Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator

Highlights

• An artificial transcription activator xyr1-cre1_b was expressed in T. reesei Rut C30.

• The mutants displayed constitutive (hemi) cellulase production from glucose.

• FPase of 2.63 IU/mL was achieved within short time (48 h) in a 7-L fermenter.

• The crude enzyme is suitable for sugar release from alkali pretreated corn stover.

Abstract

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72 IU/mL, respectively, were achieved using glucose as sole carbon source within 48 h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved.

Introduction

Production of biofuels and bio-based chemicals from lignocellulosic biomass through biorefinery is renewable and environmentally friendly, and therefore has received great attention. The major component of lignocellulose is cellulose, which can be efficiently degraded by cellulase. However, economic production of cellulase is one of the major bottlenecks for cellulosic biorefinery (Parisutham et al., 2014). Trichoderma reesei has been widely used for production of cellulase. Currently the major challenge for cellulase production from T. reesei is that expression of cellulase genes requires expensive inducers such as cellulose and lactose, and is repressed by the cellulose degradation product glucose (Lichius et al., 2014). Development of low cost process and improvement of productivity are of great interest for economic cellulase production.

Expression of cellulolytic and xylanolytic genes in T. reesei are coordinately regulated by transcription factors such as Xyr1, Ace3 and Cre1, among which Xyr1 (Xylanase regulator 1) is the key transcriptional activator (Stricker et al., 2006), whereas Cre1 is the major negative regulator mediating carbon catabolite repression (CCR). Multiple xyr1 and cre1 binding sites exist in the promoter regions of cellulase and xylanase encoding genes (Castro et al., 2014). Overexpression of xyr1 improved production of glycoside hydrolases, whereas deletion of cre1 eliminated CCR but the catabolic de-repression was not sufficient to increase glycoside hydrolases production, suggesting that hyper-production of cellulase is still inducer dependent (Nakari-Setala et al., 2009). Although in T. reesei RUT C30, the cre1 gene was truncated which results in the loss of its DNA binding ability (Seidl et al., 2008), cellulase production of this strain is still inhibited by glucose (Lichius et al., 2014). Constitutive overexpression of xyr1 has been explored previously, but cellulase activities of the recombinant strains using glucose are very low (Lv et al., 2015, Wang et al., 2013).

It was reported a chimeric transcription activator can bind to the target DNA binding domains and regulate gene expression (Su et al., 2009). Here we designed a novel chimeric transcription activator containing the Cre1 DNA-binding domain, together with the Xyr1 DNA-binding domain and effector domain, and investigated effect of this artificial transcription factor on cellulase production of T. reesei RUT C30. We reason that constitutive expression of this artificial transcription activator in T. reesei RUT C30 can lead to improved cellulase production.