Simultaneous manipulation of multiple genes within a same regulatory stage for iterative evolution of Trichoderma...

While there is growing interest in developing non-canonical filamentous fungi as hosts for producing secretory proteins, genetic engineering of filamentous fungi for improved expression often relies heavily on the understanding of regulatory mechanisms.

In this study, using the cellulase-producing filamentous fungus Trichoderma reesei as a model system, we designed a semi-rational strategy by arbitrarily dividing the regulation of cellulase production into three main stages-transcription, secretion, and cell metabolism. Selected regulatory or functional genes that had been experimentally verified or predicted to enhance cellulase production were overexpressed using strong inducible or constitutive promoters, while those that would inhibit cellulase production were repressed via RNAi-mediated gene silencing. A T. reesei strain expressing the surface-displayed DsRed fluorescent protein was used as the recipient strain. After three consecutive rounds of engineering, the cellulase activity increased to up to 4.35-fold and the protein concentration increased to up to 2.97-fold in the genetically modified strain.

We demonstrated that, as a proof-of-concept, selected regulatory or functional genes within an arbitrarily defined stage could be pooled to stimulate secretory cellulase production, and moreover, this method could be iteratively used for further improvement. This method is semi-rational and can essentially be used in filamentous fungi with little regulatory information.