Part:BBa_K4805002

BmeTC-FL-BmeTC Y167A,D373C

description

BmeTC is a tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658). BmeTC_Y167A, D373C refers to a variant of BmeTC that contains two specific mutations: Y167A and D373C. Although BmeTC can only catalyze the bicyclic structure at the end of squalene, its variant BmeTC_D373C and BmeTC_Y167A, D373C possess the ability of catalyzing both monocyclic and bicyclic structure, which can produce ambrein. So far, the synergistic effect of BmeTC_Y167A, D373C and BmeTC in the cell-free system can produce the highest yield of ambrein with squalene as substrate. (Tsutomu S. et al, 2020) Thus we tried to construct a fusion protein with a flexible linker to tie BmeTC to BmeTC_Y167A, D373C, hoping that this strategy can improve the specifity of the cyclase. Our part can help and inspire other future teams to build and perfect the pathway of producing ambrein from squalene. It belongs to the terpenoids part collection we have established for the production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012.

Usage and Biology

BmeTC and BmeTC_Y167A,D373C are both parts that our team uses to enhance the production of ambrein in S.Cerevisiae. Here we used a flexible linker to link the two enzymes together, enabling a higher rate of cyclization. We assembled the plasmid concluding the promoter, coding sequence, and terminator.

sequencing and features

Assembly Compatibility:

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

  Illegal NheI site found at 676
  Illegal NheI site found at 2581
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 79
  Illegal BamHI site found at 1984
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

references

Yamabe, Y., Kawagoe, Y., Okuno, K., Inoue, M., Chikaoka, K., Ueda, D., Tajima, Y., Yamada, T. K., Kakihara, Y., Hara, T., & Sato, T. (2020). Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein. Scientific Reports, 10(1), 19643. https://doi.org/10.1038/s41598-020-76624-y