Rational Engineering of Xylanase Hyper-producing System in Trichoderma Reesei for Efficient Biomass Degradation

Background: Filamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation.

Results: In this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310-3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone.

Conclusions: An efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.