Part:BBa_K3993010

PTDH3-SPE1-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-SPE1-TCYC1..

The profiles of every basic part are as follows

BBa_K3993000

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

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 SPE1, Lane 2 is promoter PTDH3.

Proof of function

1. Metabolite production tests

Figure 4. 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.

2. Modeling for predicting the performance of our engineered bacteria to produce tropine

Firstly, we get the polynomial linear regression-2 shown in figure 5. (Data from the published articles, according to references3/4 )

Figure 5..

The R-squared reaches 0.9855, which can be used to predict the performance of our engineered bacteria to produce tropine. Substituting the time and the OD600 value we tested in the laboratory into the model to get figures 6.

Figure 6..

The results show that when the OD600 of our engineered bacteria reaches a certain value, the output of tropine will increase sharply, indicating that our engineered bacteria have great industrial application prospects.

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

  Illegal BglII site found at 1862
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]