title: Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli.
abstract: Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.
authors.affiliation: Department of Biochemistry, University of Puerto Rico School of Medicine, P.O. Box 365067, San Juan, 00936-5067, Puerto Rico. Electronic address: delise.oyola@upr.edu.
authors.forename: Delise
authors.initials: D
authors.lastname: Oyola-Robles
authors.affiliation: Department of Biochemistry, University of Puerto Rico School of Medicine, P.O. Box 365067, San Juan, 00936-5067, Puerto Rico. Electronic address: carlos.rullan@upr.edu.
authors.forename: Carlos
authors.initials: C
authors.lastname: Rullán-Lind
authors.affiliation: Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan, 00931-3346, Puerto Rico. Electronic address: nestor.carballeira1@upr.edu.
authors.forename: Néstor M
authors.initials: NM
authors.lastname: Carballeira
authors.affiliation: Department of Biochemistry, University of Puerto Rico School of Medicine, P.O. Box 365067, San Juan, 00936-5067, Puerto Rico; Molecular Sciences Building, University of Puerto Rico, San Juan, 00926, Puerto Rico. Electronic address: abel.baerga@upr.edu.
authors.forename: Abel
authors.initials: A
authors.lastname: Baerga-Ortiz
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D056804:Biofuels; D002244:Carbon / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis; D005285:Fermentation; D005990:Glycerol / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010776:Photobacterium / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D056804:Biofuels; D003470:Culture Media; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D002244:Carbon; D064429:Fatty Acid Synthases; D005990:Glycerol
keywords: ACP; AT; Biodiesel; Bioengineering; Biofuel; DH; Dehydratase domain; ER; Escherichia coli.; FA; FAME; Fatty acids overproduction; GC; KR; KS; MS; PUFA; SFA; UFA; acyl carrier protein; acyl tranferases; dehydratase; enoyl reductase; fatty acid; fatty acid methyl ester; gas chromatography; keto-acyl reductase; keto-acyl synthase; mass spectrometry; polyunsaturated fatty acids; saturated fatty acid; unsaturated fatty acid