Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host.
The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp. PCC6803) was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter. Genes for the apoprotein (C-phycocyanin alpha subunit; cpcA) and the heterodimeric lyase (cpcE and cpcF) that catalyzes chromophore attachment were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used the cellular pool of heme to produce holo-CpcA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria. About a third of the apo-CpcA was converted to holo-CpcA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks cpcE and cpcF. This approach should permit incisive analysis of many remaining questions in phycobiliprotein biosynthesis. These studies also demonstrate the feasibility of generating constructs of these proteins in situ for use as fluorescent protein probes in living cells.
Tooley
A J
AJ
Department of Molecular and Cell Biology, University of California, 142 LSA no. 3200, Berkeley, CA 94720-3200, USA.
Cai
Y A
YA
Glazer
A N
AN
D000458:Cyanobacteria / Q000235:genetics; D004926:Escherichia coli; D015870:Gene Expression; D015394:Molecular Structure; D010641:Phenotype; D052980:Phycobilins; D010798:Phycocyanin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011758:Pyrroles / Q000737:chemistry; D045725:Tetrapyrroles
D052980:Phycobilins; D011758:Pyrroles; D045725:Tetrapyrroles; D010798:Phycocyanin; C011069:phycocyanobilin