Utilization of the small regulatory protein CP12 to engineer photosynthetic electron fluxes in cyanobacteria

Photoautotrophic microorganisms like cyanobacteria hold great potential for sustainable production of chemicals and fuels from light, CO2, and water. However, their economical applicability is limited, in part due to low product yields achieved to date. Recent insights into their metabolism, energy and metabolite fluxes, and metabolic regulation provide new opportunities for interventions to enhance product yields. For conversions that only require electrons – such as redox catalysis or the formation of hydrogen by hydrogenases - the partitioning of electrons from the photosynthetic electron transport chain (PETC) determines the yield. In these processes the Calvin-Benson-Bassham (CBB) cycle is the main competitor for electrons. Here, the small regulatory protein CP12 was used to reduce electron flux into the CBB cycle and redirect them into valuable products in the model cyanobacterium Synechocystis sp. PCC 6803. In most phototrophic organisms, CP12 is used to control the CBB cycle in dark-night cycles by binding and inhibiting the enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphofructokinase (PRK). The native CP12 of Synechocystis itself is redox-regulated, inhibiting the CBB cycle only in the dark and becoming deactivated by a reduced cell status when light is present and the PETC is running1,2. We therefore employed variants of CP12 either derived from cyanophages - which use them during infection in light as auxiliary metabolic genes yielding a more reduced status of their hosts3 - or rationally designed to be unaffected by the redox-status. By inducible expression of these CP12 variants, we were able to modulate photoautotrophic growth and shift the intracellular redox potential to a more reduced state. This altered metabolic state increased the electron flux from photosynthesis into hydrogen production and, in combination with disruption of cyclic electron flow and partial uncoupling of the proton-motif-force, enabled higher continuous hydrogen production. By utilizing CP12 variants to redirect electrons from the CBB cycle toward hydrogen production in cyanobacteria, we highlight the potential of small regulatory proteins for metabolic engineering to optimize resource allocation towards desired products.

1 McFarlane, et al. 2019, PNAS 116(42): 20984-20990.

2 Lucius et al. 2022, Front. Plant Sci. 13: 1028794.

3 Thompson et al. 2011, PNAS 108: E757-E764