Part:BBa_K4350814

Neonothopanus nambi Luciferase

This gene is a modified version of the luciferase enzyme responsible for N. nambi bioluminescence.
The sequence has been modified by the 2022 UBC-Okanagan team by codon optimization for expression in the cyanobacteria Synechocystis sp. PCC 6803, a model organism.
Furthermore, the 5' and 3' flanks of this sequence have been engineered with BbsI restriction sites for compatibility with the MoClo standard.

Usage and Biology

Although the fungal bioluminescence pathway was only elucidated in 2018, luciferase from Neonothopanus nambi has already demonstrated successful heterologous expression in Pichia pastoris (Kotlobay et al., 2018), Escherichia coli (Kotlobay et al., 2018), Nicotiana tabacum (Mitiouchkina et al., 2020), Xenopus laevis embryos (Kotlobay et al., 2018), and human cell lines (Gorokhovatsky et al., 2021). The fungal bioluminescence pathway is one of the two only bioluminescent pathways that have been fully elucidated, and the only one elucidated from eukaryotes.

Fungal luciferase catalyzes the oxidation of fungal luciferin (3-hydroxyhispidin) with molecular oxygen. This produces a high-energy endoperoxide intermediate that emits light upon decomposition into oxyluciferin (caffeoylpyruvate) (Kotlobay et al., 2018).

This fungal luciferase is a member of a novel protein family, lacking homology with other well-characterized enzymes (Beregovaya et al., 2021). The structure of luciferase has yet to be studied through crystallography. However, software predictions indicate that this luciferase likely possesses an N-terminal α-helical transmembrane domain (Beregovaya et al., 2021).

Luciferase functions at an optimum pH of 8.0 and temperature of 14 °C, inactivating at temperatures surpassing 30 °C. By extracting recombinant luciferase expressed in P. pastoris, the Michaelis-Menten constant was determined to be 1.09 +/- 0.06 µM by simulation (Gorokhovatsky et al., 2021).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 411
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

• Beregovaya, K. A., Myshkina, N. M., Chepurnykh, T. V., Kotlobay, A. A., Purtov, K. V., Petushkov, V. N., Rodionova, N. S., & Yampolsky, I. V. (2021). Rational design and mutagenesis of fungal luciferase from Neonothopanus Nambi. Doklady Biochemistry and Biophysics, 496(1), 14–17. https://doi.org/10.1134/s1607672921010026
• Gorokhovatsky, A. Y., Chepurnykh, T. V., Shcheglov, A. S., Mokrushina, Y. A., Baranova, M. N., Goncharuk, S. A., Purtov, K. V., Petushkov, V. N., Rodionova, N. S., & Yampolsky, I. V. (2021). The recombinant luciferase of the fungus neonothopanus Nambi: Obtaining and properties. Doklady Biochemistry and Biophysics, 496(1), 52–55. https://doi.org/10.1134/s1607672921010051
• Kotlobay, A. A., Sarkisyan, K. S., Mokrushina, Y. A., Marcet-Houben, M., Serebrovskaya, E. O., Markina, N. M., Gonzalez Somermeyer, L., Gorokhovatsky, A. Y., Vvedensky, A., Purtov, K. V., Petushkov, V. N., Rodionova, N. S., Chepurnyh, T. V., Fakhranurova, L. I., Guglya, E. B., Ziganshin, R., Tsarkova, A. S., Kaskova, Z. M., Shender, V., … Yampolsky, I. V. (2018). Genetically encodable bioluminescent system from fungi. Proceedings of the National Academy of Sciences, 115(50), 12728–12732. https://doi.org/10.1073/pnas.1803615115
• Mitiouchkina, T., Mishin, A. S., Somermeyer, L. G., Markina, N. M., Chepurnyh, T. V., Guglya, E. B., Karataeva, T. A., Palkina, K. A., Shakhova, E. S., Fakhranurova, L. I., Chekova, S. V., Tsarkova, A. S., Golubev, Y. V., Negrebetsky, V. V., Dolgushin, S. A., Shalaev, P. V., Shlykov, D., Melnik, O. A., Shipunova, V. O., … Sarkisyan, K. S. (2020). Author correction: Plants with genetically encoded autoluminescence. Nature Biotechnology, 38(8), 1001–1001. https://doi.org/10.1038/s41587-020-0578-0