We have engineered an Escherichia coli cell line that produces 4.5g/L/day total fatty acid in a fed-batch
fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved
unpredictable. To develop a more reliable engineering strategy, a cell-free systemwas developed that
enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using
this system,the strong dependence of fatty acid synthesis on malonyl-CoA availability and several
important phenomena in fatty acid synthesis were verified. Results from this cell-free system were
confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative
findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and
target specific steps for protein and metabolic engineering to enhance the catalytic conversion of
glucose into biodiesel.