Photoautotrophic Production of Butanol from Genetically Engineered Cyanobacteria

Abstract

The development of renewable energy solutions is essential to address rising global energy demands and the climate crisis. Cyanobacteria, oxygenic photosynthetic prokaryotes, provide a sustainable platform for biofuel production by converting solar energy, CO₂, and water into valuable chemicals. Isobutanol, a promising biofuel compatible with existing engines, was synthesized in Synechocystis sp. PCC 6803 through genetic engineering, cultivation optimization, and product recovery strategies. A synthetic 2-keto acid pathway was introduced via heterologous expression of α-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (Yqhd) under a light-inducible promoter (psbA2), yielding strain DM12 with titres of 371.8 mg L-1 in photobioreactors. Further engineering of a PHB synthase mutant (ECDM12) enhanced titres 3.8-fold, achieving 687.6 mg L-1 under simulated diurnal light. Outdoor validation confirmed 398 mg L-1 under natural sunlight. Media optimization using OFAT and RSM improved titres to 947.8 mg L-1 in photobioreactors under constant light and 892.6 mg L-1 under diurnal cycles, with outdoor titres reaching 538.3 mg L-1. Isobutanol toxicity studies revealed growth inhibition above 500 mg L-1. Integration of AmberSep® OPTIPORE L-493 resin and gas stripping mitigated toxicity, enabling 1.75 g L-1 in batch mode and 7.75 g L-1 cumulatively under continuous cultivation. This work establishes a comprehensive framework for scalable cyanobacteria-based isobutanol production, demonstrating industrial potential under natural sunlight.