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

Amit Ghosh, David Ando, Jennifer Gin, Weerawat Runguphan, Charles Denby, George Wang, Edward E. K. Baidoo, Chris Shymansky, Jay Keasling, Héctor García Martín

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Abstract

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%.
Original languageEnglish
Article number76
JournalFrontiers in Bioengineering and Biotechnology
Volume4
Number of pages10
DOIs
Publication statusPublished - 2016

Bibliographical note

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licens or are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Keywords

  • -omics data
  • 13C metabolic flux analysis
  • flux analysis
  • metabolic engineering
  • predictive biology

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