Modulating membrane composition alters free fatty acid tolerance in Escherichia coli.
Microbial synthesis of free fatty acids (FFA) is a promising strategy for converting renewable sugars to advanced biofuels and oleochemicals. Unfortunately, FFA production negatively impacts membrane integrity and cell viability in Escherichia coli, the dominant host in which FFA production has been studied. These negative effects provide a selective pressure against FFA production that could lead to genetic instability at industrial scale. In prior work, an engineered E. coli strain harboring an expression plasmid for the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase was shown to have highly elevated levels of unsaturated fatty acids in the cell membrane. The change in membrane content was hypothesized to be one underlying cause of the negative physiological effects associated with FFA production. In this work, a connection between the regulator of unsaturated fatty acid biosynthesis in E. coli, FabR, thioesterase expression, and unsaturated membrane content was established. A strategy for restoring normal membrane saturation levels and increasing tolerance towards endogenous production of FFAs was implemented by modulating acyl-ACP pools with a second thioesterase (from Geobacillus sp. Y412MC10) that primarily targets medium chain length, unsaturated acyl-ACPs. The strategy succeeded in restoring membrane content and improving viability in FFA producing E. coli while maintaining FFA titers. However, the restored fitness did not increase FFA productivity, indicating the existence of additional metabolic or regulatory barriers.
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Rebecca M
RM
Lennen
Brian F
BF
Pfleger
D000213:Acyl Carrier Protein / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D015964:Gene Expression Regulation, Bacterial; D050296:Microbial Viability / Q000235:genetics; D009154:Mutation; D056507:Paenibacillus / Q000201:enzymology / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D000213:Acyl Carrier Protein; D029968:Escherichia coli Proteins; C458522:FabR protein, E coli; D005230:Fatty Acids, Nonesterified; D005231:Fatty Acids, Unsaturated; D014157:Transcription Factors; D013869:Thiolester Hydrolases