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

碳足迹更少的烷烃生物燃料，尤其是那些可直接用于现有内燃机的烷烃生物燃料，是化石燃料更可持续、更可行的替代品。醛去甲酰基加氧酶（ADO）酶是蓝藻烷烃生物合成途径的最后一步，但它的效率低下，是生物烷烃生产的瓶颈。本研究建议开发新的高通量方法来筛选和选择ADO酶，并随后使用这些方法来识别具有更高催化效率和/或所需底物特异性的ADO变体。此外，本研究将利用这些工具，通过靶向途径中的其他酶，进一步优化代谢水平上的烷烃生物合成。本研究的总体目标是通过酶和代谢工程显著提高烷烃生物合成的滴度、生产力和产量，最终推动生物燃料的商业可行性。

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.