Edinburgh Napier University

  Fossil fuels are a finite resource, which are subject to erratic price fluctuations and are contributing to disruption of the global carbon cycle. There is a requirement to establish a renewable, sustainable and secure fuel source that can reduce worldwide dependence on oil. Industry and agriculture contribute to the vast quantity of carbohydrate-rich waste biomass continuously generated. Certain Clostridium spp. can convert the sugars found in biomass into solvents which can be used as biofuel, but there is a need to learn more about their metabolic capabilities in order to introduce them efficiently on an industrial scale. The phosphotransferase system (PTS) IS a predominant method of carbohydrate transport in Firmicutes. An in silico analysis of the complete phosphotransferase complement of C. acetobutylicum A TCC 824 revealed that the genome encodes thirteen permease proteins and a lone EIIA domain, the properties of which are discussed. The genes encoding the general PTS energy coupling proteins have an unusual genome context. In the majority of Gram positive bacteria the two genes are in an operon; in C. acetobutylicum the two genes appear to be monocistronic. Interestingly in C. acetobutylicum the gene hprK, encoding the fbp-stimulated HPr regulator HPr kinase/phosphorylase (HprKJP), is found directly upstream from a gene glpX, which appears to encode a fructose-I,6-bisphosphatase (FBPase). Here, two genes were identified encoding FBPases in C. acetobutylicum - a class II, GlpX-like, FBPase and a class III FBPase. In silico analysis revealed two systems involved with ~-glucoside utilisation. System I is comprised of three open reading frames (ORFs), which encode all functions necessary for the regulated uptake and utilisation of ~-glucosides; a transcriptional antiterminator (bgIG), an EII (bgIA) and a phospho-f-glucosidasc (bgIB). Gene spacing, regulatory elements and similarity with other PTS' s are consistent with the expression of bglGAB as an operon. Molecular cloning of the permease revealed it was specific for arbutin transport. The domains of the permease from System II were encoded on three separate ORFs, interjected by a phospho-Bvglucosidase. A divergently expressed transcriptional activator was identified upstream from the Ell. Heterologous expression revealed System II was specific for salicin. Preliminary investigation of the transport and utilisation mechanisms of the monomers present in hemicellulose revealed two putative pentose isomerases, as well as two mannose phosphotransferases. Characterisation of carbohydrate transport, metabolism and regulation could provide information to optimise future biosolvent production facilities. -