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Title: Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, themore » Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. Lastly, the effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [1] ; ORCiD logo [1] ;  [1] ;  [1] ; ORCiD logo [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States); Hubei Univ., Wuhan (People's Republic of China)
Publication Date:
Report Number(s):
NREL/JA-2700-67652
Journal ID: ISSN 1475-2859
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 16; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Lipomyces starkeyi; oleaginous yeast; heterologous expression; cellulase; cellobiohydrolase I; endoglucanase II; advanced biofuels
OSTI Identifier:
1373671

Xu, Qi, Knoshaug, Eric P., Wang, Wei, Alahuhta, Markus, Baker, John O., Yang, Shihui, Vander Wall, Todd, Decker, Stephen R., Himmel, Michael E., Zhang, Min, and Wei, Hui. Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi. United States: N. p., Web. doi:10.1186/s12934-017-0742-5.
Xu, Qi, Knoshaug, Eric P., Wang, Wei, Alahuhta, Markus, Baker, John O., Yang, Shihui, Vander Wall, Todd, Decker, Stephen R., Himmel, Michael E., Zhang, Min, & Wei, Hui. Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi. United States. doi:10.1186/s12934-017-0742-5.
Xu, Qi, Knoshaug, Eric P., Wang, Wei, Alahuhta, Markus, Baker, John O., Yang, Shihui, Vander Wall, Todd, Decker, Stephen R., Himmel, Michael E., Zhang, Min, and Wei, Hui. 2017. "Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi". United States. doi:10.1186/s12934-017-0742-5. https://www.osti.gov/servlets/purl/1373671.
@article{osti_1373671,
title = {Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi},
author = {Xu, Qi and Knoshaug, Eric P. and Wang, Wei and Alahuhta, Markus and Baker, John O. and Yang, Shihui and Vander Wall, Todd and Decker, Stephen R. and Himmel, Michael E. and Zhang, Min and Wei, Hui},
abstractNote = {Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. Lastly, the effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.},
doi = {10.1186/s12934-017-0742-5},
journal = {Microbial Cell Factories},
number = 1,
volume = 16,
place = {United States},
year = {2017},
month = {7}
}

Works referenced in this record:

Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose
journal, June 2009

Consolidated bioprocessing of cellulosic biomass: an update
journal, October 2005
  • Lynd, Lee R.; van Zyl, Willem H.; McBride, John E.
  • Current Opinion in Biotechnology, Vol. 16, Issue 5, p. 577-583
  • DOI: 10.1016/j.copbio.2005.08.009

Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters
journal, January 2000

Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production
journal, February 2007
  • Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.
  • Science, Vol. 315, Issue 5813, p. 804-807
  • DOI: 10.1126/science.1137016

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae
journal, March 1988

Bacteria engineered for fuel ethanol production: current status
journal, December 2003
  • Dien, B. S.; Cotta, M. A.; Jeffries, T. W.
  • Applied Microbiology and Biotechnology, Vol. 63, Issue 3, p. 258-266
  • DOI: 10.1007/s00253-003-1444-y

Microbial Cellulose Utilization: Fundamentals and Biotechnology
journal, December 2002
  • Lynd, L. R.; Weimer, P. J.; van Zyl, W. H.
  • Microbiology and Molecular Biology Reviews, Vol. 66, Issue 4, p. 739-739
  • DOI: 10.1128/MMBR.66.4.739.2002

Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae
journal, January 2007