Development and application of novel screening and selection strategies for the enzyme- and metabolic engineering of alkane biosynthesis

Alkane biofuels with a reduced carbon footprint, especially those directly usable in existing internal combustion engines, are more sustainable and viable alternatives to fossil fuels. The aldehyde deformylating oxygenase (ADO) enzyme catalyzes the final step in the well-studied cyanobacterial alkane biosynthesis pathway, however it is notoriously inefficient and a bottleneck to biological alkane production. This study proposes to develop novel high-throughput methods for screening and selection of the ADO enzyme and to subsequently use these approaches to identify ADO variants with improved catalytic efficiencies and/or desired substrate specificities. In addition, this study will employ these tools to further optimize alkane biosynthesis on a metabolic level by targeting other enzymes in the pathway. The overarching goal of this study is to contribute to significantly improving the titer, productivity and yield of alkane biosynthesis through enzyme and metabolic engineering, ultimately to drive drop-in biofuels toward commercial viability.