Part:BBa_K5007001

CsPT4-NoTP

This part codes for Prenyltransferase 4, responsible for the prenylation of olivetolic acid with Geranyl-diphosphate, generating cannabigerolic acid. This part has had the the first 231 nucleotides removed, which code the 77 amino acids in the protein sequence, corresponding to the transport peptide responsible for translocating the protein to the chloroplast’s plastid’s membrane.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 810
  Illegal SapI site found at 384
  

Design Choices

Native CsPT4 is a chloroplastic membrane prenyltransferase. It contains a 77 residue long transit peptide, responsible for guiding it into the chloroplast’s plastid membrane. It is not biochemically characterized with regards to catalytic and binding sites, or its crystalline structure. In order to not cause any issues with the cell machinery promoting premature protein degradation, and to get a more valid structural model, the nucleotides coding for the transit peptide in this part were removed.

Structural Modeling

This part was modeled using PyMOL’s MODELLER extension, using the modified sequence as query and a predicted AlphaFold model as template. The query and template were aligned via MUSCLE and annotated as needed in PyMOL.

Figure 1: Structural modeling of BBa_K5007001

References

Luo X, Reiter MA, d'Espaux L, Wong J, Denby CM, Lechner A, Zhang Y, Grzybowski AT, Harth S, Lin W, Lee H, Yu C, Shin J, Deng K, Benites VT, Wang G, Baidoo EEK, Chen Y, Dev I, Petzold CJ, Keasling JD. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature. 2019 Mar;567(7746):123-126. doi: 10.1038/s41586-019-0978-9. Epub 2019 Feb 27. Erratum in: Nature. 2020 Apr;580(7802):E2. PMID: 30814733.