Due to the rapidly diminishing sources of conventional fossil fuels coupled with the large evidence-base for ecocide related to increased anthropogenic atmospheric carbon dioxide, there is an urgent need for rapid transition from non-renewable hydrocarbons to alternative novel pathways in order to secure, clean and efficient ‘green’ bio-energy production. This will have a major impact on individuals and the environment at a global scale. Thermophilic Gram-positive spore-forming bacterial Geobacillus species have an intrinsic ability to utilise a wide range of substrates including renewable resources, such as cell wall hemicellulose polymers, and are of biotechnologically and industrially important producers of ethanol and isobutanol. However, tolerance to biofuel-related alcohols by the host-producing organism is often lower than that of the exogenously supplied alcohol. This arises due to the toxicity of reactive intermediates, such as aldehydes. Co-localising the steps in alcohol production in an organelle designed to protect reactive intermediates from undesirable interactions provides a route to increased yields and decreased toxicity. Bacterial MicroComPartments (MCPs) are organelles that host specific biochemical reactions for both anabolic and catabolic functions. Engineered, morphologically-diverse MCPs bearing heterologous enzymatic pathways have shown enhanced productivity for commodity chemicals, which make MCPs an important focus for metabolic engineering. Gaining control of BMC assembly and incorporation of a heterologous enzymatic cargo has yet to be achieved in thermophiles. The Fellowship aimed to find new perspectives in the development of novel bacterial strains of thermophilic Geobacillus spp. to enhance the production of second-generation biofuels at high temperatures.
The THERMCP objectives were: 1). To search for shell protein homologs in the available sequenced genomes of Geobacillus species and perform bioinformatics analysis of the pdu operon from G. thermoglucosidasius; 2). To establish the protein engineering rules and the ultimate benefit of directing the Pdc and Adh enzymes required for ethanol production to the Pdu MCP shell of thermophilic G. thermoglucosidasius; 3). To engineer a strain of G. thermoglucosidasius capable of overproducing 2-ketoisovalerate (2-kiv) as described in the literature, in a strain where fermentation pathways have been deleted; 4). To use the strain developed in 3. as a platform to engineer a novel keto-acid dehydrogenase and acylating alcohol dehydrogenase route for isobutanol production, and introduce this into the protein MCP in thermophilic G. thermoglucosidasius. The project has achieved most of its objectives and milestones for the period, with relatively minor deviations.
Furthermore, a set of project training objectives were satisfied to reintegrate Dr. Yana Wade into the research environment after a career break. She also gained a thorough grounding in microbial engineering of thermophiles to complement her existing knowledge and skills in thermophilic environmental microbiology providing a foundation for a research career in industrial applied microbiology.
