Precision engineering of the transcription factor Cre1 inHypocrea 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. To our knowledge, this is the first report demonstrating an improvement of cellulase production in fungal species 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 fungal species under CCR. AbbreviationsCCR: carbon catabolite repression; RT-qPCR: reverse transcription quantitative polymerase chain reaction; PKA: cyclic adenosine monophosphate (cAMP)-dependent protein kinase A; FP: filter paper

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

“…The authors gratefully acknowledge Prof. Luying Xun's help in revising this manuscript. The part contents in this article have been released as a pre-print at BioRxiv (Han et al, 2020).…”

Analysis of the phosphorylome of trichoderma reesei cultivated on sugarcane bagasse suggests post-translational regulation of the secreted glycosyl hydrolase Cel7A

“…Interestingly, the phosphorylation of Cre1 at S241 is required for DNA binding [8] . Recently, Han et al, in order to evaluate the influence of phosphorylation on the transcription factor Cre1 in T. reesei and its effect on the expression of cellulolytic enzymes, mutated several residues at C terminus of Cre1 to mimic dephosphorylation and observed that there was an improvement in cellulase transcription levels and activity in the presence of glucose and inhibition of carbon catabolite repression [9] .…”

Parallel proteomic and phosphoproteomic analyses reveal cellobiose‐dependent regulation of lignocellulase secretion in the filamentous fungus Neurospora crassa

“…Post‐translational modifications, particularly phosphorylation and dephosphorylation, may play a critical role in signal perception, transmission, and subsequent cellular responses during adaptation of filamentous fungi to plant cell wall polysaccharides (Horta et al, 2019; Rodriguez‐Iglesias & Schmoll, 2019; Schmoll, 2018; Xiong, Coradetti, et al, 2014). Phosphorylation of CRE‐1 has been found to be required for binding to its target sequences in Trichoderma reesei and for triggering CCR (Cziferszky et al, 2002; Han et al, 2020). Six non‐essential protein phosphatases were found to be positive or negative regulators for lignocellulase production in T .…”