Employment of light-inducible promoter in genetically engineered cyanobacteria for photosynthetic isobutanol production with simulated diurnal sunlight and CO2

Abstract

Cyanobacteria are oxygen-evolving prokaryotes that can be engineered for biofuel production from solar energy, CO_2, and water. Isobutanol (IB) has the potential to serve as an alternative fuel and important chemical feedstock. The research involves engineering Synechocystis sp. PCC 6803, for photosynthetic isobutanol production via the 2-keto-acid pathway and their cultivation in lab-scale photobioreactors. This synthetic pathway involves the heterologous expression of two enzymes, α-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (Yqhd), under a strong light-inducible promotor, psbA2, known to show increased gene expression under high light. The use of psbA2 could be a valuable strategy for isobutanol production as economic scaling up demands the utilization of natural sunlight, which also provides very high light intensity at midday, facilitating increased production. The study reports isobutanol production from engineered strains containing both pathway genes and with only kivd. In shake flask studies, the highest isobutanol titre of 75 mg L⁻¹ (12th day) was achieved from an engineered strain DM12 under optimized light intensity. DM12 was cultivated in a 2 L flat panel photobioreactor, resulting in a maximum isobutanol titre of 371.8 mg L⁻¹ (10th day) with 2 % CO₂ and 200 μmol photons m⁻² s⁻¹. Cultivation of DM12 in a photobioreactor under mimic diurnal sunlight demonstrated the highest productivity of 39 mg L⁻¹ day⁻¹ with the maximum titre of 308.5 mg L⁻¹ (9th day). This work lays the foundation for sustainable, large-scale biobutanol production using solar energy.