Divergent Biosynthetic Routes Encoded by a Single Gene Cluster in Shikimate-Derived Fungal Meroterpenoid Biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Chemistry

Background:

• Meroterpenoids are complex natural products derived from both the shikimate and terpenoid pathways.
• The biosynthesis of shikimate-derived meroterpenoids is not fully understood, with limited knowledge of the enzymes involved.
• Fungi are a rich source of diverse secondary metabolites, including meroterpenoids.

Purpose of the Study:

• To identify and characterize the biosynthetic gene cluster responsible for producing meroterpenoids in *Trichothecium crotocinigenum*.
• To elucidate the enzymatic mechanisms underlying the biosynthesis of trichothosporon A and (±)-trichothecrotocin J.
• To explore novel enzymatic strategies for generating structural diversity in meroterpenoid scaffolds.

Main Methods:

• Identification and analysis of the *tcr* biosynthetic gene cluster from *Trichothecium crotocinigenum*.
• Heterologous expression of putative biosynthetic genes in a suitable host organism.
• Biochemical characterization of key enzymes, including TcrH and TcrG, using *in vitro* assays.
• Determination of enzyme kinetics and substrate specificities.

Main Results:

• The *tcr* gene cluster was identified, encoding enzymes for the biosynthesis of trichothosporon A (1) and (±)-trichothecrotocin J (2).
• The cupin-domain enzyme TcrH and ketoreductase TcrG were shown to sequentially catalyze the dearomatization step from a common precursor.
• Biochemical characterization revealed a novel cofactor-dependent substrate specificity for the ketoreductase TcrG.
• This study provides insights into the enzymatic pathways for generating structurally distinct meroterpenoids.

Conclusions:

• The identified *tcr* gene cluster provides a platform for understanding shikimate-derived meroterpenoid biosynthesis.
• Novel enzymatic strategies involving TcrH and TcrG expand the known repertoire of enzymes in natural product synthesis.
• This research opens avenues for the engineered biosynthesis of meroterpenoid analogs with potential applications.