Skip to content
DraconiticResearch
Search Sources API
201,818 studies31 sourcesUpdated 18 min ago
Peer-reviewedCC BY 4.0The Journal of Biological Chemistry

Biochemical, bioluminescence and bioreporter properties of functionally improved firefly luciferases

  • Chadaporn Kantiwiriyawanitch1
  • Vinutsada Pongsupasa1
  • Pratchaya Watthaisong1
  • Jittima Phonbuppha1
  • Aisaraphon Phintha1
  • Daisuke Sasaki2
  • Thanyaporn Wongnate1
  • Ruchanok Tinikul3
  • Kazuki Niwa4
  • Yoshihiro Nakajima2
  • Yoshihiro Ohmiya1
  • Ubolsree Leartsakulpanich5
1 more authors
  • Pimchai Chaiyen1
5 affiliations
  1. 1School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand
  2. 2Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa, Japan
  3. 3Faculty of Science, Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Mahidol University, Bangkok, Thailand
  4. 4National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
  5. 5National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Agency (NSTDA), Pathum Thani, Thailand
Read full textDOICite

Abstract

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 kcat, 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 (FLUXVc) 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/FLUXVc 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.

Related

  1. Biochemical characterisation of fungal bioluminescence enzymes reveals substrate inhibition and secondary turnover of 3-hydroxyhispidin by H3H
    J. F. Burnett, J. Lang, A. Tumber +5
    bioRxivPreprintCC BYFull textMolecular biology & gene regulationbioRxiv
  2. Detection of Buried Landmines in an Active Minefield Through Bioreporter-Based Optoelectronic Sensing of Their Chemical Signatures.
    Yonatan Uziel, Mulkan Adgo, Natan Orlov +3
    BiosensorsPeer-reviewedCC BYFull textMolecular biology & gene regulationEurope PMC
  3. ATP-free fatty aldehyde biosynthesis enables an autonomous lux-based bioluminescence system
    Subhan Hadi Kusuma, Takeharu Nagai
    iSciencePeer-reviewedCC BY-NC-ND 4.0Full textMolecular biology & gene regulationCell PressCrossrefPMC
  4. Unveiling bioluminescence: understanding mechanisms and exploring wide-ranging applications
    Maria das Dores A. Oliveira, Chistiane M. Feitosa, Patrícia e S. Alves +3
    Results in ChemistryPeer-reviewedCC BYFull textMolecular biology & gene regulationDOAJ
  5. Development of shelf-stable reagents and assay kits for bioluminescence applications using the capillary-assisted vitrification platform stabilization technology
    Shari Radford, Yolanda Peris-Taverner, Michael Dibble +12
    PLOS OnePeer-reviewedCC BY 4.0Enzymology & metabolismMaterials & polymersPLOS

Cite

BibTeXRIS
BibTeX
@article{kantiwiriyawanitch2026biochemical,
  title = {{Biochemical, bioluminescence and bioreporter properties of functionally improved firefly luciferases}},
  author = {Chadaporn Kantiwiriyawanitch and Vinutsada Pongsupasa and Pratchaya Watthaisong and Jittima Phonbuppha and Aisaraphon Phintha and Daisuke Sasaki and Thanyaporn Wongnate and Ruchanok Tinikul and Kazuki Niwa and Yoshihiro Nakajima and Yoshihiro Ohmiya and Ubolsree Leartsakulpanich and Pimchai Chaiyen},
  journal = {The Journal of Biological Chemistry},
  year = {2026},
  month = sep,
  doi = {10.1016/j.jbc.2026.113568},
  url = {https://doi.org/10.1016/j.jbc.2026.113568},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Biochemical, bioluminescence and bioreporter properties of functionally improved firefly luciferases
AU  - Chadaporn Kantiwiriyawanitch
AU  - Vinutsada Pongsupasa
AU  - Pratchaya Watthaisong
AU  - Jittima Phonbuppha
AU  - Aisaraphon Phintha
AU  - Daisuke Sasaki
AU  - Thanyaporn Wongnate
AU  - Ruchanok Tinikul
AU  - Kazuki Niwa
AU  - Yoshihiro Nakajima
AU  - Yoshihiro Ohmiya
AU  - Ubolsree Leartsakulpanich
AU  - Pimchai Chaiyen
PY  - 2026
DA  - 2026/09/18
JO  - The Journal of Biological Chemistry
DO  - 10.1016/j.jbc.2026.113568
UR  - https://doi.org/10.1016/j.jbc.2026.113568
AB  - 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 kcat, 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 (FLUXVc) 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/FLUXVc 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.
SN  - 0021-9258
SN  - 1083-351X
ER  -