AUTHOR=Ghosh Amit , Ando David , Gin Jennifer , Runguphan Weerawat , Denby Charles , Wang George , Baidoo Edward E. K. , Shymansky Chris , Keasling Jay D. , García Martín Héctor TITLE=13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=4 YEAR=2016 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2016.00076 DOI=10.3389/fbioe.2016.00076 ISSN=2296-4185 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%.