Combinational biosynthesis and characterization of a fluorescent 82β-phycocyanin of Spirulina platensis

To biosynthesize fluorescent Spirulina platensis (Sp)β -phycocyanin (PC) in Escherichia coli, a BLASTP search for homologs of the cpeS gene, a chromophore lyase, was performed against the Synechocystis sp. PCC 6803 (S6) proteome. A highly homologous gene, slr2049, was obtained from the S6 genome. Sites 82 and 153 in -phycocyanin of Sp were modified by site-directed muta- genesis. Two recombinant expression vectors were constructed and transformed into E. coli BL21: (i) pCDF-cpcB (C153A)- slr2049-sll0583-ho1-pcyA; and (ii) pCDF-cpcB (C82I)-slr2049-sll0583-ho1-pcyA. Lyases encoded by the genes slr2049 and sll0583 catalyzed the linking of Sp 82β -PC to phycocyanobilin (PCB), and fluorescent CpcB (C153A)-PCB was generated. We present a strategy for the co-expression of multiple genes in a single expression vector to identify the function of an unknown gene. Recombinant phycobiliproteins produced on a large scale are promising fluorescent tags for diagnostics and pharmacology.

To biosynthesize fluorescent Spirulina platensis （Sp） β-phycocyanin （PC） in Escherichia coli, a BLASTP search for homologs of the cpeS gene, a chromophore lyase, was performed against the Synechocystis sp. PCC 6803 （S6） proteome. A highly homologous gene, sir2049, was obtained from the S6 genome. Sites 82 and 153 in β-phycocyanin of Sp were modified by site-directed mutagenesis. Two recombinant expression vectors were constructed and transformed into E. coli BL21： （i） pCDF-cpcB （C153A）- slr2049-sllO583-hol-pcyA; and （ii） pCDF-cpcB （C821）-slr2049-sllO583-hol-pcyA. Lyases encoded by the genes slr2049 and sl10583 catalyzed the linking of Sp 8213-PC to phycocyanobilin （PCB）, and fluorescent CpcB （C153A）-PCB was generated. We present a strategy for the co-expression of multiple genes in a single expression vector to identify the function of an unknown gene. Recombinant phycobiliproteins produced on a large scale are promising fluorescent tags for diagnostics and pharmacology.