Photosynthetic isobutanol production by integrating pathway engineering with carbon sink removal in cyanobacteria under outdoor natural sunlight.

There is significant interest in employing cyanobacteria for eco-friendly biofuel production, utilizing CO and sunlight. Recent advancements highlight the advantages of pathway engineering in cyanobacteria in enhancing the yields of biobutanol from the engineered strains. Isobutanol has excellent potential as an alternative fuel and can be blended with gasoline in ratios reaching 100% for use in existing internal combustion engines (ICE). This research focuses on the genetic engineering of Synechocystis sp. PCC 6803 to create mutant strains impaired in PHB synthesis but can biosynthesize isobutanol through an incorporated 2-keto-acid pathway in their genome. The synthesis of isobutanol is achieved through the heterologous expression of α-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (Yqhd), both driven by the strong, light-inducible psbA2 promoter. The PHB synthase mutant strain ECDM12, which produces isobutanol, showed a 3.8-fold higher titer than PHB-synthesizing strains under identical cultivation conditions. Indoor cultivation in a 2 L photobioreactor (PBR) under simulated diurnal light resulted in the highest titer of 687.6 mg L (11th day) and productivity of 64.1 mg L day. Outdoor studies in PBR under natural sunlight resulted in a maximum titer of 398 mg L (15th day) and productivity of 33.7 mg L day, marking the first photosynthetic isobutanol production under natural sunlight.