Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer

Abstract

Background: Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing. Results: We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn 2 Cys 6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer. Conclusions: This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila

“…A previous report suggested that removing the C-terminal segment of the ACE3 regulator increased cellulase production in T. reesei ( 53 ). Comparative analysis of NcCLR-2 and MtCLR-2 revealed an extra segment in the C terminus of MtCLR-2 compared with its ortholog in N. crassa after 722 aa.…”

Lignocellulolytic Biocatalysts: The Main Players Involved in Multiple Biotechnological Processes for Biomass Valorization

“…In T. reesei , for example, the transcription factor CRE1 is the main repressor of lignocellulolytic enzyme expression, while XYR1 is its main transcriptional activator. Mutations that caused the first silencing and the second’s overexpression demonstrated elevated levels of extracellular cellulase secretion, producing hypersecretory T. reesei strains [ 359 , 360 ]. The combinatorial engineering of three transcriptional activators in P. oxalicum (ClrB, XlnR, and AraR) also demonstrated the generation of a strain with an increase in lignocellulolytic enzymes production from 3.1 to 51.0-fold, in addition to a more significant release of fermentable sugars from corn fiber, when compared with the original strain enzymes [ 361 ].…”

Factors regulating cellulolytic gene expression in filamentous fungi: an overview

“…This finding was further corroborated by a recent study in which different variants of Ace3, having a common Zn2Cys6 domain in the N-terminal and different truncations in the C-terminal region were expressed in T. reesei . It was observed that a truncation of 7–17 amino acids in the C-terminal region led to a 1.5–twofold increase in total cellulase production in presence of glucose, and a threefold increase in activity in presence of lactose, whereas a longer truncation of 20–25 amino acids led to severe reduction in protein production [ 63 ]. The authors have suggested two possibilities for this interesting observation—either the presence of an inhibitory domain in this region or the occurrence of a stretch of hydrophobic residues, which could interact with other transcription factors leading to reduced expression [ 63 ].…”