Biosynthesis of Shearinine: Diversification of a Tandem Prenyl Moiety of Fungal Indole Diterpenes

Abstract

The late-stage biosynthetic pathway of the indole diterpene shearinine involving four enzymatic reactions (JanQDOJ) was elucidated by an efficient heterologous expression system using Aspergillus oryzae. Key oxidative cyclization, forming a characteristic A/B bicyclic shearinine core by flavoprotein oxidase, was studied using a substrate analogue and a buffer containing HO. These experimental data provided evidence that JanO catalyzes two-step oxidation via a hydroxylated product and that the JanO reaction involves the hydride-transfer mechanism.

Tremorgenic and neurotoxic paspaline-derived indole-diterpenes: biosynthetic diversity, threats and applications

“…1). This scaffold is further elaborated by prenylation, oxidation, reduction, cyclization and chlorination in the various members of the group (Saikia et al 2007; Tagami et al 2013; Liu et al 2016). Paspaline-derived IDTs have been classified in various ways, using historic and structural criteria.…”

“…Paspalitrem A is then further oxidized at C32 giving rise to paspalitrem B. For shearinines, the JanD dimethylallyl transferase from P. janthinellum conducts two consecutive α-selective prenylations at C21 and C22 to yield shearinine K (Nicholson et al 2015; Liu et al 2016). This intermediate undergoes a two-step oxidative cyclization sequence catalyzed by the JanO FAD-dependent oxidase that yields a bicyclic ring system fused to the paspalinine core, affording shearinine A.…”

“…Paxilline is an excellent substrate for recombinant PaxD that produces 21,22-diprenylpaxilline. While PaxD and PaxO are silent (or at least very weakly expressed) under normal fermentation conditions thus leaving the fungus to produce paxilline as its major IDT product, the P. paxilli gene cluster may nevertheless encode the biosynthesis of a janthitrem congener (Liu et al 2016). Similarly, sulpinine biosynthesis may follow a sequence analogous to that of PtmH-PtmD-PtmVI for penitrems, with the relevant dimethylallyl transferase catalyzing a γ-selective prenylation at C22 on ring A.…”

“…The only exception to date is the pair of clusters for paxilline and the shearinines (Fig. 3), where the order and orientation of the genes are the same, except for the absence of a janJ orthologue in the paxilline gene cluster (Liu et al 2016). In contrast, clusters that are derived from closely related species that produce metabolites belonging to the same IDT family are highly syntenic.…”

“…Therefore, future metabolic engineering and fermentation optimization studies may need to target the improvement of the yields of these compounds under industrial fermentation conditions (Motoyama et al 2012; Kalinina et al 2017). The construction of various fungal synthetic biology platforms for the heterologous production of IDTs with potential biomedical significance is also on the agenda (Tagami et al 2014; Liu et al 2015; Tang et al 2015; Oikawa et al 2016; Liu et al 2016). …”