A Synthetic Biology Approach to Enhancing Metabolic Engineering of Clostridium Acetobutylicum
Transition from fossil carbon to sustainable fuels is crucial for the long-term energy security. n-Butanol is a natural product of the fermentation of carbohydrates by Clostridium acetobutylicum and can be used as biofuel and/or biofuel additive with superior properties over other bio-derived alcohols. Formerly, C. acetobutylicum was used in the industrial Acetone-Butanol-Ethanol (ABE) fermentation to produce solvents in a ratio of 3 parts acetone, 6 parts butanol to 1 part ethanol. However, further strain engineering and process optimization is needed to make production of bio-butanol economically feasible.
Metabolic engineering of C. acetobutylicum had been hampered for many years due to lack of suitable genetic tools, which some were developed very recently (Heap et al., 2007; Heap et al., 2007; Heap et al., 2012). Libraries of expression control elements (promoters and RBSs) with known characteristics are important to allow construction of synthetic/heterologous metabolic pathways that perform predictably, and similarly to allow rational modification of existing pathways. To generate such libraries, reporter constructs are used to measure strengths of elements. However, classical fluorescent reporter proteins are oxygen-dependent and inactive in anaerobic bacteria. Therefore, we tested several oxygen-independent fluorescent reporter systems such as Light-Oxygen-Voltage proteins and SNAP/CLIP-tag protein fusions as well as enzymatic reporters phoZ and gusA. Among them gusA was found to give the highest sensitivity and the lowest background signal and was used to expand our existing toolkit of synthetic parts for controlling gene expression.
Subsequently, we attempted to create a high-yield strain of C. acetobutylicum by employing synthetic regulatory elements to develop and implement a system for metabolic flux redistribution. By using recently developed tools for genetic manipulation of Clostridia, we up-regulated and down-regulated expression of enzymes involved in the carbohydrate fermentation to improve production of solvents. This strategy will also be employed in the future to create high-yield strains of C. acetobutylicum and other industrially relevant strains for production of a wide range of commodity chemicals.
Heap, J.T., Pennington, O.J., Cartman, S.T., Carter, G.P. and Minton, N.P. (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J. Microbiol. Methods. 70: 452-464
Heap, J.T., Pennington, O.J., Cartman, S.T. and Minton, N.P. (2009) A modular system for Clostridium shuttle plasmids. J. Microbiol. Methods. 78: 79-85
Heap, J.T., Ehsaan, M., Cooksley, C.M., Ng, Y.K., Cartman, S.T., Winzer, K. and Minton, N.P. (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acid Res. 40:e59