Precision Engineering of the Transcription Factor Cre1 in Hypocrea jecorina (Trichoderma reesei) for Efficient Cellulase Production in the Presence of Glucose

Abstract

In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1 S387V , Tr_Cre1 S388V , Tr_Cre1 T389V , and Tr_Cre1 T390V , respectively. Transcription of cel7a in Tr_ Cre1 S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1 S388V were significantly increased by up to 2-to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR.

Network Analysis Reveals Different Cellulose Degradation Strategies Across Trichoderma harzianum Strains Associated With XYR1 and CRE1

“…It has been reported that cellulase gene expression can be regulated by the dynamics of protein phosphorylation and dephosphorylation, which involve protein kinases and phosphatases, respectively ( Schmoll et al, 2016 ). Furthermore, in filamentous fungi, phosphorylation is a prerequisite for CRE1 activity ( Cziferszky et al, 2002 ; Han et al, 2020 ; de Assis et al, 2021 ). Therefore, in T. harzianum , it is important to elucidate the role of kinases and phosphatases in the regulation of CRE1 function.…”

“…Upon glucose depletion, the concentration of CRE1 in the nucleus rapidly decreases, and CRE1 is recycled into the cytoplasm ( Lichius et al, 2014 ). Furthermore, the phosphorylation of CRE1 plays an essential role in signal transduction to achieve CCR ( Horta et al, 2019 ; Han et al, 2020 ). CRE1 is the only conserved TF throughout the fungal kingdom, suggesting a conserved mechanism for CCR in fungi ( Adnan et al, 2017 ).…”

Carbon catabolite repression involves physical interaction of the transcription factor CRE1/CreA and the Tup1–Cyc8 complex in Penicillium oxalicum and Trichoderma reesei

“…The presence of carbon sources e.g., glucose and related sugars represses the transcription of certain genes. As a sequence-specific TF, CRE1/CreA plays a central role in CCR and is essential for the adaptation and survival of several species, such as Aspergillus , Penicillium , and Trichoderma [ 8 , 13 , 15 ]. In T. reesei , TrCRE1 rapidly shifts from cytoplasmic to nuclear with glucose addition [ 14 ] and represses the expression of glucose-repressible cellulolytic genes (such as cbh2 or egl1 ) [ 51 ].…”

“…Cellulolytic gene induction requires a release from CCR. Therefore , the deletion, truncation, or multisite-directed mutagenesis of gene cre1 / creA can alleviate CCR and thus improve the expression level of prominent cellulolytic genes in various carbon sources, such as glucose, lactose, sophorose, cellulose, or a mixture of plant polymers [ 5 , 15 – 18 ]. For example, either the deletion or truncation of cre1 in T. reesei wild-type strain QM6a leads to de-repressed production of cellulase and hemicellulase, when the mutants are cultivated in glucose-containing media [ 19 ].…”

Induction and Repression of Hydrolase Genes in Aspergillus oryzae

“…However, none of the substitutions of these phosphorylation sites with alanine had a significant effect on CreA degradation under de-repressing conditions ( de Assis et al, 2021 ). A recent study showed that replacement of S388 at the T. reesei Cre1 C-terminus with valine releases CCR ( Han et al, 2020 ). This serine residue is conserved in the Aspergillus CreA proteins.…”