Reported yields (percentage of FA from a supplied carbon source) in the literature from FFA-overproducing E.
coli acetyl-CoA carboxylase (ACC) has been shown to improve fatty acid yields in some metabolically engineered strains ( 19, 52) but to have little impact in others ( 48). The additional overexpression of the native E.
Deletion of fadD and/or fadE eliminates catabolism of fatty acids by the aerobic β-oxidation pathway ( 43, 65). Overexpression of an acyl-ACP thioesterase depletes the level of acyl-ACP intermediates, which inhibit via feedback enzymes of fatty acid biosynthesis ( 19, 36). In each, the key strain modifications included overexpression of one or more cytosolic acyl-acyl carrier protein (ACP) thioesterases and deletion of fadD, or both fadD and fadE, which encode an acyl-coenzyme A (CoA) synthetase and acyl-CoA dehydrogenase, respectively. Several studies have demonstrated FFA overproduction in Escherichia coli ( 19, 48, 52, 81, 83). The physical and chemical properties of the resulting products are dependent on chain length and hydrophobicity however, medium-chain-length (8- to 14-carbon) methyl esters, olefins, and alkanes exhibit many properties analogous to those of diesel and jet fuel and are therefore potential drop-in replacements ( 44, 61). These pathways can either be exploited in their native host or heterologously expressed in a genetically pliable microorganism ( 3). Alternatively, enzymatic pathways exist for intracellular conversion to esters ( 42, 81), olefins ( 10, 59, 75), alkanes ( 78), or fatty aldehydes and fatty alcohols ( 22, 81, 82). FFAs can be extracted from culture medium and catalytically converted to esters or alkanes ( 48, 55).
#Biorad cfx manager adjust baseline free
Microbially derived free fatty acids (FFAs) are attractive intermediates for producing a wide range of high-energy-density biofuels from sustainable carbon sources, such as biomass ( 34). The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. 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. 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. 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. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. 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. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. 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. 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.