Expression of an endoglucanase-cellobiohydrolase fusion protein in and .

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 eg2 (EGII) 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: and . Our initial assays with the secretome expressing differing EGII domains fused to a chimeric - CBHI (CBHI) showed that the complete EGII 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 (EGII-CBHI, Fusion 3) had an increased secretion level of at least threefold in compared to the expression level of the chimeric CBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in and . Digestion of pretreated corn stover with the secretomes of and showed that conversion was much better using secretomes (50% versus 29%, respectively). In , CBHI performed better than the fusion construct. Furthermore, expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, NP-cellobiose. No activity was observed for the NP-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 was significantly higher than that of the GH7 domain in CBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just CBHI. We conclude that the strategy of leading CBHI expression with a linked EGII module significantly improved the expression of active CBHI in .