Expressing 2-keto acid pathway enzymes significantly increases photosynthetic isobutanol production

Cyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO₂ and solar energy in a direct process.

Heterologous expression of α-ketoisovalerate decarboxylase (Kivd^S286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192^OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L^-1 in short-term screening experiments. Through modulating kivd^S286T copy numbers as well as the composition of the 5'-region, a final Synechocystis strain HX47 with three copies of kivd^S286T showed a significantly improved isobutanol production of 144 mg L^-1, an 177% increase compared to the previously reported best producing strain under identical conditions.

This work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.