Utilization of the key regulatory protein CP12 to re-direct reductive power for production in cyanobacteria

Cyanobacteria as phototrophic microorganisms bear great potential to produce chemicals and fuels from sustainable resources such as light, CO2, and water. The recently gained knowledge on internal electron and carbon fluxes and the regulation of cellular metabolism brings about opportunities to channel the energy towards product formation to overcome low efficiencies in product formation that hinder economical application to date. For instance, intrinsic regulators might pose interesting engineering targets to tune metabolic fluxes. In case of reductive processes like hydrogen production or redox catalysis, the partition of redox equivalents determines the efficiency. Here, the small regulatory protein CP12 controlling the major competitor for electrons – the Calvin-Benson-Bassham (CBB) cycle – was utilized to minimize electron drain in the model cyanobacterium Synechocystis sp. PCC 6803. In particular, we made use of phage-based CP12 variants to downregulate the CBB cycle. Unlike the native CP12 of Synechocystis, which only inhibits the CBB cycle in the dark and gets deactivated by a reduced cell status^1,2, CP12 homologs from cyanophages appear to be active when the infected host cell is exposed to light. This, together with other metabolic interventions, lead to an enhanced electron availability for phage reproduction³. To simulate this circumstance, we constructed Synechocystis strains that encode those CP12 homologs. In addition, we used rational protein design targeting the elimination of redox-control from the CP12 variant native to Synechocystis. In total, 6 different CP12 variants were inducibly expressed in Synechocystis. Intriguingly, impairment of growth upon induction of CP12 expression could be detected in different strengths for all variants. For promising variants, titratability of this effect could be demonstrated and their impact on the metabolism and hydrogen production was analysed. With this, we demonstrated that small proteins can be used to re-direct metabolic capacities in cyanobacteria.

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