Abstract
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.
Highlights
One of the most promising routes for producing renewable substitutes for petrodiesel makes use of intermediates derived from fatty acid biosynthesis in microbes such as Escherichia coli
We investigated the impact of BTE expression on unsaturated membrane lipid content, cell lysis, free fatty acids (FFA) titers, and fabA and fabB expression levels in the absence of FabR, eliminating the mechanism for feedback repression of unsaturated fatty acid biosynthesis
Effect of fabR deletion on unsaturated membrane-bound fatty acids To verify that the elevated levels of unsaturated C16 and C18 fatty acids found in BTE expressing E. coli cultures [11,12] were located in membrane lipids, we compared fatty acid methyl ester (FAME) profiles prepared by acid and base catalysis
Summary
One of the most promising routes for producing renewable substitutes for petrodiesel makes use of intermediates derived from fatty acid biosynthesis in microbes such as Escherichia coli. The released free fatty acids (FFA) can either be separated from the culture medium and catalytically decarboxylated to alkanes [4,5], or they can be directed into heterologous pathways that produce products that include fatty acid ethyl esters, fatty alcohols, alkanes, olefins, methyl ketones, and polyhydroxyalkanoates [6,7,8,9,10]. Past reports have indicated that heterologous expression of the acyl-ACP thioesterase from Umbellularia californica (BTE) in E. coli results in greatly elevated levels of unsaturated and cyclopropane phospholipids, which are derived from unsaturated acyl-ACPs [11,12]. The heightened unsaturated membrane lipid content was postulated to result from altered long-chain acyl-
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
