Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1301-y) contains supplementary material, which is available to authorized users.

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

“…The recombinant TeTr CBH I enzymes were purified and used for further characterization. Our prior work with the CBH I-endoglucanase II fusion protein showed variability in the active protein expression levels and in biomass deconstruction efficacy when expressed in different yeast species [ 26 ]. To better understand the basis of these differences in CBH I performance, without interference from the fusion partner, we planned a series of experiments investigating the yields of purified and active TeTr CBH I enzyme, sample heterogeneity (e.g., glycosylation and charge differences), thermal and proteolytic stability, and performance.…”

“…The chimeric T. emersonii – T. reesei cbh1 ( TeTrcbh1 ) gene was expressed in L. starkeyi driven by the L. starkeyi native pyruvate kinase (pyk) promoter detailed previously [ 25 ]. Wild-type (WT) L. starkeyi NRRL Y-11557 used as the transformation host was acquired from the ARS Culture Collection (NRRL) and was transformed as previously described [ 25 , 42 – 43 ]. Briefly, a single colony of L. starkeyi was inoculated into YPD medium and incubated at 30 °C and shaken at 225 rpm until OD 600 reached 8.0.…”

“…To enable CBP capabilities in yeast, significant secretion levels of cellulases, particularly cellobiohydrolase I (CBH I) are needed [ 1 , 18 ]. S. cerevisiae [ 7 , 19 – 21 ], Y. lipolytica [ 22 – 24 ], L. starkeyi [ 25 ], or all three hosts [ 26 ] are often studied examples of yeast CBP organisms. Still, low secretion levels and yields of active enzymes have affected their usability [ 27 – 29 ].…”

“…Another strategy employed for fungal cellulase expression is making chimeric enzymes from domains of different fungal species that may be more easily secreted, such as the Talaromyces emersonii catalytic domain connected to the T. reesei linker peptide and carbohydrate binding module (CBM1) forming a chimeric TeTr CBH I [ 24 ]. Similarly, a fusion protein constructed with an easily secreted leading enzyme, endoglucanase II from T. reesei , followed by the more difficult to secrete TeTr CBH I was constructed and shown to be effective in improving cellulase secretion in L. starkeyi and Y. lipolytica [ 26 ]. Recently, this approach was taken further by introducing a translational fusion partner protein and screening for cellulases that were shown to have better secretion and improved ethanol production in S. cerevisiae [ 35 ].…”

“…Although, there are some reports about characterization of heterologously expressed CBH I enzymes in a specific yeast host, most are limited to some individual property relevant to that specific host, and few of them have involved systematic analysis using various approaches across a diversity of yeast. A notable exception is our prior work comparing the endoglucanase II TeTr CBH I fusion construct in three yeasts [ 26 ]. While the goal of the fusion construct work was mainly aimed at understanding the benefits of the fusion, the results suggested significant TeTr CBH I expression and activity variations between yeasts.…”