Part:BBa_K3887000:Design
Fre-L3
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Fre-L3 is a type of flavin reductase (Fre). It is commonly used as a fusion enzyme. In our experiment, it is partnered with SttH. It can be highly expressed as a soluble form in E. coli and also regenerate FADH2 effectively. The expression SttH fusion enzyme is lower when using SttH alone, because it was affected by N-terminal domains. Fre was effective as a soluble N-terminal tag for SttH. Fre-L1–SttH, Fre-L2–SttH and Fre-L3– SttH are three fusion enzymes using three different linker sequence, resulting in different flexibility in fusing.
During the experiment, ∆tnaA Fre-L3-SttH shows the highest concentration when conversion 6-Br-Trp from Trp. It is 39 times and 2.5 times higher than that of ΔtnaA SttH and ΔtnaA SttH+Fre, respectively. To test if other protein will increase the 6-Br-Trp production, Fre-L3 is compared with maltose-binding protein (MBP). MBP-L3-SttH also shows an increase in production of 6-Br-Trp. However, ∆tnaA MBP-L3-SttH shows 1.4 times increase compared to ∆tnaA SttH, while ∆tnaA Fre-L3-SttH produces 15-fold more. This experiment proves Fre efficiently enhanced SttH activity.
References
Lee, J., Kim, J., Song, J.E. et al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol 17, 104–112 (2021). https://doi.org/10.1038/s41589-020-00684-4