Fig. 3.

E. coli-Clostridium shuttle vector for the inducible genomic integration of an acetone biosynthesis gene cluster. a The vector pIM-SLIC contains a multiple cloning site (MCS) and a SwaI restriction site to allow directed and undirected insertion of cassettes. b A cluster comprising the four genes necessary for acetone biosynthesis (adc, thlA, ctfA-ctfB) was inserted by Gibson assembly via the SLIC sites (dark blue triangles) between the ptb promoter (P-ptb, yellow) and the fdx terminator (fdx-T, orange) resulting in pIM-Ace#22. c In C. ljungdahlii acetyl-CoA is derived from syngas (CO, CO₂, H₂) via the Wood–Ljungdahl pathway. Acetyl-CoA is used as a starting material for acetone biosynthesis by the engineered pathway. Acetone biosynthesis starts with the condensation of two acetyl-CoA molecules to acetoacetyl-CoA and the release of one molecule of CoA by acetyl-CoA acetyltransferase (ThlA). The CoA is then transferred to an acetate molecule by the CoA-transferase (CtfA/CtfB) to yield acetoacetate. Finally, acetone is generated via a decarboxylation reaction catalyzed by acetoacetate decarboxylase (Adc). However, the obtained acetone gets reduced by an endogenous primary secondary alcohol dehydrogenase (psAdh) to the secondary alcohol isopropanol