Part:BBa_K3993011

PTDH3-speB-TCYC1

Usage and Biology

Kl tropane alkaloids (TAs) refers to a kind of alkaloids containing the tropane alkyl skeleton formed by the combination of pyrrole ring and piperidine ring in structure. It is a natural product of plant and has a long history and important medicinal value. tropane alkaloids have great market demand and often appear in global shortages. A method that can produce Tas in scale is expected. Using synthetic biology to create a microbial cell factory to produce TAs is a highly potential strategy.

The Tropane alkaloid (TAs) is obtained by a series of chemical reactions through the formation of Putrescine (1, 4-butylenediamine, Putrescine) from Arginine. Putrescine is an essential polyamine for ribosomal biogenesis and mRNA translation, but is regulated by polyamines and remains at low concentrations during normal cell growth. In this study, by overexpressing the natural genes involved in arginine metabolism and polyamine biosynthesis, the regulatory mechanism of polyamine biosynthesis is adjusted, so as to engineer the production of excessive putrescine strains.

Figure1. Principle diagram of TAs..

Construct design

The Tropine part of Tropane alkaloids (TAs) is obtained from arginine to putrescine (1,4-butanediamine, putrescine), and then through a series of chemical reactions. In this project, natural genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The engineer strains that produced excess putrescine. (Figure 2).

Figure 2. DNA sequence map of plasmid PTDH3-SpeB-TCYC1..

BBa_K3993001

Usage and Biology

This protein is involved in step 1 of the subpathway that synthesizes putrescine from agmatine. This subpathway is part of the pathway putrescine biosynthesis via agmatine pathway, which is itself part of Amine and polyamine biosynthesis. The expression of AUH activity is antagonistically regulated by cyclic AMP and agmatine. In the presence of the cAMP receptor protein, cAMP represses the expression of AUH, while agmatine induces it.

BBa_K3993003

Usage and Biology

Yeast CEN/ARS vector (Leu2) that contains multiple cloning site ( MCS ) and TDH3 promoter.

BBa_K3993004

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems, and can be used for modulating gene expression in yeast.

Experimental approach

1. Fragments PCR products Electrophoresis

Figure 3. Gel electrophoresis of amplified fragments..

Lane 1 is target gene SpeB, Lane 2 is promoter PTDH3.

References

4. Prashanth Srinivasan & Christina D. Smolke. Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11588-w

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 986
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 986
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1462
  Illegal BamHI site found at 1375
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 986
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 986
  Illegal AgeI site found at 1397
  
• 1000
  COMPATIBLE WITH RFC[1000]