After cycles of mutagenesis and selection, the accumulated library of function-retained and functionlost. Based on the functional landscape of the amino acid residues in EcFbFP variants, crucial sites that determine EcFbFP function can be categorized into three groups. The first group is located on the direct interaction site of the FMN molecule for facilitating binding, the second group is located on turns and loops of the protein where conformational changes may occur, and the third group is located at spatially clustered residues near the Glu56-Lys97 salt bridges. Taken together, this approach allows the analysis of thousands of mutations with functional phenotype correlation by single highthroughput sequencing. Detailed analysis of the amino acid substitutions in variants of FR and FL libraries provided a landscape of the effect of each mutation on the EcFbFP function. All the variants in the FR library were considered to have non-deleterious mutations. On the other hand, each variant in the FL library contains both mutations that have non-deleterious effect and those that cause loss of function, because the variants in FR library were generated by repeated random mutagenesis of FR variant DNAs obtained from each cycle. The number of mutation occurrences in amino acid sequences of variants was 329 and 563 in the FR and FL libraries, respectively. Among those, 304 amino acid substitutions were detected in both libraries, which were tolerant to the loss of function. A total of 259 unique mutations were identified from the variants in the FL library, which were expected to be crucial amino acid residues responsible for the EcFbFP function. In total, the accumulated mutations indicate that each amino acid residue in EcFbFP has an average of 108 mutations; however, the pattern of enriched mutations differ so that some residues are enriched with FR mutations and some residues are enriched with FL mutations. To analyze this difference in the mutation enrichment pattern, we quantitatively compared the mutation frequency of each amino acid residue in FR and FL variants. The ratio of FL mutations over total mutations in each residue was calculated. In this study, the ratio of enrichment of FL mutations is referred to as the positional effect. The resulting heatmap of the positional effect indicates that positions tolerant to the deleterious effect on the protein function have a value close to zero and positions sensitive to the deleterious effect on the protein function have a value close to one. To further analyze the functionally sensitive positions, we used the 15 FMN-binding sites identified from previous study as a standard to determine the cutoff value for the most sensitive residues of EcFbFP. After applying the cutoff based on the positional effect, we determined 25 functionally sensitive residues, which included ten FMN-binding sites used in the standard.