Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica
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 express...
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Frontiers Media S.A.
2021-12-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2021.757741/full |
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| author | Hui Wei Wei Wang Eric P. Knoshaug Xiaowen Chen Stefanie Van Wychen Stefanie Van Wychen Yannick J. Bomble Michael E. Himmel Min Zhang |
| author_facet | Hui Wei Wei Wang Eric P. Knoshaug Xiaowen Chen Stefanie Van Wychen Stefanie Van Wychen Yannick J. Bomble Michael E. Himmel Min Zhang |
| author_sort | Hui Wei |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-24T11:15:17Z |
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| id | doaj.art-df6778c97c404075affb97a7a6d936ab |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-24T11:15:17Z |
| publishDate | 2021-12-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-df6778c97c404075affb97a7a6d936ab2022-12-21T16:58:24ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2021-12-011210.3389/fmicb.2021.757741757741Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolyticaHui Wei0Wei Wang1Eric P. Knoshaug2Xiaowen Chen3Stefanie Van Wychen4Stefanie Van Wychen5Yannick J. Bomble6Michael E. Himmel7Min Zhang8Biosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesBiosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesBiosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesNational Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, United StatesBiosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesNational Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, United StatesBiosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesBiosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesBiosciences Center, National Renewable Energy Laboratory, Golden, CO, United StatesYarrowia 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.https://www.frontiersin.org/articles/10.3389/fmicb.2021.757741/fullYarrowia lipolyticacellobiohydrolase Iendoglucanase IIlipid metabolismATP citrate lyasediacylglycerol acyltransferase |
| spellingShingle | Hui Wei Wei Wang Eric P. Knoshaug Xiaowen Chen Stefanie Van Wychen Stefanie Van Wychen Yannick J. Bomble Michael E. Himmel Min Zhang Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica Frontiers in Microbiology Yarrowia lipolytica cellobiohydrolase I endoglucanase II lipid metabolism ATP citrate lyase diacylglycerol acyltransferase |
| title | Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica |
| title_full | Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica |
| title_fullStr | Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica |
| title_full_unstemmed | Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica |
| title_short | Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica |
| title_sort | disruption of the snf1 gene enhances cell growth and reduces the metabolic burden in cellulase expressing and lipid accumulating yarrowia lipolytica |
| topic | Yarrowia lipolytica cellobiohydrolase I endoglucanase II lipid metabolism ATP citrate lyase diacylglycerol acyltransferase |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2021.757741/full |
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