Engineering bacteria for biofuel production

摘要

This thesis addresses the need for environmentally and socially responsible sources of energy. Biofuels, made from organic matter, have recently become a viable alternative to petroleum-based fossil fuel. Sugar and starch make up the majority of feedstock used in biofuel production as it is easily digested. However, the use of these feedstocks is problematic as they consume resources with negative implications. By using a bacterium able to utilise five and six carbon sugars, such as the thermophile Geobacillus thermoglucosidans, organic lignocellulosic waste material can be used as a feedstock. ududThe aim of this project was to investigate and utilise key genetic regulators of fermentation in G. thermoglucosidans and to construct genetic engineering tools that enable strain development for second generation biofuel production. We have focused on the redox-sensing transcriptional regulator Rex, widespread in Grampositive bacteria, which controls the major fermentation pathways in response to changes in cellular NAD+/NADH ratio. Following the identification of several members of the Rex regulon via bioinformatics analysis, ChIP-seq and qRT-PCR experiments were performed to locate genome-wide binding sites and controlled genes in G. thermoglucosidans. Initial electromobility shift assay experiments were performed to demonstrate the potential for use of Rex from Clostridium thermocellum as an orthogonal regulator. To further this research, novel in vivo synthetic regulatory switches were designed and tested with the aim of controlling gene expression in response to changes in cellular redox state. In addition, new tools for the efficient genetic engineering of G. thermoglucosidans were produced and optimised, including an E. coli-G. thermoglucosidans conjugation method for plasmid transfer and gene disruption.