Deep Mutational Scanning of an Oxygen-Independent Fluorescent Protein CreiLOV for Comprehensive Profiling of Mutational and Epistatic Effects.

Abstract

Oxygen-independent, flavin mononucleotide-based fluorescent proteins (FbFPs) are promising alternatives to green fluorescent protein in anaerobic contexts. Deep mutational scanning performs systematic profiling of protein sequence-function relationships but has not been applied to FbFPs. Focusing on CreiLOV from Chlamydomonas reinhardtii, we created and analyzed two comprehensive mutant collections: (1) single-residue, site-saturation mutagenesis libraries covering all 118 residues; and (2) a full combinatorial metagenesis library among 20 mutations at 15 residues, where mutation and residue selection was based on single-site mutagenesis results. Notably, the second type of library is indispensable to study higher-order epistasis but underrepresented in the literature. Using optimized FACS-seq assays, 2,185 (>92.5%) out of 2,360 possible single-site mutants and 165,428 (>89.7%) out of 184,320 possible combinatorial mutants were reliably assigned with fitness values. We constructed statistical and machine-learning models to analyze the CreiLOV data set, enabling accurate fitness prediction of higher-order mutants using lower-order mutagenesis data. In addition, we successfully isolated CreiLOV variants with improved fluorescence quantum yield and thermostability. This work provides new empirical data and design rules to engineer combinatorial protein variants.