Genome Mining of the Biocontrol Agent Trichoderma afroharzianum Unearths a Key Gene in the Biosynthesis of Anti-Fungal Volatile Sesquiterpenoids

The volatile organic compounds (VOCs) in Trichoderma afroharzianum ACCC 33109 have the biological activities of both hydrolytic enzymes and antimicrobial peptides to mitigate attack by phytopathogens and spread over long distances in soil. However, the biosynthesis pathway of anti-fungal VOCs has not been elucidated yet. In this study, we identified 15 genes (TaTS1–15) coding for putative terpene synthase with low identities (<79.54%) to functionally characterized homologs through genome mining. Upon Fusarium induction, the relative expression levels of nine TaTS genes were up-regulated by up to 2793-fold (TaTS9). To verify the contribution of TaTS9 to the synthesis of anti-fungal VOCs, the TaTS9 knockout mutant strain was constructed and characterized by its antagonistic activities, transcript profiles, and VOC metabolomes. Heterologous expression of TaTS9 in Escherichia coli produced the target gene product, which converted the precursor farnesyl pyrophosphate (FPP) into β-cubenene (>90%) and γ-amorphene. Thus, TaTS9 was confirmed as the first β-cubenene synthase of Trichoderma, which catalyzes the biosynthesis of various sesquiterpenes with anti-fungal activities. This study provides insight into the key terpene synthase gene in the biosynthesis of anti-fungal sesquiterpenoids for potential applications in the agriculture and food industries.