Biosynthesis of cyanobacterial phycobiliproteins in E. coli: examination of allophycocyanin subunit biosynthesis and creation of unique fluorescent phycobiliproteins containing phycoerythrobilin

Phycobiliproteins are soluble light‐harvesting proteins and are highly fluorescent due to the addition of a bilin attached at specific Cys residues, usually by enzymes called bilin lyases. An in vivo heterologous E. coli multiplasmid co‐expression system was created to test different phycobiliprotein bilin lyases and to produce large amounts of holo‐phycobiliproteins. Co‐expression of the bilin biosynthetic genes ho1 encoding heme oxygenase from Synechocystis sp. PCC 6803 and pcyA from Synechococcus sp. PCC 7002 (pPcyA) resulted in the production of large amounts of phycocyanobilin (PCB) from heme in E. coli cells as evidenced by the blue color of cells. The CpcS/CpcU bilin lyase was shown to recognize and attach PCB to HT‐ApcD/ApcB and to HT‐ApcF. However, ApcE (the allophycocyanin domain 1‐228) fused to glutathione S transferase had autocatalytic bilin lyase activity in the presence of PCB (pPcyA). A plasmid expressing ho1 and pebS to produce phycoerythrobilin (PEB) in E. coli was used to test the substrate specificity of the bilin lyase CpcS/CpcU from the cyanobacterium Synechococcus sp. PCC 7002 and of the bilin lyase CpcE/CpcF from Synechocystis sp. PCC 6803. We used this system to make unique phycobiliproteins containing PEB. This research was supported by National Science Foundation grant to W. M. S. (MCB‐0133441).