Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3

Abstract

Background: The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low. Results: In this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20-30% improvement to that of the parental strain PC-3-7. Conclusions: This work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Penicillium Janthinellum NCIM-1366 Shows Improved Biomass Hydrolysis Performance Over Trichoderma Reesei RUT-C30, and Secretome Analysis Reveals a Larger Number of CAZymes with Higher Induction Levels.

“…Lignocellulose degrading enzymes are critical in biomass conversion to biofuels and lamentous fungi are typically used for the production of these enzymes because of their ability to synthesize and secrete a wide array of plant cell wall degrading enzymes [13]. T. reesei RUT-C30 is the most widely used fungus for cellulase production, in the past several decades, and efforts for improving its enzyme production are still at large [14,15]. P. janthinellum NCIM 1366 is a mutant strain developed at the National Chemical Laboratory, Pune, India through classical mutagenesis and which exhibited enhanced cellulase production compared to the parent strain NCIM 1171 [9].…”

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30

“…This is primarily due to the fact that T. reesei produces the highest known titers of enzymes, the extracellular protein concentrations reaching as high 100g/L [19]; and there is a wealth of information accumulated on its genetics and gene regulation through works spanning several decades [20]. However, there are still efforts targeted at improving its enzyme production [21,22] as the cost of cellulases cannot yet be considered as economical for bio-re nery operations. P. janthinellum NCIM 1366 is a mutant strain developed at the National Chemical Laboratory, Pune, India through classical mutagenesis and which exhibited enhanced cellulase production compared to the parent strain NCIM 1171 [9].…”

“…Proteomic approaches have been widely used in lamentous fungi for the identi cation of both intracellular and extracellular proteins [21]. The genome of T. reesei QM6a, which is the parent strain of RUT-C30 was rst sequenced in 2008 giving insight into its CAZyme system [22]. T. reesei is known to encode at least 10 cellulases, 16 hemicellulases and a total of around 400 CAZymes in its genome.…”

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

“…The 11-amino acid C-terminal truncation is an SNP that arose from random mutagenesis in the NG14 strain [4]. The C-terminus has been shown to be of importance for ACE3 and some other Zn finger regulators [23,33]. This led us to further map important residues at the ACE3 C-terminus by generating a set of serial truncations.…”

“…The authors also proposed that ACE3 and XYR1 may interact through their corresponding C-termini, forming homodimers and/or a heterodimer complex. Interestingly, an 11-amino acid truncation due to a premature stop codon at the ACE3 C-terminus that arose in strain NG14 during a mutagenesis experiment by Sheir-Ness and Monentencourt [4] has been shown to account for increased cellulase production in strain NG14 and its derivative strains of RUT C-30 and RL-P37 [23]. In addition, there is an atypically large intron near the 5′ end of the ace3 gene.…”