The unprecedented global interest in sustainable and renewable biofuels using cutting-edge techniques is increasing enormously. The development of sustainable routes to the bio-manufacture of gaseous hydrocarbons will contribute widely to future energy needs. Their realisation would contribute towards minimising over-reliance on fossil fuels, improving air quality, reducing carbon footprints and enhancing overall energy security. Alkane gases (propane, butane and isobutane) are efficient and clean-burning fuels that are suitable for the development of low carbon footprint fuel and energy policies. In the last few decades, synthetic biology techniques have been used to manipulate existing life forms or create a new life that never occurred in nature to achieve novel functions. Synthetic biology approaches have contributed to the evolution of new bacterial strains that are able to synthesise a wide range of biofuels. Despite some success, the yields, productivity and titres of these biofuels remain to be improved and their pilot-scale production requires optimisation in terms of both the host and pathways used. In this context, this thesis describes the exploitation of synthetic biology approaches to develop novel microbial cell factories for generating bio-LPG blends. Current progress in bio-alkane gas production has been reviewed, and the potential for implementation of scalable and sustainable commercial bioproduction hubs has been highlighted. As natural biosynthetic routes to these short chain alkanes have not been discovered, de novo pathways have been engineered. These pathways incorporate one of two enzymes, either aldehyde deformylating oxygenase (ADO) or Chlorella variabilis fatty acid photodecarboxylase (CvFAP), to catalyse the final step that leads to gas formation. Multiple next-generation pathways that use amino acids (valine, leucine and isoleucine) as fuel precursors were designed in E. coli and ultimately integrated into the Halomonas chromosome for the production of clean-burning bio-LPG (propane, butane and isobutane, respectively). Halomonas production strains showed gas production stability for up to 7 days under non-sterile conditions. This approach paves the way to utilise widely available and inexpensive feedstock precursors such as amino acids and volatile fatty acids (VFAs) found in many industrial waste streams for the production of gaseous bio alkanes. As such, this project represents a foundation upon which further stable production strains can be investigated for upscaling the production of next-generation gaseous biofuels in the field under non-sterile conditions following process optimisation.
