A high performance Trichoderma reesei strain that reveals the importance of xylanase III in cellulosic biomass conversion.

The ability of the Trichoderma reesei X3AB1strain enzyme preparations to convert cellulosic biomass into fermentable sugars is enhanced by the replacement of xyn3 by Aspergillus aculeatus ß-glucosidase 1 gene (aabg1), as shown in our previous study. However, subsequent experiments using T. reesei extracts supplemented with the glycoside hydrolase (GH) family 10 xylanase III (XYN III) and GH Family 11 XYN II showed increased conversion of alkaline treated cellulosic biomass, which is rich in xylan, underscoring the importance of XYN III. To attain optimal saccharifying potential in T. reesei, we constructed two new strains, C1AB1 and E1AB1, in which aabg1 was expressed heterologously by means of the cbh1 or egl1 promoters, respectively, so that the endogenous XYN III synthesis remained intact. Due to the presence of wild-type xyn3 in T. reesei E1AB1, enzymes prepared from this strain were 20-30% more effective in the saccharification of alkaline-pretreated rice straw than enzyme extracts from X3AB1, and also outperformed recent commercial cellulase preparations. Our results demonstrate the importance of XYN III in the conversion of alkaline-pretreated cellulosic biomass by T. reesei.

Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation.

Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

A comprehensive analysis of the effects of the main component enzymes of cellulase derived from Trichoderma reesei on biomass saccharification.

The aim of this study was a comprehensive analysis of the effects of the component enzymes of cellulase derived from Trichoderma reesei strain PC-3-7 on biomass saccharification. We used cellulases with deleted CBH I, CBH II, or EG I, which contain all other component enzymes, for saccharification of differently pretreated biomasses of rice straw, Erianthus, eucalyptus, and Japanese cedar. We found that CBH I was the most effective in saccharification of all pretreated cellulosic biomasses, although the effect was weaker in saccharification of sulfuric acid- and hydrothermally pretreated rice straw than of others; CBH II was more effective for rice straw than for eucalyptus, and was the most effective at the early stages of biomass degradation; EG I had little effect on pretreated biomasses, in particular, it had no effect on steam-exploded Japanese cedar. Thus, the effects of the main component enzymes depend on the biomass source and pretreatment. These findings will likely help to improve cellulase for industrial use.

Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterparts.

Although the capabilities of Trichoderma reesei cellulases have been greatly improved, these enzymes are still too costly for commercial use. The aim of this research was to assess the biomass saccharification capability of JN11, a recombinant cellulase, compared with that of the commercially available cellulases Accellerase 1500 and Cellic CTec. The activities of JN11, Accellerase 1500, and Cellic CTec were compared by using various types of cellulosic biomass, including rice straw, Erianthus, eucalyptus, and Japanese cedar. JN11 had higher saccharification capability for rice straw, Erianthus, eucalyptus, and Japanese cedar compared with the commercial cellulases. The JN11 saccharification of cellulosic biomasses, including hemicellulose (NaOH-pretreated biomasses), resulted in high glucose and xylose yields because of the high xylanase/xylosidase activity of JN11. Moreover, even JN11 saccharification of hemicellulose-free biomasses (sulfuric acid-, hydrothermally, and steam exploded-pretreated biomasses) resulted in high glucose yields. The cellulase activity of JN11, however, was comparable to that of its commercial counterparts. These findings indicate that the saccharification ability of cellulase is unrelated to its cellulase activity when measured against Avicel, CMC, pNP-lactoside, and other substrates. JN11 showed high activity for all types of pretreated cellulosic biomasses, indicating its usefulness for saccharification of various cellulosic biomasses.

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus ß-glucosidase 1 for efficient biomass conversion.

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus ß-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher ß-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous ß-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and ß-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.