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Title: 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.
Authors:
 [1] ;  [2] ;  [2] ;  [3] ;  [2] ;  [2] ;  [2] ;  [4] ;  [5] ;  [2]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Indian Inst. of Technology (IIT), Kharagpur (India). School of Energy Science and Engineering
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathum Thani (Thailand)
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering; Technical Univ. of Denmark, Horsholm (Denmark). Novo Nordisk Foundation Center for Biosustainability
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Frontiers in Bioengineering and Biotechnology
Additional Journal Information:
Journal Volume: 4; Journal ID: ISSN 2296-4185
Publisher:
Frontiers Research Foundation
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; flux analysis; 13C metabolic flux analysis; -omics data; predictive biology; metabolic engineering
OSTI Identifier:
1393590

Ghosh, Amit, Ando, David, Gin, Jennifer, Runguphan, Weerawat, Denby, Charles, Wang, George, Baidoo, Edward E. K., Shymansky, Chris, Keasling, Jay D., and García Martín, Héctor. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids. United States: N. p., Web. doi:10.3389/fbioe.2016.00076.
Ghosh, Amit, Ando, David, Gin, Jennifer, Runguphan, Weerawat, Denby, Charles, Wang, George, Baidoo, Edward E. K., Shymansky, Chris, Keasling, Jay D., & García Martín, Héctor. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids. United States. doi:10.3389/fbioe.2016.00076.
Ghosh, Amit, Ando, David, Gin, Jennifer, Runguphan, Weerawat, Denby, Charles, Wang, George, Baidoo, Edward E. K., Shymansky, Chris, Keasling, Jay D., and García Martín, Héctor. 2016. "13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids". United States. doi:10.3389/fbioe.2016.00076. https://www.osti.gov/servlets/purl/1393590.
@article{osti_1393590,
title = {13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids},
author = {Ghosh, Amit and Ando, David and Gin, Jennifer and Runguphan, Weerawat and Denby, Charles and Wang, George and Baidoo, Edward E. K. and Shymansky, Chris and Keasling, Jay D. and García Martín, Héctor},
abstractNote = {Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.},
doi = {10.3389/fbioe.2016.00076},
journal = {Frontiers in Bioengineering and Biotechnology},
number = ,
volume = 4,
place = {United States},
year = {2016},
month = {10}
}

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