Part:BBa_K4011003

Fre-SttH

Fre-SttH is a fusion protein used to halogenate the 6th carbon of tryptophan (Trp) in the dye production of 6, 6’dibromoindigo (tyrian purple) and 6, 6’dichloroindigo (tyrian red), with the help of another fusion protein: TnaA-FMO. Fre-SttH is composed of two separate domains: Fre and SttH, fused together with a rigid linker. This is a part in a part collection where we enable the production of indigo, tyrian purple, and related dyes from tryptophan in E. coli.

The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. BBa_K4011003 and BBa_K4011012 . Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. BBa_K4011004 BBa_K4011005 BBa_K4011013 BBa_K4011014 BBa_K4011015 and BBa_K4011019 .

Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in E. coli, adding to the collection.

Usage and Biology

Fre-SttH is a fusion protein used to halogenate the 6th carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains: Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp and halogen ions into 6-X-trp, and is highly insoluble in E.coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E.coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added. We express Fre-SttH in ptac system and measured its enzymatic activity using HPLC (Lee et al, 2021).

Characterization

SDS-PAGE

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 576
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 948
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 424