Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin.  These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase-isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase-isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy and tandem mass spectrometry. Our results show that MpeV is the lyase-isomerase that covalently attaches a doubly-linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB).  MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82).  Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon).  MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase-isomerases.

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Marine Synechococcus efficiently harvest available light for photosynthesis using complex antenna systems, called phycobilisomes, composed of an allophycocyanin core surrounded by rods, which in the open ocean are always constituted of phycocyanin and two phycoerythrin (PE) types: PEI and PEII. These cyanobacteria display a wide pigment diversity primarily resulting from differences in the ratio of the two chromophores bound to PEs, the green-light absorbing phycoerythrobilin and the blue-light absorbing phycourobilin. Prior to phycobiliprotein assembly, bilin lyases post-translationally catalyze the ligation of phycoerythrobilin to conserved cysteine residues on α- or β-subunits, whereas the closely related lyase-isomerases isomerize phycoerythrobilin to phycourobilin during the attachment reaction. MpeV was recently shown in Synechococcus sp. RS9916 to be a lyase-isomerase which doubly links phycourobilin to two cysteine residues (C50 and C61; hereafter C50, 61) on the β-subunit of both PEI and PEII. Here we show that Synechococcus sp. WH8020, which belongs to the same pigment type as RS9916, contains MpeV that demonstrates lyase-isomerase activity on the PEII β-subunit but only lyase activity on the PEI β-subunit. We also demonstrate that occurrence of a histidine at position 141 of the PEI β-subunit from WH8020, instead of a leucine in its counterpart from RS9916, prevents the isomerization activity by WH8020 MpeV, showing for the first time that both the substrate and the enzyme play a role in the isomerization reaction. We propose a structural-based mechanism for the role of H141 in blocking isomerization. More generally, the knowledge of the amino acid present at position 141 of the β-subunits may be used to predict which phycobilin is bound at C50, 61 of both PEI and PEII from marine Synechococcus strains.

Synthetic biology (SynBio) aims to rationally engineer or modify traits of an organism or integrate the behaviors of multiple organisms into a singular functional organism through advanced genetic engineering techniques. One objective of this research was to determine the environmental persistence of engineered DNA in the environment. To accomplish this goal, the environmental persistence of legacy engineered DNA building blocks were targeted that laid the foundation for SynBio product development and application giving rise to “post-use products.” These building blocks include genetic constructs such as cloning and expression vectors, promoter/terminator elements, selectable markers, reporter genes, and multi-cloning sites. Shotgun sequencing of total DNA from water samples of pristine sites was performed and resultant sequence data mined for frequency of legacy recombinant DNA signatures. Another objective was to understand the fate of a standardized contemporary synthetic genetic construct (SC) in the context of various chassis systems/genetic configurations representing different degrees of “genetic bioavailability” to the environmental landscape. These studies were carried out using microcosms representing different environmental matrices (soils, waters, wastewater treatment plant (WWTP) liquor) and employed a novel genetic reporter system based on volatile organic compounds (VOC) detection to assess proliferation and persistence of the SC in the matrix over time.