Significance: Cyanobacteria have been increasingly explored as a biotechnological platform, although their economic feasibility relies in part on the capacity to maximize their photosynthetic, solar-to-biomass energy conversion efficiency. Here we show that cyanobacterial photosynthetic capacity can be increased by diverting cellular resources toward heterologous, energy-storing metabolic pathways and by reducing electron flow to photoprotective, but energy-dissipating, oxygen reduction reactions. We further show that these heterologous sinks can partially contribute to photosystem I (PSI) oxidation, suggesting an engineering strategy to improve both energy storage capacity and robustness by selective diversion of excess photosynthetic capacity to productive processes.

Photosynthesis holds the promise of sustainable generation of useful products using light energy. Key to realizing this potential is the ability to rationally design photosynthesis to redirect energy and reductant derived from photons to desired products. Cytochrome P450s (P450s), which catalyze a broad array of reactions, have been engineered into a variety of photosynthetic organisms, where their activity has been shown to be photosynthesis-dependent, thus acting as heterologous sinks of electrons derived from photosynthesis. Furthermore, the addition of P450s can increase the photosynthetic capacity of the host organism. In this study, we developed this technology further using a P450 (CYP1A1) expressed in the cyanobacterium Synechococcus sp. PCC 7002. We show that rationally engineering photosynthesis by the removal of a competing electron sink, the respiratory terminal oxidase cytochrome c oxidase, increased the activity of CYP1A1. We provide evidence that this enhanced CYP1A1 activity was facilitated via an increase in the flux of electrons through Photosystem I. We also conducted a transcriptomic analysis on the designed strains to gain a more holistic understanding of how the cell responds to rational engineering. We describe a complex response including changes in expression of genes involved in photosynthesis and electron transfer linked to respiration. Specifically, the expression of CYP1A1 resulted in the reduction in expression of other natural electron dissipation pathways. This study emphasizes the potential for engineering photosynthetic organisms in biotechnology but also highlights the need to consider the broader impacts on cellular metabolism of any rationally induced changes.

Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous β-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD₇₃₀ (1.03 ± 0.47 mmol mol^–1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.

All known cyanobacteria contain cytochrome c₆, a small soluble electron carrier protein whose main function is to transfer electrons from cytochrome b₆f complex to photosystem I, although it is also involved in respiration. We have previously described a second isoform of this protein, the cytochrome c₆-like, whose function remains unknown. Here we describe a third isoform of cytochrome c₆ (here called cytochrome c_6-3), which is only found in heterocyst-forming filamentous cyanobacteria. Cytochrome c_6-3 is expressed in vegetative cells but is specifically repressed in heterocysts cells under diazotrophic growth conditions. Although there is a close structural similarity between cytochrome c_6-3 and cytochrome c₆ related to the protein general folding, cytochrome c_6-3 presents differential electrostatic surface features as compared with cytochrome c₆, its expression is not copper-dependent and has a low reactivity towards photosystem I. According to the different expression pattern, functional reactivity and structural properties, cytochrome c_6-3 has to play a regulatory role, yet-to-be-defined, related to heterocyst differentiation.

Cytochrome c_M was first described in 1994 and its sequence has been found in the genome of manifold cyanobacterial species ever since. Numerous studies have been carried out with the purpose of determining its function, but none of them has given place to conclusive results so far. Many of these studies are based on the assumption that cytochrome c_M is a soluble protein located in the thylakoid lumen of cyanobacteria. In this work, we have reevaluated the sequence of cytochrome c_M, with our results showing that its most probable 3D structure is strongly similar to that of the C subunit of the bacterial nitric oxide reductase. The potential presence of an α-helix tail, which could locate this protein in the thylakoid membrane, further supports this hypothesis, thus providing a new, unexpected role for this redox protein.