Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host

doi:10.1073/pnas.181340998

Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host

Department of Molecular and Cell Biology, University of California, 142 LSA no. 3200, Berkeley, CA 94720-3200

Contributed by Alexander N. Glazer

Abstract

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 α 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.

Footnotes

↵ * Present address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.
↵ † To whom reprint requests should be addressed at: Natural Reserve System, University of California System, 1111 Franklin Street, 6th Floor, Oakland, CA 94607-5200. E-mail: alexander.glazer{at}ucop.edu.
Abbreviations:

CpcA,

α subunit of C-phycocyanin;

CpcE and CpcF,

subunits of the heterodimeric phycocyanin α-subunit phycocyanobilin lyase;

HO1,

heme oxygenase 1;

HT,

His6-tag;

PCB,

phycocyanobilin;

PcyA,

3Z-phycocyanobilin:ferredoxin oxidoreductase;

Sp,

spectinomycin;

Km,

kanamycin