title: Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica.
abstract: Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Shuobo
authors.initials: S
authors.lastname: Shi
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Department of Cell and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, SE-405 30 Gothenburg, Sweden.
authors.forename: Haichuan
authors.initials: H
authors.lastname: Ji
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
authors.forename: Verena
authors.initials: V
authors.lastname: Siewers
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2970 Hørsholm, Denmark nielsenj@chalmers.se.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D005227:Fatty Acids / Q000096:biosynthesis*; D015723:Gene Library; D005820:Genetic Testing; D057166:High-Throughput Screening Assays / Q000379:methods; D060847:Metabolic Engineering; D010078:Oxazines / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013194:Staining and Labeling; D025062:Yarrowia / Q000235:genetics*
chemical_list: D005227:Fatty Acids; D010078:Oxazines; D011994:Recombinant Proteins; C044808:nile red
keywords: Nile red; Saccharomyces cerevisiae; Yarrowia lipolytica; cDNA library; fatty acids; high-throughput screening