Metabolic engineering for improved solvent production by Clostridium acetobutylicum

Completed

Not Energy Related
Renewable Energy Sources(Bio-Energy, Production of transport biofuels (incl. Production from wastes))
Renewable Energy Sources(Bio-Energy, Other bio-energy)

Basic and strategic applied research

BIOLOGICAL AND AGRICULTURAL SCIENCES (Biological Sciences)

Not Cross-cutting

Prof N Minton
Centre for Biomolecular Sciences
University of Nottingham

Research Grant

BBSRC

01 October 2007

31 March 2011

42 months

£643,519

Pharmaceuticals and Biotechnology

East Midlands

Prof N Minton, Centre for Biomolecular Sciences, University of Nottingham

Dr K Winzer, Centre for Biomolecular Sciences, University of Nottingham

Objectives not supplied

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

07/10/13