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Deletion of sll1541 in Synechocystis sp. Strain PCC 6803 Allows Formation of a Far-Red-Shifted holo-Proteorhodopsin In Vivo

ABSTRACT In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of β-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal. IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.