Abstract
BackgroundThe conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear.ResultsThe T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed.ConclusionsThe T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of β-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0550-3) contains supplementary material, which is available to authorized users.
Highlights
The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits
Recombinant T. reesei strain TRB1 shows β‐glucosidase hyper‐production and faster cellulase induction Gene bgl1 from T. reesei was cloned into plasmid pDht/sk under a modified CBH1 promoter [21], resulting in the plasmid construction pBGL (Fig. 1a). pBGL was transformed into T. reesei RUT-C30 by Agrobacterium tumefaciens-mediated transformation (AMT) method and five T. reesei transformants were obtained: TRB1, TRB2, TRB3, TRB4 and TRB5
CMC activity), the the CBH activity (pNPCase) activity and the the filter paper activity (FPase) activity in strain TRB1 on day 3 were increased by 23, 8.7, 3.3- and 22.4fold, respectively
Summary
The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Extracellular enzyme mixture, mainly consisting of three synergistic enzymes participating in the degradation of cellulose: endoglucanase (EG, EC3.2.1.4), exoglucanase (or cellobiohydrolase, CBH, EC 3.2.1.91), and β-glucosidase (BGL, EC 3.2.1.21) [1]. T. reesei has low β-glucosidase activity, which reduces efficiency in biomass degradation and compromises its industrial application [4]. There are ongoing interests in research to increase β-glucosidase amount in the cellulase complex from T. reesei. The supplementation of β-glucosidase produced by other fungi to the T. reesei cellulase preparations has been employed to increase the enzyme efficiency of hydrolyzing cellulosic substrates [6, 7]. In spite of all the genetic efforts which have been done, there is still no available T. reesei strain which could produce cellulase with optimal amounts for different components
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