Discovery, identification of tricyclic sesquiterpene avermitilol synthase, and its heterologous production in plasmid-free Corynebacterium glutamicum.

Abstract

Tricyclic sesquiterpenes are bioactive natural products with broad applications in pharmaceuticals, fragrances, and sustainable aviation biofuels. However, the repertoire of characterized tricyclic sesquiterpene synthases (tSTSs) remains limited. To systematically expand this enzyme class, we developed a multi-step bioinformatics workflow to identify genes encoding for cyclic sesquiterpene synthase integrating BLAST-based homology filtering, conserved motif validation, AlphaFold2-based structural modeling, and molecular docking. This workflow progressively refined an initial set of 1,063 tSTS candidates to six putative enzymes that satisfied a C1-C10 distance criterion. Heterologous expression of all six candidates in Corynebacterium glutamicum JP-2 overexpressing a methylerythritol 4-phosphate pathway gene module confirmed biosynthetic activity, yielding five known cyclic sesquiterpenes and tricyclic avermitilol produced by a tSTS (AtTPS) identified from Actinokineospora terrae by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. To improve industrial feasibility, the chemical-inducible promoter was replaced with constitutive leaderless synthetic σB promoters, and fusion of avermitilol synthase with farnesyl pyrophosphate synthase via a GGGGS linker enhanced the local concentration of intermediates between enzymes. In addition, a plasmid-free, antibiotic selection-free strain was constructed via the CRISPR-associated transposons, enabling dual chromosomal integrations of the avermitilol synthase expression cassette and achieving 81.52 mg/L avermitilol production. The chromosomally integrated strain maintained stable production over serial passages, whereas the plasmid-based strain exhibited greater than 90% loss of productivity. Fed-batch fermentation of AVM-int02cp in a 2-L bioreactor achieved a final avermitilol titer of 100.99 mg/L. This work demonstrates a sequence- and 3D structure-assisted gene discovery-to-production workflow for a tricyclic sesquiterpene through the identification of an uncharacterized synthase gene in a microbial host.