Biosynthesis of a Fluorescent Cyanobacterial Phycoerythrocyanin 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 apo-(protein)(Phycoerythrocyanin α subunit;pecA) were expressed on a second plasmid pETDuet-pecA.Genes for the heterodimeric lyase(pecE and pecF) that catalyzes chromophore attachment were expressed on a third plasmid pCOLADuet-pecE and a fourth plasmid pCDFDuet-pecF.Upon induction,recombinant E.coli used the cellular pool of heme to produce holo-PecA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria.However,unlike the other biliproteins,isolated PEC shows a pronounced reversible photochemistry,which has been related to the α-subunit(α-PEC).About 0.1% of the apo-PecA was converted to holo-PecA-PCB in a similarly engineered E.coli strain that lacks pecE and pecF.