Improved N-alkanes Production in Escherichia coli By Spatial Organization of Alkane Biosynthetic Pathway Enzymes

Linear
hydrocarbons, especially n-alkanes, are the major constituents of
gasoline and jet fuel with ever increasing demand as alternative biofuels. However,
the conventional n-alkanes production process based on expensive raw
materials (coal, hydrogen and cobalt) increases the overall production cost of n-alkanes.
To meet the growing demand, efforts have been made to engineer microbial systems
for the economical production of n-alkanes. Nevertheless, the microbial
productions of n-alkanes are far below a commercial threshold. Biologically, n-alkanes are produced from
fatty acyl-ACPs with the help of acyl-ACP reductases (AAR) and aldehyde
deformylating oxygenases (ADO). One of the major challenges in the biological n-alkanes
production process is a slow catalytic turnover rate of ADO. To
increase the n-alkanes production, we controlled the spatial arrangement
and stoichiometric ratio of enzymes. First, a chimeric protein of AAR and ADO was synthesized. Second, the enzymes were arranged on a DNA scaffold with
various ratios. As the result, production of n-alkanes was increased (4.4-fold) by the chimeric fusion of ADO-AAR compared to
a control strain expressing wild type AAR and ADO. Furthermore, when the ratio
of ADO to AAR was 3 to 1, n-alkanes production was increased (8.8-fold) compared to the control strain. Our results showed that the spatial organizations of enzymes using
protein chimera and DNA scaffolds are applicable for establishing an efficient n-alkanes
biosynthetic pathway in Escherichia
coli.