Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: Need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpr ession, were employed to examine the effects of two genetic modifications o n the fermentation characteristics of Clostridium acetobutylicum. Inactivat ion of the butyrate kinase gene (buk) was examined using strain PJC4BK, whi le the combined effect of buk inactivation and overexpression of the aad ge ne-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formati on-was examined using strain PJC4BK(pTAAD). The two strains were characteri zed in controlled pH greater than or equal to 5.0 fermentations, and by a r ecently enhanced method of metabolic flux analysis. Strain PJC4BK was previ ously genetically characterized, and fermentation experiments at pH greater than or equal to 5.5 demonstrated good, but not exceptional, solvent-produ ction capabilities. Here, we show that this strain is a solvent superproduc er in pH greater than or equal to 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol . Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butan ol toxicity limit, without any selection for butanol tolerance. Work with s train PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4B K while the second antibiotic selected for the pTAAD presence. Overexpressi on of aad from pTAAD resulted in increased ethanol production but did not i ncrease butanol titers, thus indicating that AAD did not limit butanol prod uction under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in ace tate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recom binant strains, up to 300% and 400%, respectively. Onset of solvent product ion occurred during the exponential-growth phase when the culture optical d ensity was very low and when total and undissociated butyric acid levels we re <1 mM. Butyrate levels were low throughout all fermentations, never exce eding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomeno logical model to explain solvent formation. (C) 2000 John Wiley & Sons, Inc .