Construction of a Hydrogenase minus Mutant of Clostridium acetobutylicum : a Platform Strain for the Continuous Production of Fuels and Chemicals

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Currently, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce bulk chemicals like 1, 3 propanediol and fuels like n-butanol. To develop commercial processes via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism of C. acetobutylicum and its regulation. Here we developed an improved and biochemically validated genome-scale model, (iCac967) and apply a quantitative system-scale analysis to acidogenic, solventogenic and alcohologenic steady-state C. acetobutylicum cells to report for the first time quantitative transcriptomic, proteomic and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism. Based on these data a metabolic engineering strategy was developed to engineer C. acetobutylicum for the continuous production of several bulk chemicals at high yield. A new method was first developed to simultaneously inactivate several genes. Using this method we first engineered a â??hydrogenase minus platform strainâ? producing lactate as the major product without any production of either butyrate, acetate, acetone, ethanol or butanol. When used in continuous culture a titer of 28 g/l with a yield on glucose of 0.92 g/g and a productivity of 1.4 g/l.h were obtained.

This platform strain was further engineered to continuously convert raw glycerin to 1, 3 propanediol at a yield of 0.61 g/g, a titer of 60g/l and a productivity of 3 g/l.h with acetate as the only by-product. Finally the platform strain was engineered to produce ethanol as the major product. Used in continuous culture a titer of 39 g/l with a yield on glucose of 0.48 g/g and a productivity of 2 g/l.h were obtained. Stability of the three processes was demonstrated for several months and the cultures were only stopped due to biofilmsâ?? formation on probes and problems of pH and level control.

Work in our laboratory is currently concentrating on engineering the â??hydrogenase minus platform strainâ? strain for the continuous production of n-butanol at high yield.