Investigating Key Diversity Generating Enzymes in Indole Diterpene Biosynthesis
The indole diterpenes (IDTs) are a wide class of secondary metabolites found in filamentous fungi that are composed of an indole ring fused to a twenty-carbon geranylgeranyl terpene chain that can be variably cyclised and modified. Members of this group are notable for their possession of significant bioactivities that have the potential to be developed as drugs or agrichemicals. Owing to their complex structures, synthetic efforts to deliver commercial quantities of these compounds have not yet been successful. This challenge and their chemical complexity have led to considerable efforts to understand their biosynthesis in their host organisms.
The biosynthesis of IDTs may be assigned to the action of two groups of enzymes: the ‘core’ and the ‘decoration’ enzymes. The core enzymes are responsible for the generation of the first cyclised architecture of the IDTs and the genes encoding these enzymes are conserved across all known IDT biosynthetic clusters. The decoration enzymes, on the other hand, introduce various modifications such as prenylations, oxidations, halogenations among others onto the core skeleton resulting in the generation of tremendous diversity. These enzymes, however, are not ubiquitous across IDT biosynthesis and are highly pathway specific. Of the universal core enzymes, the epoxidase (idtM) and the cyclase (idtB) are involved in the cyclisation of the geranylgeranyl terpene chain and are responsible for delivering a large amount of the structural diversity observed among the IDTs.
The work described herein bolsters the understanding of these structure-diversifying enzymes, by uncovering the biosynthetic origins of the emindoles SA (31), DA (29), and DB (30) – IDTs predicted to be generated from a novel cyclisation route. Interrogation of the genomes of the fungi producing these IDTs – emindole SA (31) from Aspergillus striata and emindoles DA (29) and DB (30) from Aspergillus desertorum – revealed the presence of biosynthetic gene clusters possessing copies of the ubiquitous core genes.
A. striata possessed two putative epoxidases, estM1 and estM2, and three putative cyclases, estB1, estB2, and estB3. A. desertorum, on the other hand, possessed only one putative epoxidase, desM, and one putative cyclase, desB. Heterologous expression of the cyclases in a Penicillium paxilli host strain revealed that estB1 and desB delivered emindole SA (31) and emindole DA (29) /DB (30), respectively. The discovery of these cyclases links this fundamental IDT biochemistry to sequence information for the first time.
To further understand diversity generation originating from the epoxidases and cyclases, particularly with regard to the generation of the tetrahydropyran (THP) ring observed in certain IDT core structures, non-natural combinations of these genes were investigated. Surprisingly, it was observed that a monoepoxidase from A. striata, estM1, was not only able to catalyse two epoxidation reactions on a non-emindole SB core but was also able to generate unusual stereochemistry on this core. Finally, in an effort to aid future testing of epoxidase-cyclase combinations, a ΔpaxMΔpaxB double mutant P. paxilli strain was generated using CRISPR-Cas9.
