Use of fusion transcription factors to reprogram cellulase transcription and enable efficient cellulase production in Trichoderma reesei

Abstract

Background Trichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production. Results In this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of Δcre1 strain. Conclusions A new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.

Precision engineering of the transcription factor Cre1 inHypocrea jecorina (Trichoderma reesei)for efficient cellulase production in the presence of glucose

“…The primers used for the construction of cre1 mutation replacement cassettes are listed in Supplemental Table S1. The transformation of pUG-S387V, pUG-S388V, pUG-T389V, and pUG-T390V replacement cassettes into T. reesei was performed using previously described methods (Wang et al, 2019). The transformants were selected on plates containing minimal medium (MM) supplemented with 2% glucose and 200 µg/mL hygromycin.…”

“…The transformants were selected on plates containing minimal medium (MM) supplemented with 2% glucose and 200 µg/mL hygromycin. The composition of MM was based on a protocol described elsewhere (Wang et al, 2019). A schematic diagram of cre1 replacement cassettes is shown in Fig.…”

“…S1. Propagation of transformant strains was performed based on a protocol published previously (Wang et al, 2019).…”

“…Carbon catabolite repression (CCR) is a global regulatory system that is found in nearly all heterotrophic hosts (Gorke and Stulke, 2008). Notably, the presence of monosaccharides, like D-glucose, during cellulolytic enzyme production triggers CCR, which is regulated mainly by the cellulase transcriptional repressor Cre1 and activators Xyr1, Clr1, Clr2, etc., and significantly downregulates the transcription of cellulolytic enzymes in T. reesei (Wang et al, 2019). Endogenous Cre1, which belongs to the group of C2H2-type zinc finger proteins, binds to the promoters of target genes, such as cel7a, and inhibits the transcription of encoded cellulase genes (Mello-de-Sousa et al, 2014).…”

“…Moreover, it was shown that the disruption of cre1 in T. reesei, or its replacement with a cre1 mutant, inhibits biomass accumulation and leads to a decrease in cellulase production (Nakari-Setä lä et al, 2009;Wang et al, 2019). Furthermore, it was reported that CCR is achieved with the phosphotransferase system (Wang et al, 2017;Liu et al, 2020).…”