title: Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.
abstract: Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, 41296, Sweden, bouke@chalmers.se.
authors.forename: Bouke Wim
authors.initials: BW
authors.lastname: de Jong
authors.forename: Shuobo
authors.initials: S
authors.lastname: Shi
authors.forename: Juan Octavio
authors.initials: JO
authors.lastname: Valle-Rodríguez
authors.forename: Verena
authors.initials: V
authors.lastname: Siewers
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D000106:Acetate-CoA Ligase / Q000235:genetics / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D056804:Biofuels / Q000600:supply & distribution; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis* / Q000737:chemistry; D050356:Lipid Metabolism; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C492820:ACB1 protein, S cerevisiae; D056804:Biofuels; D002352:Carrier Proteins; D005227:Fatty Acids; D029701:Saccharomyces cerevisiae Proteins; D000105:Acetyl Coenzyme A; C509911:ADH2 protein, S cerevisiae; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+); D000217:Acyltransferases; C079165:long-chain-alcohol O-fatty-acyltransferase; D002262:Carboxy-Lyases; D000106:Acetate-CoA Ligase