Repurposing the cellulase workhorse Trichoderma reesei as a ROBUST chassis for efficient terpene production†

The comprehensive utilization of agro-industrial residues poses a persistent global challenge. Microbial fermentation is an efficient way to convert agro-industrial residues into valuable products. Trichoderma reesei is a traditional cellulase and other protein producer using agro-industrial residues as substrates. The potential of T. reesei as a chassis to produce small natural products remains untapped. Here, we successfully employed T. reesei to efficiently synthesize different terpene types. To optimize the chassis for metabolite synthesis, we deleted major (hemi-)cellulase genes along with the global regulator Lae1 to improve the efficiency of secondary metabolite biosynthesis, and overexpressed the constitutively activated transcriptional factor XYR1^A824V in MC3 (a uridine auxotrophic strain derived from T. reesei Rut-C30) to alleviate glucose repression. Through glucose, lactose, and corn steep as substrates, the production of ophiobolin F using the modified chassis was increased to 1187.06 mg L⁻¹ in shake flask fermentation and up to 3072.45 mg L⁻¹ under fed-batch fermentation. We further demonstrated the versatility of the Δlae1::xyr1/MC3-Δ10 chassis by successfully producing other fungal and plant terpenes. Collectively, our results demonstrated the potential of the Reducing Outflow and Broadened Upstream Substrate Type (ROBUST) T. reesei chassis for efficient terpene production utilizing agro-industrial residues, with important implications for terpene biosynthesis and sustainable biofabrication.