title: 13C Metabolic Flux Analysis for Systematic Metabolic Engineering ofS. cerevisiaefor Overproduction of Fatty Acids.
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 inSaccharomyces cerevisiae. We combined13C 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 fromYarrowia lipolyticaas 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 toS. cerevisiaeWRY2, 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.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA; Indian Institute of Technology (IIT), School of Energy Science and Engineering, Kharagpur, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Ando
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Gin
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA; National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathum Thani, Thailand.
authors.forename: Weerawat
authors.initials: W
authors.lastname: Runguphan
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Denby
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA; Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Shymansky
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA; Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA, USA; Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA; Novo Nordisk Foundation Center for Biosustainability, Technical University Denmark, Horsholm, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Lawrence Berkeley National Laboratory, Biological Systems and Engineering Division, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA.
authors.forename: Héctor
authors.initials: H
authors.lastname: García Martín
keywords: -omics data; 13C metabolic flux analysis; flux analysis; metabolic engineering; predictive biology