Biosynthesis of a Fluorescent Cyanobacterial C-phycocyanin Holo-α Subunit in Escherichia coli

The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium(Anabaena sp.PCC7120)was reconstituted in Escherichia coli.Cyanobacterial genes for enzymes ho1 and pcyA were expressed from a plasmid pACYCDuet-ho1-pcyA.Genes for the apoprotein(C-phycocyanin α subunit;cpcA)were expressed on a second plasmid pETDuet-cpcA.Genes for the heterodimeric lyase(cpcE and cpcF)that catalyzes chromophore attachment were expressed on a third plasmid pCOLADuet-cpcE-cpcF.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 0.2% of the apo-CpcA was converted to holo-CpcA-PCB in a similarly engineered E.coli strain that lacks cpcE and cpcF.