Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV both in vitro and in fission yeast. We introduced SynPCB2.1, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB2.1. These tools not only enable the easy use of multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in awider range of living organisms.

Automated summary

The study demonstrates that phycocyanobilin (PCB) functions as a brighter chromophore for near-infrared fluorescent protein (iRFP) than biliverdin (BV), increasing the brightness of iRFP fluorescence. By introducing a PCB biosynthesis system and developing an endogenous gene tagging system along with integration plasmids carrying iRFP-fused marker proteins, multiplexed live-cell imaging in fission yeast becomes more accessible.