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.
AbstractThe development of renewable alternatives to diesel and jet fuels is highly desirable for the heavytransportation sector, and would offer benefits over the production and use of short-chain alcoholsfor personal transportation. Here, we report the development of a metabolically engineered strainof Escherichia coli that overproduces medium-chain length fatty acids via three basicmodifications: elimination of β-oxidation, overexpression of the four subunits of acetyl-CoAcarboxylase, and expression of a plant acyl–acyl carrier protein (ACP) thioesterase fromUmbellularia californica (BTE). The expression level of BTE was optimized by comparing fattyacid 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 anegative control strain (lacking β-oxidation), with a composition dominated by C12 and C14saturated and unsaturated fatty acids. Next, a strategy for producing undecane via a combinationof biotechnology and heterogeneous catalysis is demonstrated. Fatty acids were extracted from aculture of an overproducing strain into an alkane phase and fed to a Pd/C plug flow reactor, wherethe extracted fatty acids were decarboxylated into saturated alkanes. The result is an enrichedalkane 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.
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