(234f) Engineering Escherichia Coli for Hydrocarbon Production

Biological hydrocarbons offer many advantages over cellulosic ethanol including higher energy density, lower water solubility, and compatibility with current engines. Biodiesel, the methyl or ethyl esters of fatty acids, is currently the second leading biofuel to ethanol. Similar to corn-based ethanol, the production of biodiesel is limited by the availability of oil crops. Metabolic engineering offers the tools link cellulosic technologies with the production of more desirable fuels. For instance, the microbial conversion of fatty acids to biodiesel via esterification of short alcohols has recently been reported with promising but suboptimal yields. Increasing the amount of substrate available for these final conversions should increase yields as has been seen in other metabolic engineering projects. One potential platform for the synthesis of diesel-like hydrocarbons, is the production of lipids in bacteria such as Escherichia coli for subsequent reduction to hydrocarbon fuels. Here we present progress towards engineering fatty acid production in E. coli.

Fatty acid biosynthesis is a tightly regulated process. The initial development of a fatty acid overproducing strain will be reported. To bypass known regulatory steps, we have combined three approaches ? eliminating b-oxidation, pushing flux into fatty acid biosynthesis, and pulling on biosynthetic pathways. The current strain features: (1) deletion of fadD, which encodes an acyl-CoA synthetase necessary for beta-oxidation; (2) overexpression of the four subunits of acetyl-CoA carboxylase (ACC), which converts acetyl-CoA to malonyl-CoA, a known bottleneck in fatty acid biosynthesis; and (3) heterologous expression of a codon-optimized plant medium chain acyl-acyl carrier protein thioesterase (BTE). Initial tests indicate an approximately ten-fold increase in production of C8 to C18 fatty acids in E. coli MG1655 ΔfadD when overexpressing ACC and BTE on plasmids. The predominant fatty acid chain length also shifts dramatically from C16 to C12 when BTE is expressed. Ongoing work includes optimization of ACC expression and identification of new metabolic and regulatory bottlenecks.