Improved Butanol Production Using FASII Pathway in <i>E. coli</i>

Abstract

<i>n</i>-Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e., butanol. We first assembled pathway genes, i.e., <i>car</i> encoding carboxylic acid reductase from <i>Mycobacterium marinum</i>, <i>sfp</i> encoding phosphopantetheinyl transferase from <i>Bacillus subtilis</i>, and <i>adh2</i> encoding alcohol dehydrogenase from <i>S. cerevisiae</i>, responsible for bioconversion of butyrate to butanol in three different configurations (Operon, Pseudo-Operon, and Monocistronic) to achieve optimum expression of each gene and compared with the clostridial solventogenic pathway for <i>in vivo</i> conversion of butyrate to butanol under aerobic conditions. An <i>E. coli</i> strain harboring <i>car</i>, <i>sfp</i>, and <i>adh2</i> in pseudo-operon configuration was able to convert butyrate to butanol with 100% bioconversion efficiency when supplemented with 1 g/L of butyrate. Further, co-cultivation of an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting) at different inoculation ratios was investigated, and an optimized ratio of 1:4 (upstream strain: downstream strain) was found to produce ∼2 g/L butanol under fed-batch fermentation. Further, a mono-cultivation approach was applied by transforming a plasmid harboring <i>tesBT</i> gene into the downstream strain. This approach produced 0.42 g/L in a test tube and ∼2.9 g/L butanol under fed-batch fermentation. This is the first report where both mono- and co-cultivation approaches were tested and compared for butanol production, and butanol titers achieved using both strategies are the highest reported values in recombinant <i>E. coli</i> utilizing FASII pathway.