Year-end Sale % OFF 
Abstract
The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii–T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII–TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.
Highlights
Cellulose and lignin present in biomass are the most abundant forms of organic carbon on Earth
We reported previously that the secretion level of T. reesei endoglucanase II (TrEGII) in L. starkeyi is much higher than that of a chimeric cellobiohydrolase I (CBHI) generated by the fusion of the catalytic module from Talaromyces emersonii CBHI with the linker peptide and cellulosebinding module from T. reesei CBHI (TeTrCBHI) [16]
We proposed that the full-length TrEGII or its individual domains could be fused with CBHI at the N-terminus to generate fusion proteins
Summary
Cellulose and lignin present in biomass are the most abundant forms of organic carbon on Earth. The primary bottleneck in the production of lignocellulosic biofuels is the high cost associated with release of monomeric sugars for fermentation due to recalcitrance of the plant cell wall. To overcome this difficulty, consolidated bioprocessing (CBP) has been proposed in which cellulose degradation and biofuel production are combined in a single microorganism [1, 2]. Previous research in developing yeast CBP organisms has focused on S. cerevisiae [1, 11, 12], Y. lipolytica, [13,14,15], and L. starkeyi [16] These previous studies of the expression of fungal CBHIs in yeast have encountered major challenges, such as low secretion, yield, and activity of the recombinant proteins which directly discourages the application of these yeast to CBP [17, 18]. We reported previously that the secretion level of TrEGII in L. starkeyi is much higher than that of a chimeric CBHI generated by the fusion of the catalytic module from Talaromyces emersonii CBHI with the linker peptide and cellulosebinding module from T. reesei CBHI (TeTrCBHI) [16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
