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

Abstract

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:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1373671
Report Number(s):
NREL/JA-2700-67652
Journal ID: ISSN 1475-2859
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 16; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Lipomyces starkeyi; oleaginous yeast; heterologous expression; cellulase; cellobiohydrolase I; endoglucanase II; advanced biofuels

Citation Formats

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., 2017. 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. Mon . "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},
issn = {1475-2859},
number = 1,
volume = 16,
place = {United States},
year = {2017},
month = {7}
}

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Figures / Tables:

Fig. 1 Fig. 1: Diagram of gene cassettes and vector used for cellulase expression in L. starkeyi. a Cellulase gene expression cassette; b the base vector, in which the star denotes where genes of interest are cloned into. Bla, beta-lactamase gene for ampicillin resistance; PGK1 trm, 3-phosphoglycerate kinase gene’s terminator; pPYK, promotermore » of L. starkeyi pyk (pyruvate kinase gene); spAMY, signal peptide of L. starkeyi amylase; spDEX1, signal peptide of L. starkeyi dextranase 1; spDEX2, signal peptide of L. starkeyi dextranase 2; spPRO, signal peptide of lipolytica protease; TDH3 pro, glyceraldehyde-3-phosphate dehydrogenase gene’s promoter; tGAL1, terminator of L. starkeyi gal1 (galactokinase 1 gene); 2μ, . cerevisiae 2-micron origin of replication. Gene, cellulase gene without native signal peptide. Size of various signal peptides is described in Table 2, while the size of chimeric CBH I and EG II is 500 and 397 in amino acid residues, respectively; the size of fusion genes that encode signal peptide-cellulase can be deduced accordingly« less

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Works referenced in this record:

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    Works referencing / citing this record:

    Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi [Supplemental Data]
    dataset, July 2017

    • Xu, Qi; Knoshaug, Eric; Wang, Wei
    • figshare-Supplementary information for journal article at DOI: 10.1186/s12934-017-0742-5, 1 PDF file; 1 XLSX file
    • DOI: 10.6084/m9.figshare.c.3834790

    MOESM1 of Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi
    dataset, July 2017

    • Xu, Qi; Knoshaug, Eric; Wang, Wei
    • Figshare-Supplementary information for journal article at DOI: 10.1186/s12934-017-0742-5, 1 xlsx file (1.73 MB)
    • DOI: 10.6084/m9.figshare.c.3834790_d1

    MOESM2 of Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi
    dataset, July 2017

    • Xu, Qi; Knoshaug, Eric; Wang, Wei
    • figshare-Supplementary information for journal article at DOI: 10.1186/s12934-017-0742-5, 1 PDF file (195.75 kB)
    • DOI: 10.6084/m9.figshare.c.3834790_d2