Part:BBa_K3875011

Usage and Biology

Design: Contained genes: trpE (BBa_K3875009), trpG ( BBa_K3875003), AntrpC ( BBa_K3875001), trpB ( BBa_K187029), p4h ( BBa_K3875004), pcd ( BBa_K3875005)
In E. coli, chorismate is a branching point for the synthesis of aromatic amino acids, and we intend to insert four related genes that facilitate the four-step transformation of this substance into 5-HTP (as Figure 1).

e study of Yuheng Lin et al. shows that the presence of pterin in E. coli is in the form of MH4, which is the coenzyme of P4H [1]. Using PCD and P4H with E. coli endogenous DHMR, an artificial pterin cycle (MH4 cycle) can be formed to ensure the final step of 5-HTP synthesis.
Based on our vision of integrating five genes into one plasmid, we decide to construct two plasmids first (composite part: BBa_K3875006, BBa_K3875010), and then use PCR to obtain two expression frames lac-trpEG-AntrpC-T1 and lac-trpB-p4h-pcd-T1 respectively. Finally, the two frames are integrated into the pSA74 plasmid by homologous recombinase (as Figure 2.).

Method: lac-trpB-pcd-p4h-T1 was obtained by PCR amplification. pSA74- lac-trpE-trpG-AntrpC-T1 and a had the same cohesive end by restriction endonuclease, and then they were connected by T4 ligase. Finally, the linked plasmid was transformed into E. coli DH5a (failed).
In order to detect the expression of recombinant genes, two plasmids containing lac-trpB-pcd-p4h-T1 and lac-trpE-trpG-AntrpC were transformed into E. coli DH5a for fermentation.
Result: It is possible that the synthesis path of 5-HTP is too long, so the yield of 5-HTP is not high.
The peak of 5-HTP production of E. coli Nissle 1917 appeared at 24h of fermentation, and the yield was about 2mg/L. Technically, blocking chorismate's entry into the branching pathway for synthesis of other substances could further increase the yield of 5-HTP. It’s also a question we needs to solve in the future.

Reference: [1] Lin, Y. , Sun, X. , Yuan, Q. , & Yan, Y. . (2014). Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan. Acs Synthetic Biology, 3(7), 497.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 616
  Illegal BglII site found at 677
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4168
  Illegal AgeI site found at 5106
  
• 1000
  COMPATIBLE WITH RFC[1000]