title: Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid short- and branched-chain alkyl esters biodiesel.
abstract: BACKGROUND
  Biodiesel is a mixture of fatty acid short-chain alkyl esters of different fatty acid carbon chain lengths. However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties. Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties. Hence, microbial production as a renewable, nontoxic and scalable method to produce fatty acid esters with branched-chain alcohol moieties from biomass is critical.
  
  
  RESULTS
  We engineered Saccharomyces cerevisiae to produce fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters using endogenously synthesized fatty acids and alcohols. Two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences. To boost the ability of S. cerevisiae to produce the aforementioned esters, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted to increase flux towards fatty acyl-CoAs. In addition, five isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, and Adh7) targeted into the mitochondria were overexpressed to enhance production of alcohol precursors. By combining these engineering strategies with high-cell-density fermentation, over 230 mg/L fatty acid short- and branched-chain alkyl esters were produced, which is the highest titer reported in yeast to date.
  
  
  CONCLUSIONS
  In this work, we engineered the metabolism of S. cerevisiae to produce biodiesels in the form of fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters. To our knowledge, this is the first report of the production of fatty acid isobutyl and active amyl esters in S. cerevisiae. Our findings will be useful for engineering S. cerevisiae strains toward high-level and sustainable biodiesel production.
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore, 117597 Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456 Singapore.
authors.forename: Wei Suong
authors.initials: WS
authors.lastname: Teo
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore, 117597 Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456 Singapore.
authors.forename: Hua
authors.initials: H
authors.lastname: Ling
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore, 117597 Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456 Singapore.
authors.forename: Ai-Qun
authors.initials: AQ
authors.lastname: Yu
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore, 117597 Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456 Singapore.
authors.forename: Matthew Wook
authors.initials: MW
authors.lastname: Chang
keywords: Biodiesel; Biofuel; Fatty acid branched-chain alkyl esters; Fatty acid short-chain alkyl esters; Metabolic engineering; Synthetic biology; Yeast