Discovery of Novel <i>Pseudomonas putida</i> Flavin-Binding Fluorescent Protein Variants with Significantly Improved Quantum Yield
Sanghwan Ko, Hyunwoo Jeon, Sanghan Yoon, Munsu Kyung, Hyungdon Yun, Jung‐Hyun Na, Sang Taek Jung
Abstract
Oxygen-independent, flavin-binding fluorescent proteins (FbFPs) are emerging as alternatives to green fluorescent protein (GFP), which has limited applicability in studying anaerobic microorganisms, such as human gastrointestinal bacteria, which grow in oxygen-deficient environments. However, the utility of these FbFPs has been compromised because of their poor fluorescence emission. To overcome this limitation, we have employed a high-throughput library screening strategy and engineered an FbFP derived from Pseudomonas putida (SB2) for enhanced quantum yield. Of the resulting SB2 variants, KOFP-7 exhibited a significantly improved quantum yield (0.61) compared to other reported engineered FbFPs, which was even higher than that of enhanced GFP (EGFP, 0.60), with significantly enhanced tolerance against a strong reducing agent.