Sustainable production of photosynthetic isobutanol and 3-methyl-1-butanol in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Background: Cyanobacteria are emerging as green cell factories for sustainable biofuel and chemical production, due to their photosynthetic ability to use solar energy, carbon dioxide and water in a direct process. The model cyanobacterial strain Synechocystis sp. PCC 6803 has been engineered for the isobutanol and 3-methyl-1-butanol production by introducing a synthetic 2-keto acid pathway. However, the achieved productions still remained low. In the present study, diverse metabolic engineering strategies were implemented in Synechocystis sp. PCC 6803 for further enhanced photosynthetic isobutanol and 3-methyl-1-butanol production.

Results: Long-term cultivation was performed on two selected strains resulting in maximum cumulative isobutanol and 3-methyl-1-butanol titers of 1247 mg L^-1 and 389 mg L^-1, on day 58 and day 48, respectively. Novel Synechocystis strain integrated with a native 2-keto acid pathway was generated and showed a production of 98 mg isobutanol L^-1 in short-term screening experiments. Enhanced isobutanol and 3-methyl-1-butanol production was observed when increasing the kivd^S286T copy number from three to four. Isobutanol and 3-methyl-1-butanol production was effectively improved when overexpressing selected genes of the central carbon metabolism. Identified genes are potential metabolic engineering targets to further enhance productivity of pyruvate-derived bioproducts in cyanobacteria.

Conclusions: Enhanced isobutanol and 3-methyl-1-butanol production was successfully achieved in Synechocystis sp. PCC 6803 strains through diverse metabolic engineering strategies. The maximum cumulative isobutanol and 3-methyl-1-butanol titers, 1247 mg L^-1 and 389 mg L^-1, respectively, represent the current highest value reported. The significantly enhanced isobutanol and 3-methyl-1-butanol production in this study further pave the way for an industrial application of photosynthetic cyanobacteria-based biofuel and chemical synthesis from CO₂.

Keywords: 2-Keto acid pathway; 3-Methyl-1-butanol; Cyanobacteria; Isobutanol; Metabolic engineering; Solar fuel; Synechocystis sp. PCC 6803.