Alkane and biodiesel production by engineered escherichia coli using a dna scaffold system

Abstract

Fatty acid-based biofuels, especially biodiesel and n-alkanes, are the major constituents of diesel and petroleum fuels, with an increasing demand as alternative biofuels. However, the conventional process for the production of biodiesel and n-alkanes are based on the expensive raw materials (vegetable oil, coal, hydrogen and cobalt) and extensive downstream processing steps, that increases their overall production cost. Efforts have been made to engineer microbial systems for the economical production of biodiesel and n-alkanes from cost effective raw materials such as lignocellulosic sugars. Nevertheless, the microbial production of these biofuels is far below a commercial threshold. The biodiesel and n-alkanes in Escherichia coli are produced from fatty acyl-ACPs with the help of enzymes, thioesterase B (TesB), wax synthase (WS), acyl-ACP reductases (AAR), aldehyde deformylating oxygenases (ADO). One of the major challenges in the microbial production of fatty acid biofuel is the low catalytic efficiency of the pathway enzymes and toxicity of intermediates to the host. More specifically, the toxicity of free fatty acids and the slow catalyzing enzyme (ADO) are the major bottlenecks in the efficient production of biodiesel and n-alkanes in E. coli, respectively. To solve these problems, a concept of “protein complex formation” was applied, to enhance the catalysis of pathway enzymes. In the case of n-alkanes production, first, a chimeric protein of AAR and ADO was synthesized and studied. Second, diverse combinations of protein complexes of the pathway enzymes were constructed with the help of DNA scaffolds. As the result, production of n-alkanes was 4.4-fold (24 mg/L) increased with the chimeric fusion of ADO-AAR compared to a control strain expressing wild type AAR and ADO. By utilizing DNA scaffolds, when the ratio of ADO to AAR was 3 to 1, the n-alkanes production was 8.8-fold increased (44 mg/L) compared to the control strain (5 mg/L). Our results demo...