High-yield trichodermin production in Trichoderma taxi via in silico-driven exporter identification and systems...

Trichodermin, a sesquiterpenoid with notable antifungal, plant growth-regulating, and antitumor activities, holds broad application potential in medicine and agriculture. However, its low native production in Trichoderma presents a significant bottleneck, hindering the large-scale utilization and development of high-value pharmaceutical derivatives. In this study, a systematic metabolic engineering framework was established in the native producer T. taxi to enhance trichodermin production. A foundational genetic toolbox was first developed, enabling the robust overexpression, knockout, and knockdown of genes. This system was validated using the tri5 gene as a model, facilitating iterative rounds of strain engineering. A subsequent key outcome was the identification of the ABC transporter G5728 as a central trichodermin efflux pump, achieved through an integrated in silico strategy combining homology mining, transcriptomics, and molecular docking. Overexpression of G5728 resulted in a 70.9% increase in trichodermin production, achieving a titer of 2.0 g/L. Building on this, four key modules were systematically engineered: enhancement of acetyl-CoA precursor supply, MVA pathway flux, and trichodermin biosynthesis, along with the downregulation of competing pathways. This integrated approach led to the construction of the high-yield T. taxi strain LH58, which achieved a trichodermin titer of 7.22 g/L in a 5-L bioreactor. This represents a substantial advancement in productivity, highlighting its potential for industrial-scale biomanufacturing. This work provides a reliable platform for the sustainable production of trichodermin and offers a transferable strategy for optimizing terpenoid biosynthesis in microbial hosts.