## 3 Results and Discussion
### Attempted Purification of Recombinant and Native Synechocystis Z-ISO  
The maize Z-ISO has previously been purified as a recombinant His-tagged MBP-fusion from detergent-solubilized _E. coli_ membranes and shown to contain a heme B cofactor [5; 35]. We generated a construct similar to that described by [PERSON] [6] to produce N-terminally His-MBP-tagged maize Z-ISO, and a second construct in which the maize _Z-ISO_ gene was replaced with slr1599 (fusion gene sequences are provided in Table S3). Following the protocol described by [PERSON] and [PERSON] [35] (see Section 2.4), we were able to isolate the maize protein and demonstrate similar spectroscopic characteristics to those reported previously (Figure S2) [5]. However, we were unable to purify Slr1599 using the same approach; unlike with the maize enzyme, the _E. coli_ pellets and membranes from cells carrying the slr1599 construct were not pink/red, indicating that significant quantities of a heme-containing protein were not produced, and the protein was not detected by SDS-PAGE or immunoblot analysis. Attempts to produce the _Synechocystis_ protein using a second construct with the gene cloned into the commercial pMAL-c5X plasmid (Table S1) were also unsuccessful.  
The maize Z-ISO and other plant enzymes are predicted to have seven TMHs with an N-terminus in/C-terminus out orientation [5; 12], whereas the _Synechocystis_ enzyme is predicted to contain one less TMH with both the N- and C-termini in the periplasm/thylakoid lumen (Figure 2). While cytoplasmic MBP is compatible with the N-terminal orientation of the maize enzyme, we hypothesized that the presence of MBP may prevent the localization of the N-terminus of the _Synechocystis_ enzyme to the periplasm, preventing the proper folding, orientation, and insertion of Z-ISO into the membrane. To retain MBP and the N-terminus of Z-ISO in the cytoplasm and periplasm, respectively, we fused a single TMH protein, YnhF [45], between MBP and Z-ISO. Despite repeated attempts to produce and purify the fusion protein, we were unable to do so. We also inserted slr1599 into the commercial pMAL-p5x vector to N-terminally fuse _Synechocystis_ Z-ISO to an MBP variant that is directed to the periplasm so that the N-terminus of Z-ISO is in the correct 'out' orientation (Table S1), but this also failed to yield detectable quantities of protein.  
Due to the inability to isolate recombinant Slr1599 from _E. coli_, we changed tack and tried to purify the enzyme from the native cyanobacterial host. To attempt to immuno-precipitate Slr1599 from _Synechocystis_, we expressed the gene encoding the enzyme with a 3 xFLAG tag from the _psbAII_ locus in the Aslr1599 background, generating the strain Aslr1599 FLAG-Slr1599. It was only possible to perform this with the FLAG tag at the N-terminus as we were unable to isolate _Synechocystis_ colonies for the C-terminally-tagged equivalent, which is consistent with previous findings that the C-terminus is important for the function of plant Z-ISO [6]. FLAG-tagged Slr1599 was functional as the Aslr1599 FLAG-Slr1599 strain produced \(\beta\)-carotene during LAHG (Figure S1); however, we were unable to purify the tagged enzyme from detergent-solubilized membranes from the cells grown under these conditions or under constant illumination. We speculate that the enzyme is quickly degraded in the light, either during growth under illumination, where it is not required and may be inactivated by the proposed redox-dependent ligand-switching mechanism [5], or during the subsequent cell breakage and purification process, even though these steps were performed under dim light. Consistent with this hypothesis, Slr1599 was not identified in our recent quantitative proteomics analysis of _Synechocystis_ cells grown under constant illumination, conditions where PDS, ZDS, and CRT-ISO were all detected [46]. Our inability to isolate Slr1599 following LAHG suggests that the enzyme is present in low amounts even under conditions where we have shown that it is required for carotenoid biosynthesis (Figure 4).