Abstract
To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6–2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.
Highlights
One of the main challenges facing industrial bioethanol production from lignocellulose is the large amount of cellulase enzymes required for the hydrolysis of cellulose[4, 7]
Each gene encoding the mature part of the cellulase was amplified using polymerase chain reaction (PCR) using the primers indicated in Supplementary Table S1, and cloned into the YGaTFPn vectors harbouring 24 different translational fusion partners (TFP) (Table 2) under the control of the GAL10 promoter and GAL7 terminator on a URA3-selectable episomal multi-copy vector, by in vivo recombination (Fig. 2a)
The following transformants were identified as showing the best secretion of cellulases: TFP 13 for ClCBH2, HgCBH1, NfCBH1, CfCex, TrCBH1, TrCBH2, PaCel[1], and PaCel[2]; TFP 8 for TeCBH1; the
Summary
One of the main challenges facing industrial bioethanol production from lignocellulose is the large amount of cellulase enzymes required for the hydrolysis of cellulose[4, 7]. Fan et al.[16] accomplished bioethanol production through the use of mini-cellulosomes, including EGL, CBHs, and BGL, on the cell surface in S. cerevisiae, whereas a low ethanol production yield (about 27%) was obtained from Avicel, with similar crystallinity of pre-treated cellulosic biomass[17]. This cellulase secretion system could reduce the total production cost for the cellulosic bioethanol and be useful for yeast biorefineries to produce valuable biochemicals from lignocellulosic biomass
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