Fig. 2

Designed pathway for butanol and hexanol biosynthesis and the corresponding plasmids. A Schematic representation of the heterologous butanol and hexanol biosynthesis pathway. ThlA1/2: thiolase A1/2; Hbd1/2: hydroxybutyryl-CoA-dehydrogenase 1/2; Crt1/2: crotonase 1/2; Bcd-EtfA/B complex = Bcd 1/2: butyryl-CoA-dehydrogenase 1/2; EtfA/B 1/2: electron transferring protein A/B 1/2 (all genes from C. kluyveri); AdhE2: bifunctional aldehyde-alcohol-dehydrogenase from C. acetobutylicum. Two molecules of acetyl-CoA are condensed by acetyl-CoA acetyltransferase (ThlA) to form acetoacetyl-CoA, which is then reduced to 3-hydroxybutyryl-CoA by 3-hydroxybutyryl-CoA dehydrogenase (Hbd) with NADH as the electron donor. In the next step, 3-hydroxybutyryl-CoA is dehydrated by crotonase (Crt) to form crotonyl-CoA, which is then reduced with NADH and oxidized ferredoxin (Fd) by a Flavin-based electron bifurcating butyryl-CoA dehydrogenase (Bcd) to form butyryl-CoA. From here, butanol can be produced via two reduction steps with NADH as the electron donor, catalyzed by a bifunctional aldehyde dehydrogenase/alcohol dehydrogenase (AdhE2). Alternatively, for the generation of hexanol, a third acetyl group derived from acetyl-CoA is added to the butyryl-CoA to produce 3-ketohexanoyl-CoA. Analogous to the steps in which acetoacetyl-CoA is converted to butyryl-CoA, 3-ketohexanoyl-CoA is again reduced with NADH, dehydrated, and reduced again by a bifurcating butyryl-CoA dehydrogenase to form hexanoyl-CoA. A bifunctional aldehyde dehydrogenase/alcohol dehydrogenase then catalyzes the final reduction steps to form hexanol, with NADH as the electron donor. Butyrate and caproate are potential side products and can be derived from butyryl-CoA and hexanoyl-CoA, respectively, with butyryl phosphate and caproyl phosphate as the corresponding intermediates. After reduction of the acids butyrate and caproate to the respective aldehydes via the native AOR (aldehyde:ferredoxin oxidoreductase) the molecules can be further converted to the alcohols butanol and hexanol. These alternative pathways allow the conservation of energy in form of ATP production via substrate level phosphorylation and are shown in grey (adapted from [26]) B Schematic representation of pIM Hex#15. The integration cassette consisting of a butanol-hexanol biosynthesis cluster and the adjacent ermC sequence is flanked by the mycomar sites (ITR–inverted terminal repeats) which allow integration catalyzed by the xylose-inducible Himar1 transposase. C Schematic representation of the Ccar1 cassette with the butanol/ hexanol biosynthesis cluster of C. carboxidivorans (pIM Ccar1)