Constitutive Overexpression of CRZ1 in <i>Trichoderma reesei</i> Reveals Its Ability to Enhance Recombinant Lipase Production

The filamentous fungus <i>Trichoderma reesei</i> is extensively utilized in the realm of recombinant protein expression owing to its well-established protein production systems. However, the potential for efficient and convenient protein production in <i>T. reesei</i> has not been fully harnessed. To further increase the production of recombinant lipase <i>Candida antarctica</i> lipase B (CalB), we overexpressed seven transcription activators and found that overexpression of the calcineurin CRZ1 could significantly enhance CalB production by 2.08-fold, and then through employing a synthetic strong promoter Pcbh7IR and knocking out <i>ace1</i>, a competitor of <i>crz1</i>, CalB production was further increased 4.2-fold. Furthermore, we found that increasing the Ca²⁺ concentration enabled the enhancement of CalB production in the CRZ1-overexpressing strains. Additionally, transcription analysis revealed a significant upregulation of calcium-related pathway genes and ER chaperone genes in <i>T. reesei</i>, revealing that overexpression of CRZ1 can increase the production of the recombinant protein by activation of the calcium signaling pathway. In this study, by systematically manipulating the recombinant protein expression module and its regulatory transcription activators, we found a positive effect of <i>crz1</i>-mediated calcium channels in boosting the expression of the recombinant protein.