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Title: Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone

Abstract

Yarrowia lipolytica, known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica. Three fungal cellulases, Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract-peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L -1, respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ~4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain,more » suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. In conclusion, this is the first study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains.« less

Authors:
ORCiD logo [1];  [1];  [2];  [1]; ORCiD logo [1];  [1];  [1];  [1]; ORCiD logo [1];  [1];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. The Univ. of Texas at Austin, Austin, TX (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:
1490107
Alternate Identifier(s):
OSTI ID: 1491724
Report Number(s):
NREL/JA-2700-73065
Journal ID: ISSN 1664-302X
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article: Published Article
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Volume: 9; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; fungal cellulolytic enzymes; Yarrowia lipolytica; cellulosic biofuel; cellobiohydrolase I; endoglucanase II; lipid metabolism; endoplasmic reticulum stress; chemical chaperone

Citation Formats

Wei, Hui, Wang, Wei, Alper, Hal S., Xu, Qi, Knoshaug, Eric P., Van Wychen, Stefanie, Lin, Chien -Yuan, Luo, Yonghua, Decker, Stephen R., Himmel, Michael E., and Zhang, Min. Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone. United States: N. p., 2019. Web. doi:10.3389/fmicb.2018.03276.
Wei, Hui, Wang, Wei, Alper, Hal S., Xu, Qi, Knoshaug, Eric P., Van Wychen, Stefanie, Lin, Chien -Yuan, Luo, Yonghua, Decker, Stephen R., Himmel, Michael E., & Zhang, Min. Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone. United States. doi:10.3389/fmicb.2018.03276.
Wei, Hui, Wang, Wei, Alper, Hal S., Xu, Qi, Knoshaug, Eric P., Van Wychen, Stefanie, Lin, Chien -Yuan, Luo, Yonghua, Decker, Stephen R., Himmel, Michael E., and Zhang, Min. Wed . "Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone". United States. doi:10.3389/fmicb.2018.03276.
@article{osti_1490107,
title = {Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone},
author = {Wei, Hui and Wang, Wei and Alper, Hal S. and Xu, Qi and Knoshaug, Eric P. and Van Wychen, Stefanie and Lin, Chien -Yuan and Luo, Yonghua and Decker, Stephen R. and Himmel, Michael E. and Zhang, Min},
abstractNote = {Yarrowia lipolytica, known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica. Three fungal cellulases, Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract-peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L-1, respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ~4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain, suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. In conclusion, this is the first study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains.},
doi = {10.3389/fmicb.2018.03276},
journal = {Frontiers in Microbiology},
issn = {1664-302X},
number = ,
volume = 9,
place = {United States},
year = {2019},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.3389/fmicb.2018.03276

Figures / Tables:

FIGURE 1 FIGURE 1: | Pedigree of transformants expressing single or multiple cellulases and the experimental outline for transformant characterization. The details for these strains are described in Table 1. CBH, cellobiohydrolase; EG, endoglucanase; FAME, fatty acid methyl esters.

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