A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes

Abstract

The development of renewable alternatives to diesel and jet fuels is highly desirable for the heavy transportation sector, and would offer benefits over the production and use of short-chain alcohols for personal transportation. Here, we report the development of a metabolically engineered strain of Escherichia coli that overproduces medium-chain length fatty acids via three basic modifications: elimination of β-oxidation, overexpression of the four subunits of acetyl-CoA carboxylase, and expression of a plant acyl–acyl carrier protein (ACP) thioesterase from Umbellularia californica (BTE). The expression level of BTE was optimized by comparing fatty acid production from strains harboring BTE on plasmids with four different copy numbers. Expression of BTE from low copy number plasmids resulted in the highest fatty acid production. Up to a seven-fold increase in total fatty acid production was observed in engineered strains over a negative control strain (lacking β-oxidation), with a composition dominated by C12 and C14 saturated and unsaturated fatty acids. Next, a strategy for producing undecane via a combination of biotechnology and heterogeneous catalysis is demonstrated. Fatty acids were extracted from a culture of an overproducing strain into an alkane phase and fed to a Pd/C plug flow reactor, where the extracted fatty acids were decarboxylated into saturated alkanes. The result is an enriched alkane stream that can be recycled for continuous extractions. Complete conversion of C12 fatty acids extracted from culture to alkanes has been demonstrated yielding a concentration of 0.44 g L−1 (culture volume) undecane.

Modular optimization of multi-gene pathways for fatty acids production in E. coli

“…Further steps to improve fatty acid production in E. coli include cytosolic expression of multifunctional fatty acyl-CoA synthetase (tesA 0 ) 4 and plant-derived fatty acyl-ACP thioesterases 6 to alleviate the feedback inhibition of b-ketoacyl-ACP synthase by the accumulated fatty acyl-ACPs 23 . Crude fatty acids were extracted, derivatized and analysed by gas chromatography mass spectrometry (GC-MS) (a representative chromatographic profile can be found in Supplementary Fig.…”

“…Early metabolic engineering studies on fatty acid production have been dedicated to optimizing precursor supply and the specificity of plant fatty acyl-ACP thioesterase 4,6,10,30,31 . Engineering the reversal of b-oxidation pathway 7 and a dynamic sensor-regulator system 32 have also led to the production of fatty-acid-derived long chain chemicals and fuels.…”

“…With physicochemical properties closely resembling those of petroleum-based fuels, fatty acid-based fuels offer some unique advantages as compared with other fuel supplements (ethanol or butanol) such as lower hygroscopicity, miscibility with diesel fuels, lower downstream purification costs and compatibility with existing infrastructure 3 . Therefore, a number of fatty-acid-derived fuel molecules including free fatty acids 4 , alkane/alkene hydrocarbons 5,6 , fatty alcohols 7 , alkyl ketones 8 and triacylglyceride 9 have been produced from the fatty acid biosynthetic pathway. For instance, manipulation of precursor malonyl-CoA availability along with the expression of specific plant acyl-ACP thioesterases have resulted in fatty acid production at 2.5 g l À 1 in E. coli 4,10 .…”

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

“…A key prerequisite is to maximize the specific activity of the fatty acid synthase (FAS) in the biotransforming microbe. Indeed, recent efforts have shown that genetic manipulation of the E. coli FAS can improve its productivity of free fatty acids and their derivatives (7,16,19,20). Notwithstanding these early successes, considerably greater improvements in atom economy and volumetric productivity are warranted, if fatty acid derivatives are to emerge as prominent fuels in the marketplace.…”

Synthesis and scavenging role of furan fatty acids

“…Fatty acids also help maintain viability in response to temperature and environmental changes and can be targets for modification by reactive oxygen species or membrane-active agents (2)(3)(4)(5)(6)(7)(8). Fatty acids, or the products derived from them, are valuable as food additives, specialty chemicals, and petroleum substitutes (9)(10)(11)(12). Thus, there is considerable interest in understanding the suite of fatty acids that can be made by native or engineered pathways.…”