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Title: 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 themore » 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.« less

Authors:
 [1];  [1]; ORCiD logo [1]; ORCiD logo [1];  [1];  [1];  [1];  [1];  [1]
  1. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1500069
Report Number(s):
NREL/JA-2700-71429
Journal ID: ISSN 1754-6834
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; fusion protein; oleaginous yeast; CBHI; consolidated bioprocessing; cellulase; cellobiohydrolase

Citation Formats

Xu, Qi, Alahuhta, Petri M, Wei, Hui, Knoshaug, Eric P, Wang, Wei, Baker, John O, VanderWall, Todd A, Himmel, Michael E, and Zhang, Min. Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. United States: N. p., 2018. Web. doi:10.1186/s13068-018-1301-y.
Xu, Qi, Alahuhta, Petri M, Wei, Hui, Knoshaug, Eric P, Wang, Wei, Baker, John O, VanderWall, Todd A, Himmel, Michael E, & Zhang, Min. Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. United States. doi:10.1186/s13068-018-1301-y.
Xu, Qi, Alahuhta, Petri M, Wei, Hui, Knoshaug, Eric P, Wang, Wei, Baker, John O, VanderWall, Todd A, Himmel, Michael E, and Zhang, Min. Mon . "Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi". United States. doi:10.1186/s13068-018-1301-y. https://www.osti.gov/servlets/purl/1500069.
@article{osti_1500069,
title = {Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi},
author = {Xu, Qi and Alahuhta, Petri M and Wei, Hui and Knoshaug, Eric P and Wang, Wei and Baker, John O and VanderWall, Todd A and Himmel, Michael E and Zhang, Min},
abstractNote = {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.},
doi = {10.1186/s13068-018-1301-y},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 11,
place = {United States},
year = {2018},
month = {12}
}

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