Biosynthesis of cyanobacterial phycobiliproteins in Escherichia coli: chromophorylation efficiency and specificity of all bilin lyases from Synechococcus sp. strain PCC 7002.
Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and alpha-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter(-1) of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (alpha(AP-B)) and ApcF (beta(18)). The N-terminal, allophycocyanin-like domain of ApcE (L(CM)(99)) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of beta-phycocyanin.
Biswas
Avijit
A
Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148, USA.
Vasquez
Yasmin M
YM
Dragomani
Tierna M
TM
Kronfel
Monica L
ML
Williams
Shervonda R
SR
Alvey
Richard M
RM
Bryant
Donald A
DA
Schluchter
Wendy M
WM
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008190:Lyases / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D052980:Phycobilins / Q000378:metabolism; D052979:Phycobiliproteins / Q000096:biosynthesis*; D010798:Phycocyanin / Q000737:chemistry / Q000378:metabolism; D046940:Synechococcus / Q000201:enzymology*
D001426:Bacterial Proteins; D052980:Phycobilins; D052979:Phycobiliproteins; C002497:allophycocyanin; D010798:Phycocyanin; C011069:phycocyanobilin; D010088:Oxidoreductases; C442236:phycocyanobilin ferredoxin oxidoreductase, Synechocystis; D008190:Lyases