Coding

Part:BBa_K3993000

Designed by: Nixue Song   Group: iGEM21_SHSID   (2021-10-11)


SPE1

Profile

Name: SPE1

Base Pairs: 1401bp

Origin: Saccharomyces cerevisiae, genome

Properties: Catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine

Usage and Biology

his protein is involved in step 1 of the subpathway that synthesizes putrescine from L-ornithine. Catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine, which is the precursor for the polyamines, spermidine and spermine. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Homodimer and only the dimer is catalytically active, as the active sites are constructed of residues from both monomers

Figure1. Principle diagram of TAs..

Experimental approach

1. Fragments PCR products Electrophoresis

Figure 2. Gel electrophoresis of amplified fragments..

Lane 1 is target gene SPE1, Lane 2 is promoter PTDH3.

Proof of function

1. Metabolite production tests

Figure 3. The peak of putrescine detected by LC-MS..

Plasmids pYES2-AsADC-SPE1-SpeB were transferred to BY4741 chemically competent yeast cells and screened on YPD/Hyg plates. Transformants were picked into YPD/Hyg medium for activation. Metabolite production tests were carried out in YNB-SC fermentation medium (containing 10 times arginine raw material). 48h metabolites (supernatant) were collected and the putrescine analyze metabolites by LC-MS. The results showed that at 32 min, the peak pattern of putrescine appeared (as shown in the figure 3), indicating that the engineered strain we constructed successfully produced putrescine.

References

1. Srinivasan, P., Smolke, C.D. Biosynthesis of medicinal tropane alkaloids in yeast. Nature 585, 614–619 (2020).

2. Srinivasan, P., Smolke, C.D. Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids. Nat Commun 10, 3634 (2019).

3. Prashanth Srinivasan & Christina D. Smolke. Biosynthesis of medicinal tropane alkaloids in yeast.Nature | Vol 585 | 24 September 2020 | 614-619

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
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1189
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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