Application of metabolic flux and transcript analyses to understanding the physiology of engineered geobacillus thermoglucosidasius

Geobacillus thermoglucosidasius is a metabolically versatile thermophilic facultative anaerobe, able to grow on a wide range of monomeric, dimeric and oligomeric carbohydrates derived from lignocellulose. It naturally carries out a mixed acid fermentation producing lactate, formate, acetate and ethanol as fermentation end products. In recent years this fermentation pathway has been redirected by metabolic engineering to produce ethanol almost exclusively, which has enabled TMO Renewables to scale-up and commercialise their cellulosic bioethanol process. Nevertheless, there are a number of potential areas for further improvement and the metabolic engineering has generated some unanswered physiological questions. Through a previous CASE studentship the group at Imperial College have developed a small scale (45ml) continuous culture system providing pH and temperature control as well as redox measurement for the economic study of metabolic flux using 13C labelling. Maintenance of a fixed redox potential and metabolic profiling of cultures at different redox potentials in continuous culture have proved to be valuable tools for reproducible physiological studies of G. thermoglucosidasius under fermentative conditions. In this project we propose to extend physiological studies of mutant and wild type strains through combined metabolic flux and transcript analysis. Selective transcriptome analysis will be done either using microarrays based on genome sequence information which is currently being assembled, or through transcriptome sequence analysis on high throughput platforms available at Imperial College or the BBSRC funded advanced genome centre. Current metabolic flux analysis uses the programme Fiatflux to generate information on flux ratios, but with the availability of genome sequence information (the TMO production strain has been sequenced and a similar strain,SB2, which is being worked on at Imperial College, is being sequenced) it is envisaged that the CASE student would build full metabolic models necessary for determining absolute fluxes. Using these approaches the initial focus would be to explore a range of issues (eg additional nutrient requirements) associated with approaching true anaerobic growth in wild type and engineered strains. In particular, we find that the knockout of pyruvate formate lyase, with or without upregulation of pyruvate dehydrogenase produces some undefined nutritional requirements. Additionally the student will investigate the regulation of the utilisation of multiple carbohydrates in G. thermoglucosidasius, which may require developing new interpretation methods based on the Fiatflux platform. Information arising from these analyses will then guide metabolic engineering strategies for strain improvement as part of an iterative programme.