Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Abstract

Microbial lipid metabolism is an attractive route for producing aliphatic chemicals, commonly referred to as oleochemicals. The predominant metabolic engineering strategy centers on heterologous thioesterases capable of producing fatty acids of desired size. To convert acids to desired oleochemicals (e.g. fatty alcohols, ketones), metabolic engineers modify cells to block beta-oxidation, reactivate fatty acids as coenzyme-A thioesters, and redirect flux towards termination enzymes with broad substrate utilization ability. These genetic modifications narrow the substrate pool available for the termination enzyme but cost one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. In this work, we demonstrate an alternative acyl-transferase strategy for producing medium-chain oleochemicals. Through bioprospecting, mutagenesis, and metabolic engineering, we developed strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones using the transacylase strategy.

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

“…Finally, this work provides P. putida strains that were able to achieve FFA titers of up to 670.9 mg/L total FFAs and 253.6 mg/L C8 FFA; titers comparable with those achieved using conventional hosts such as Escherichia coli 38 and Yarrowia lipolytica 40 . Additionally, when grown on diluted sorghum hydrolysate these P. putida strains produced medium-chain FFAs at titers greater than 450 mg/L.…”

“…Microbial oleochemical production benefits from the use of a host unable to catabolize fatty acids. Many oleochemical final products or intermediates can be degraded via β-oxidation 37 , resulting in a decreased ability to accumulate the final product 25,38 . By removing several CoA ligases that initiate β-oxidation, we engineered P. putida strains unable to catabolize medium- and long-chain FFAs.…”

“…As an example, one could potentially limit the production of unwanted straight-chain FFAs in E. coli that have been engineered to produce branched-chain FFAs 39 by introducing an engineered CoA ligase that is able to ligate straight-chained FFAs while avoiding the desired branch-chained final products. The overexpression of strategic CoA ligases can be used in conjunction with other chain-length control measures such as engineered thioesterases 30 , acyl-ACP:CoA transacylases 38 , and engineered fatty acid synthases 40 to further control final product profiles.…”

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

“…Microbial oleochemical production benefits from the use of a host unable to catabolize fatty acids. Many oleochemical final products or intermediates can be degraded via β-oxidation 37 , resulting in a decreased ability to accumulate the final product 27,38 . By removing several CoA ligases that initiate β-oxidation, we engineered P. putida strains unable to catabolize medium-and long-chain FFAs.…”

“…As an example, one could potentially limit the production of unwanted straight-chain FFAs in E. coli that have been engineered to produce branched-chain FFAs 39 by introducing an engineered CoA ligase that is able to ligate straight-chained FFAs while avoiding the desired branch-chained final products. The overexpression of strategic CoA ligases can be used in conjunction with other chain-length control measures such as engineered thioesterases 40 , acyl-ACP:CoA transacylases 38 , and engineered fatty acid synthases 41 to further control final product profiles.…”