Fig. 3

Introduction of genes for IPA production in M. thermoacetica. A Schematic representation of the synthetic IPA operon encoding enzymes for IPA production from acetyl-CoA. The reactions by enzymes derived from corresponding genes are shown in Fig. 1. B Schematic representation of the plasmid construction and introduction of the synthetic IPA operon in place of pduL2. C Agarose gel electrophoresis, following PCR amplification of the pduL2 region of the host and recombinant strains. M, DNA size marker; 1, the ∆pyrF strain; 2, the plasmid pHM71 (positive control); 3–8, candidates of the pduL2::IPA strain. The size of the amplified region was shifted to 6.5 kb in the pduL2::IPA strain, consistent with the DNA construct, which was originally 0.9 kb in the ∆pyrF strain. A candidate clone (lane No. 3) showed a faint band with the same migration as the ∆pyrF strain, which indicated a mixed population. This clone was excluded. The other clones showed the same culture profile in the following fermentation analysis