ABSTRACTClostridium acetobutylicumnaturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineeredC. acetobutylicumcan be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene fromClostridium beijerinckiiNRRL B-593 (i.e.,adhB-593) inC. acetobutylicumATCC 824. To increase the total alcohol titer, a synthetic acetone operon (actoperon;adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in thebukgene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged byin situremoval of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.
Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov.
