Fig. 1

Design and construction of acetone-producing Moorella thermoacetica strains. a Acetone production pathway. Two molecules of Ac-CoA are converted to one molecule of acetone via three reactions using one molecule of acetate. The reactions release a CoA molecule, an Ac-CoA molecule, and a CO₂ molecule, in addition to an acetone molecule. Acetate pathway from Ac-CoA is also shown. Ac-CoA is converted to acetyl phosphate by phosphotransacetylase that is encoded by pduL1 as well as pduL2, followed by conversion to acetate. b Schematic representation of the synthetic acetone production operon. Genes and promoters are shown by block and fine arrows, respectively. c, e Schematic representations of the introduction of the synthetic thermophilic acetone operon by homologous recombination into the pyrF (c) and the pduL2 (e) region. The gray boxes highlight DNA regions used for recombination, and the line arrows represent primers used for PCR. The primer set to amplify the pyrF region is JK226 and JK227 and that for the pduL2 region is 1181-up-F and 1181-dw-R. d, f Verification of the presence of the thermophilic acetone operon in the pyrF (d) and the pduL2 (f) region. The genomic region of each gene was amplified by PCR, and the size shift due to the insertion was confirmed. The size of the PCR product of the pyrF region shifted from 0.5 to 4.8 kb by introducing the thermophilic acetone operon and the selection marker (d). Similarly, the PCR product of the pduL2 region shifted from 1.0 to 4.9 kb (f)