Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica
Abstract
Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 ( Snf1 ) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase ( ACL ) and diacylglycerol acyltransferase 1 ( DGA1 ) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases- URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth andmore »
- Authors:
- Publication Date:
- Research Org.:
- National Renewable Energy Lab. (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
- OSTI Identifier:
- 1837321
- Alternate Identifier(s):
- OSTI ID: 1841967
- Report Number(s):
- NREL/JA-2700-81552
Journal ID: ISSN 1664-302X; 757741
- Grant/Contract Number:
- AC36-08GO28308
- Resource Type:
- Published Article
- Journal Name:
- Frontiers in Microbiology
- Additional Journal Information:
- Journal Name: Frontiers in Microbiology Journal Volume: 12; Journal ID: ISSN 1664-302X
- Publisher:
- Frontiers Media SA
- Country of Publication:
- Switzerland
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS; ATP citrate lyase; cellobiohydrolase I; consolidated bioprocessing; diacylglycerol acyltransferase; endoglucanase II; fungal cellulolytic enzymes; lipid metabolism; Snf1 deletion; sucrose non-fermenting 1 gene; Yarrowia lipolytica
Citation Formats
Wei, Hui, Wang, Wei, Knoshaug, Eric P., Chen, Xiaowen, Van Wychen, Stefanie, Bomble, Yannick J., Himmel, Michael E., and Zhang, Min. Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica. Switzerland: N. p., 2021.
Web. doi:10.3389/fmicb.2021.757741.
Wei, Hui, Wang, Wei, Knoshaug, Eric P., Chen, Xiaowen, Van Wychen, Stefanie, Bomble, Yannick J., Himmel, Michael E., & Zhang, Min. Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica. Switzerland. https://doi.org/10.3389/fmicb.2021.757741
Wei, Hui, Wang, Wei, Knoshaug, Eric P., Chen, Xiaowen, Van Wychen, Stefanie, Bomble, Yannick J., Himmel, Michael E., and Zhang, Min. Thu .
"Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica". Switzerland. https://doi.org/10.3389/fmicb.2021.757741.
@article{osti_1837321,
title = {Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica},
author = {Wei, Hui and Wang, Wei and Knoshaug, Eric P. and Chen, Xiaowen and Van Wychen, Stefanie and Bomble, Yannick J. and Himmel, Michael E. and Zhang, Min},
abstractNote = {Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 ( Snf1 ) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase ( ACL ) and diacylglycerol acyltransferase 1 ( DGA1 ) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases- URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.},
doi = {10.3389/fmicb.2021.757741},
journal = {Frontiers in Microbiology},
number = ,
volume = 12,
place = {Switzerland},
year = {Thu Dec 23 00:00:00 EST 2021},
month = {Thu Dec 23 00:00:00 EST 2021}
}
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