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

Abstract

Background 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. Results 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. Conclusions 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.

Engineering cellulases for conversion of lignocellulosic biomass

“…Perhaps the most remarkable (well-documented) example of strain engineering is the development of the RUT-C30 cellulase hyperproducer prototype strain, which is derived from strain QM6a, and shows a 20-fold increase in extracellular protein levels, reaching about 30 g/L on a lactose-containing growth medium ( Bischof et al , 2016 ). Recent tools such as RNAi-mediated gene silencing ( Sun et al , 2022 ), inducer-free expression systems ( Arai et al , 2022 ), CRISPR/Cas9-based methods ( Rantasalo et al , 2019 , Fonseca et al , 2020 ) and synthetic expression systems with engineered transcription factors and promoters ( Rantasalo et al , 2018 ) provide new tools for engineering cellulase production strains ( Mojzita et al , 2019 ). For example, Arai et al (2022) reported an inducer-free expression system for T. reesei that enables protein overproduction in glucose– containing media without inducers like cellulose, lactose and sophorose.…”

Improved Cellulase Production of Trichoderma reesei by Regulating Mycelium Morphology

“…Furthermore, the OCdc42 strain generated larger and Multiple transcription factors are responsible for the regulation of cellulase and xylanase genes [58]. In cellulase-hyperproducing strains of T. reesei, the expression of transcription factors and genes involved in cellulase production is usually upregulated, resulting in high cellulase production [59]. For example, the transcript abundance of xyr1 and cbh1 was significantly higher in the industrial cellulase-hyperproducing strain T. reesei CL847 than in its parent strain RUT C30 [60].…”

Combining manipulation of integration loci and secretory pathway on expression of an Aspergillus niger glucose oxidase gene in Trichoderma reesei

“…This, in turn, suggests that these regulatory stages can be engineered to improve cellulase production in T. reesei . Indeed, the plethora of knowledge has enabled successful improvement of cellulase production [ 10 , 11 ]. The heterologous genes that can be functionally expressed in T. reesei are rapidly expanding in these years [ 3 , 12 – 16 ].…”