Metabolic engineering for improved solvent production by Clostridium acetobutylicum

The production of the chemical solvents acetone-butanol (AB) by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol (alcohol) production. Since its development, however, its fortunes have shown considerable fluctuations. From the peak of activity in between the first and second world wars, there has been a steep decline as new technologies made it more economic to produce these chemicals from fossil fuels. In recent years, with current concerns over global warming and severe rises in the costs of crude oil, there has been resurgence in interest cumulating in the announcement by BP/Dupont to begin biobutanol (that is butanol produced by a biological process) production in 2007. Butanol is used primarily as an industrial solvent, but it is also a replacement for petrol as a fuel. Currently, the use of ethanol as a petrol additive is widespread in the developed world. The development of alternatives to petroleum as fuels is essential if we are to reduce our reliance on finite crude oil resources. However, butanol has many properties that make it far superior to ethanol. It has a higher energy content than ethanol, and its low vapour pressure and its tolerance to water contamination in petrol blends facilitate its use in existing petrol supply and distribution channels. Moreover, butanol can be blended into petrol at higher concentrations than existing biofuels, without the need to make expensive modifications to car engines. It also gives better fuel economy than petrol-ethanol blends. A key stage in the re-establishment of the AB process will be the generation of stable strains engineered to maximise butanol production. This can be achieved by making mutations in genes that lead to the production of products other than butanol. However, despite many decades of intense research, progress has been hampered by our inability to make the necessary mutations. Due to these limitations, a complete and thorough mutational analysis of all the key steps in the fermentation process responsible for butanol production has never been undertaken and the design of butanol hyperproducing strains by means of genetic manipulation has been impossible. In recent months, University of Nottingham scientists have developed a highly effective gene tool, the ClosTron. In proof of principle studies, over a dozen genes have now been inactivated in three different clostridial species, including 7 in C. acetobutylicum. Typically, the number of mutants obtained per experiment number in the 100s, and from start to finish are generated within 8-14 days. To date the system has been 100% effective, and opens up the possibility of revolutionizing metabolic engineering in Clostridium, through both gene inactivation and gene addition. We are, therefore, uniquely able to now undertake large scale targeted mutagenesis in C. acetobutylicum with very high efficiency. This will enable us to manipulate the AB fermentation pathway in a way that will either increase or abolish the generation of unwanted products. Constructing such a series of mutants is not only an essential prerequisite for the development of effective industrial strains, but will also allow us to identify the signals that control solvent formation. This should allow industry to more effectively control the production of butanol. Upon completion of ours studies we will, therefore, have generated a prototype production strain in which the yields of butanol have been maximised. Our analysis will have also have identified those signals which control solvent production. The results obtained will provide valuable information and strains useful to the fledgling biobutanol industry.

Clostridium acetobutylicum undertakes a complex biphasic fermentation: first it generates acids and then solvents, such as acetone and butanol. The project aims to generate metabolically engineered strains with altered fermentation characteristics, with the ultimate goals of (i) generating butanol hyperproducing strains and (ii) identifying the signals that govern the shift from acid to solvent formation. Single and multiple mutants of all major fermentation genes will be constructed in the sequenced strain ATTC 824. These will be characterised, initially in batch culture, with respect to their growth and fermentation characteristics. Mutants of interest will be studied in more detail: The expression of all fermentation and key glycolysis genes will be followed using quantitative RT-PCR over the entire growth period, and the concentration of relevant metabolites will be determined by LC-MS and GC-MS. The activity of key fermentation enzymes will also be monitored. Gene array analyses will be performed for key mutants at key stages of their fermentation to obtain an overall picture of the transcriptional changes occurring. In combination, this will allow us to link the individual induction time points and relative expression level of fermentation genes with the observed mutant phenotypes and to identify metabolic bottlenecks. For key mutants, the analyses described above will also be applied to chemostat-grown cultures. This will allow us to study the transcriptional, biochemical, and metabolic changes associated with the shift from acid to solvent formation under highly reproducible conditions and to dissect the specific effects of putative shift-inducing signals such as metabolite pool level, growth rate, pH, and acid concentration. Based on the above findings, a set of multiple mutants will be generated where most branches of the fermentation pathway have been inactivated (and limiting functions been over-expressed), leading to maximised butanol formation