## 1 Introduction
The silencing of _Z-ISO_ mRNA in a tomato plant results in a reduction in lycopene and an increase in phytoene, phytofluene, and \(\zeta\)-carotene [14] and a non-functional splice variant of Z-ISO is associated with the yellow pigmentation of the \"Finalate\" sweet orange mutant, which accumulates unusual proportions of 9,15,9\({}^{\prime}\)-tri-_cis_- and 9,9\({}^{\prime}\)-di-_cis_-\(\zeta\)-carotene, a phenotype partially restored by exposure to high light [12]. Carotenoid biosynthesis has been extensively characterized in model cyanobacteria such as _Synechocystis_ sp. PCC 6803 (hereafter _Synechocystis_), where carotenoids play important functional and structural roles in photosystems (PS) I and II [15; 16], and are also found in the cytochrome _bgf_ complex [17; 18] and the NADH dehydrogenase-like photosynthetic complex I [19; 20]. _Synechocystis_ mutants lacking _pds_ (_crtP_, slr1254) or _zds_ (_crtQ_, slr0940) accumulate phytoene or \(\zeta\)-carotene, respectively, display very slow glucose-dependent growth, are acutely light sensitive, and lack PSII [21]. Conversely, the deletion of sll0033 (_crtH_), which encodes the CRT-ISO enzyme that converts 7,9,7,9\({}^{\prime}\)-tetra-_cis_-lycopene (prolylcopene) to all-_trans_-lycopene, results in a strain that can produce mature carotenoids in the light, albeit in different ratios to the WT organism, due to the photo-isomerization of polycopene [22; 23; 24]. However, the \(\Delta\sigma tH\) strain predominantly accumulates _cis_-isomers of carotenes and does not make \(\beta\)-carotene under light-activated heterotrophic growth (LAHG) conditions (darkness with a short pulse of light every 24 h), and therefore it cannot assemble PSII [25]. CRT-ISO isomerizes the adjacent 7,9 and 7\({}^{\prime}\)_qci_-pairs of double bonds in 7,9,7\({}^{\prime}\),9\({}^{\prime}\)-tetra-_cis_-lycopene but does not act on the 15,15\({}^{\prime}\)_cis_ double bond of 9,15,9\({}^{\prime}\)-tri-_cis_-\(\zeta\)-carotene [26]. This 15-_cis_ double bond is also photo-labile and can be photo-isomerized, but this appears to be less efficient than the enzyme-catalyzed reaction [7; 8]. As Z-ISO is essential for carotenogenesis in dark plant tissues such as the roots, etiolated leaves, and endosperm [7], we predicted that the same would be true for carotenoid biosynthesis in cyanobacteria during periods of darkness. Here, we verify the \(\zeta\)-carotene isomerase activity of the _Synechocystis_ Z-ISO homolog (encoded by the slr1599 locus) by heterologous production of Slr1599 in a 9,15,9\({}^{\prime}\)-tri-_cis_-\(\zeta\)-carotene-accumulating strain of _Escherichia coli_. We also show that Z-ISO is needed for carotenoid biosynthesis in the absence of light in a _Synechocystis_\(\Delta\)slr1599 mutant, which to our knowledge is the first characterization of a cyanobacterial _Z-ISO_ deletion strain. Despite repeated attempts, we were unable to purify the _Synechocystis_ Z-ISO for spectral analysis and structure determination, but a comparison of AlphaFold structural models of the _Synechocystis_ and plant enzymes shows conservation of putative heme-ligating residues and allows us to predict the 9,15,9\({}^{\prime}\)-tri-_cis_-\(\zeta\)-carotene-binding site.