(686f) Driving Force and Oxygen Tolerance As Design Principles to Enable Efficient Photosynthetic n-Butanol Biosynthesis in Cyanobacteria

Direct chemical and fuel production from CO₂ has emerged as a promising approach for sustainability and CO₂ mitigation. However, metabolic engineering of photosynthetic microorganism such as cyanobacteria for the production of fuels or chemicals is challenging, particularly when the pathway involves oxygen-sensitive enzymes and/or unfavorable thermodynamics. We aim to take Clostridium n-butanol fermentation pathway as an example to demonstrate some of the design principles for engineering cyanobacteria. Two of the major obstacles for using Clostridium pathway to produce n-butanol in cyanobacteria are the thermodynamically unfavorable condensation of acetyl-CoA and the oxygen sensitivity of its enzymes. To address the issue of unfavorable acetyl-CoA condensation to acetoacetyl-CoA, the first and committed step of the Clostridium pathway, we incorporated energy of ATP hydrolysis into this reaction by utilizing malonyl-CoA dependent formation of acetoacetyl-CoA. Using this strategy, we effectively push carbon flux against the natural thermodynamic barrier of the Clostridium pathway and achieved n-butanol synthesis in cyanobacterium Synechococcus elongatus PCC 9742. To address the difficulty of oxygen sensitivity, we characterized six oxygen tolerant aldehyde dehydrogenases for their aerobic activity toward butyryl-CoA. Replacing Clostridium aldehyde dehydrogenase with its oxygen tolerant counterpart in the n-butanol synthesis pathway resulted in 20 fold increase of n-butanol production. Together, these results demonstrate the importance of driving force and oxygen tolerance for a successful pathway design in cyanobacteria.