Biochemical, bioluminescence and bioreporter properties of functionally improved firefly luciferases

Firefly luciferase (Fluc) is one the most widely used bioluminescent reporters for biological imaging and gene expression analysis, yet its limited stability and high assay cost remain major constraints limiting broader applications. Here, we engineered Fluc variants (N116H, M118L and N116H/M118L) with improved catalytic performance, thermostability, and soluble expression. The N116H variant exhibited the most pronounced enhancement, including a 4.6-fold increase in k_cat, a 2.7-fold higher soluble expression yield, a 3.5-fold longer half-life at 37 °C, and 1.25-fold enhanced quantum yield relative to wild-type Fluc (WT). Notably, N116H required substantially less coenzyme A (CoA) to mitigate inhibition by the dehydroluciferyl-adenylate intermediate, enabling significant reduction of assay costs. Leveraging these properties, we developed a low-cost dual-luciferase reporter platform using Fluc variants and flavin-based bacterial luciferase from Vibrio campbellii (FLUX^Vc) as a substitute for the conventional Fluc/Renilla luciferase (Rluc) system. The engineered systems generated comparable reporter signals while reducing assay costs by approximately 50 to 150-fold relative to commercial kits. Functional validation using the Keap1-Nrf2-ARE signaling pathway and carnosic acid demonstrated robust and expected reporter responses, with the N116H/FLUX^Vc system producing the strongest luminescence output. Furthermore, N116H enabled significantly improved real-time monitoring and single-cell imaging, producing up to 6.3-fold higher cumulative bioluminescence and increasing detectable cell numbers by approximately 4 to 5-fold compared with WT. Our findings establish N116H as a highly efficient and cost-effective luciferase variant for dual-reporter assays, real-time live-cell monitoring, and sensitive single-cell bioluminescence imaging applications.