis.

Biocatalytic and chemical derivatization of the fungal meroditerpenoid chevalone E

“…To achieve the structural diversification of chevalones, we decided to focus on the modification of the A-ring, since many fungal meroterpenoids possess modified or rearranged A-ring systems. For example, in several fungal meroterpenoid pathways, the A-ring is expanded to a seven-membered lactone, 16,[18][19][20] and could also be rearranged to provide highly complicated structures, such as the spiro-lactone of 15-deoxyoxalicines, 15 the spirocyclopentanone moiety of funiculolide D, 21 and the ketal scaffold of talaromyolides 22 (Fig. 1B).…”

“…11 Chevalones are fungal meroditerpenoids 12,13 derived from the polyketide pyrone triacetic acid lactone (TAL) and geranylgeranyl pyrophosphate (GGPP). Unlike many other fungal meroditerpenoids that possess a highly rearranged backbone skeleton, [14][15][16] chevalones harbor a relatively simple scaffold, and therefore, these molecules could serve as a good starting point for structural derivatization to obtain a series of new chevalone analogues with improved biological activities.…”

Discovery of branching meroterpenoid biosynthetic pathways in Aspergillus insuetus: involvement of two terpene cyclases with distinct cyclization modes

“…To characterize the functions of InsA7 and InsB2, we heterologously expressed insA7 and insB2 along with insA2, insA5, insA1, and insA4, which are the genes involved in the biosynthesis of the putative substrate of the two cyclases, in A. oryzae NSAR1, 20 a powerful platform for the refactoring of natural product biosynthesis in fungi. [21][22][23][24] The metabolites of the A. oryzae transformants were analyzed using highperformance liquid chromatography (HPLC). The HPLC analysis revealed the existence of different major products 2 and 3 in the two strains (Figure 3A), of which 2 was also detected in A. insuetus CBS 107.25 (Figure S2).…”

Branching and converging pathways in fungal natural product biosynthesis

“…12) [89, A methylated analogue of TAL (97), 5-methyl TAL (105), is often found in fungal meroterpenoid structures, and in recent years, the complete biosynthetic pathways of several 5-methyl TAL-derived meroterpenoids have been revealed (Fig. 13) [91][92][93]. In the biosynthesis of setosusin ( 106), 105 undergoes a series of reactions similar to those in TAL-derived meroterpenoid pathways to provide a precyclized intermediate 107, which is accepted by the terpene cyclase SetH to give 108 with a tricyclic terpenoid portion [93].…”

“…13) [91][92][93]. In the biosynthesis of setosusin ( 106), 105 undergoes a series of reactions similar to those in TAL-derived meroterpenoid pathways to provide a precyclized intermediate 107, which is accepted by the terpene cyclase SetH to give 108 with a tricyclic terpenoid portion [93]. This cyclized product is also used as the precursor for other fungal metabolites, such as brevione E (109) [94], although brevione E biosynthetic genes have not been reported.…”