title: Membrane stresses induced by overproduction of free fatty acids in Escherichia coli.
abstract: Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Lennen
authors.forename: Max A
authors.initials: MA
authors.lastname: Kruziki
authors.forename: Kritika
authors.initials: K
authors.lastname: Kumar
authors.forename: Robert A
authors.initials: RA
authors.lastname: Zinkel
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Burnum
authors.forename: Mary S
authors.initials: MS
authors.lastname: Lipton
authors.forename: Spencer W
authors.initials: SW
authors.lastname: Hoover
authors.forename: Don R
authors.initials: DR
authors.lastname: Ranatunga
authors.forename: Tyler M
authors.initials: TM
authors.lastname: Wittkopp
authors.forename: Wesley D
authors.initials: WD
authors.lastname: Marner
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000032:analysis; D005230:Fatty Acids, Nonesterified / Q000096:biosynthesis*; D020869:Gene Expression Profiling; D050296:Microbial Viability / Q000187:drug effects; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013312:Stress, Physiological
chemical_list: D029968:Escherichia coli Proteins; D005230:Fatty Acids, Nonesterified