Towards biobutanol production in a thermophile using synthetic biology principles

• Main Author: Sheng, Lili
• Published: University of Nottingham 2014
• Subjects: 662
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.716494

Driven by the diminishing total reserve of fossil fuels and growing concerns about energy security and environmental issues, there is an increasing interest in developing microbial-based processes for the production of biofuel. As most natural fermentation processes do not yield desirable products at an economically viable scale, it is often required to genetically engineer, including deletion and/or insertion of necessary pathways to optimize yield or get new products. Thus it is essential to have the necessary genetic tools, and such was developed, to both improve and innovate upon existing methods, for the process organism of TMO Renewables Ltd - G. thcrmoglucosidasius NCIBM 11955, during this study, with the aim of ultimately creating a thermophilic butanol producing strain. Taking a synthetic biology approach, adopting the clostridial modular shuttle vector format, a new shuttle vector for G. thermoglucosidasius - pMTL86551 was constructed. Based on pMTL86551, along with the development of an appropriate media, a G. thermoglucosidasius pyrE' strain was generated. Utilizing of the pyrE' phenotype both as a positive and negative selection marker allowed successful implementation of rapid procedures for gene knock-in and knock-out by allelic exchange. While the re-creation of the TMO production strain 1 \95SAldhApflpdfp within two month proved the rapidity and feasibility of the new genetic methods, the possibility of developing a thermophilic butanol producing strain, motivated by the superior fuel properties of butanol compared to ethanol, was also opened up. However, implementation of a functional ABE fermentation pathway from a thermophilic clostridium was met with a multitudes of barriers. Moreover, the production strain failed to show the desired fermentation profile. Further research efforts will be required to tackle these problems for progression.