Artificial gene clusters designed for simultaneous multi-gene expression enable reconstruction of metabolite biosynthesis pathways in Trichoderma reesei

The filamentous fungus Trichoderma reesei represents a highly promising chassis cell for the efficient synthesis of both protein and non-protein metabolites. Despite the availability of diverse genetic manipulation tools for T. reesei , the engineering of chassis cells frequently requires coordinated regulation of multiple genes, a process that remains laborious and time-consuming with current methods. Here, we designed a modular and inducible artificial gene cluster (AGC) by repurposing functional DNA elements from the quinic acid (QA)-responsive gene cluster in T. reesei . This AGC is capable of expressing up to six functional genes for the assembly of genetic circuits. Under culture conditions utilizing a mixed carbon source of glycerol and QA, both robust cellular vegetative growth and activation of the gene cluster are compatible. Moreover, we identified qai1 as a gene encoding a putative transcriptional activator that governs the expression of the qai gene cluster. Notably, its overexpression alone is sufficient to bypass QA-sensing induction. Leveraging this insight, we engineered a QA-independent, constitutively expressed version of the AGC. As a proof of concept, we reconstructed the biosynthetic pathways for ilicicolin H and erythritol in T. reesei using the above QA-dependent and QA-independent AGCs, successfully achieving the synthesis of ilicicolin H and enhanced erythritol production in the T. reesei chassis cells. In summary, our study establishes novel genetic manipulation tools for engineering T. reesei chassis cells, allowing for rapid, multiplexed gene expression regulation to facilitate metabolite biosynthesis.