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

Abstract

Abstract Background 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. Results Heterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) 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 (Slr1192OP), 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. Conclusions 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. Graphic abstract

Keywords

• Synechocystis PCC 6803
• Isobutanol production
• 2-Keto acid pathway
• α-Ketoisovalerate decarboxylase
• Metabolic engineering