Abstract
BackgroundFilamentous 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.ResultsIn 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.ConclusionsAn 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.
Highlights
Filamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions
Effects of XYR1 overexpression and ace1 disruption on xylanase production In T. reesei, the induction of xylanase was triggered by cellulose, xylan and its polysaccharides
To enhance the expression of different xylanolytic genes and increase xylanase production, we choose two well-characterized transcription factors, XYR1 and ACE1, which act as an activator and repressor for cellulase and xylanase production, respectively
Summary
Effects of XYR1 overexpression and ace disruption on xylanase production In T. reesei, the induction of xylanase was triggered by cellulose, xylan and its polysaccharides. The cellulase and xylanase activity of C30OExyr was higher than that of C30OExyr1Δace (Fig. 2d, e), which was similar to the trend in Avicel medium, suggesting that the negative effect of ace disruption while XYR1 overexpression is carbon source independent. For strain C30OExyr1Δace that lacks intact ace, the upregulated transcription level of xyn, bxl and axe were detected both at 24 h and 48 h, which was consistent with the enzyme activity data (Fig. 2a, b). C30OExyr1Δace1/xyn2Δcbh shows the upregulation of seven selected xylanolytic genes (Fig. 5a), especially in xyn and xyn, which means a significant increase in GH11 endo-xylanase activity. The transcription level of xyn, bxl, axe and abf was increased by at least onefold in C30OExyr1/xyn2Δcbh (Fig. 5a), suggesting the upregulation of different xylanolytic genes. Further increase in xylanase proportion gave a total 15% decrease in glucose yield, which might be attributed to the unbalanced proportion of cellulase and xylanase
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