Free fatty acid production in Escherichia coli under phosphate-limited conditions

Abstract

Microbially synthesized fatty acids are an attractive platform for producing renewable alternatives to petrochemically derived transportation fuels and oleochemicals. Free fatty acids (FFA) are a direct precursor to many high-value compounds that can be made via biochemical and ex vivo catalytic pathways. To be competitive with current petrochemicals, flux through these pathways must be optimized to approach theoretical yields. Using a plasmid-free, FFA-producing strain of Escherichia coli, a set of chemostat experiments were conducted to gather data for FFA production under phosphate limitation. A prior study focused on carbon-limited conditions strongly implicated non-carbon limitations as a preferred media formulation for maximizing FFA yield. Here, additional data were collected to expand an established kinetic model of FFA production and identify targets for further metabolic engineering. The updated model was able to successfully predict the strain's behavior and FFA production in a batch culture. The highest yield observed under phosphate-limiting conditions (0.1 g FFA/g glucose) was obtained at a dilution rate of 0.1 h(-1), and the highest biomass-specific productivity (0.068 g FFA/gDCW/h) was observed at a dilution rate of 0.25 h(-1). Phosphate limitation increased yield (∼45 %) and biomass-specific productivity (∼300 %) relative to carbon-limited cultivations using the same strain. FFA production under phosphate limitation also led to a cellular maintenance energy ∼400 % higher (0.28 g/gDCW/h) than that seen under carbon limitation.

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…Recent interest in microbial production of renewable fuels has led to successful synthesis of a variety of next-generation biofuels with improved properties over ethanol 12,14,15,[21][22][23][24][25][26][27] . Microbial synthesis of these reduced chemical species by de novo designed pathways can potentially lead to more efficient production strains, which are necessary for next-generation targets to achieve commercial relevance.…”

“…Microbial synthesis of these reduced chemical species by de novo designed pathways can potentially lead to more efficient production strains, which are necessary for next-generation targets to achieve commercial relevance. Predominantly, these pathways have employed reconstitution of natural pathways in new hosts (acetone-butanol-ethanol 5,6 ) or modified termination of natural pathways (fatty acid synthesis (FAS) 11,23,24 , amino acid synthesis 12,28 , isoprenoid synthesis 22 ).…”

Engineering Escherichia coli for odd straight medium chain free fatty acid production

“…FFAs can be produced by introducing an acyl-acyl carrier protein (ACP) thioesterase (TE) gene into E. coli. The presence of the acyl-ACP TE breaks the fatty acid elongation cycle and releases FFAs (Lu et al 2008;Li et al 2012a;Zhang et al 2011;Youngquist et al 2013). Ingram and colleagues (1977) used propionate to induce synthesis of odd-chain-length fatty acids by wild type Escherichia coli K-12.…”

Solvent‐Enabled Nonenyzmatic Sugar Production from Biomass for Chemical and Biological Upgrading

“…In this context, soluble carbohydrates are an attractive intermediate for biomass upgrading. By weight, structuralp olysaccharides typically represent between 60 and 80 wt %o f lignocellulosic biomass.I na ddition, many chemical [1][2][3][4] and biological [5][6][7][8] processes exist for upgrading carbohydrates to fuels and chemicals.Different strategies exist for deconstructing biomass hemicellulose (a polymer of C 5 sugars; mostly xylose) and cellulose (a polymer of cellobiose, ad imer of glucose) to their soluble counterparts. Concentrated mineral acids such as sulfuric or hydrochloric acid have been used to hydrolyze hemicellulose and cellulose to soluble oligomers almost quantitatively.…”

“…In this context, soluble carbohydrates are an attractive intermediate for biomass upgrading. By weight, structuralp olysaccharides typically represent between 60 and 80 wt %o f lignocellulosic biomass.I na ddition, many chemical [1][2][3][4] and biological [5][6][7][8] processes exist for upgrading carbohydrates to fuels and chemicals.…”