Team:Paris Bettencourt/Project/TMAU

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BACKGROUND

Trimethylamine (TMA) is a volatile compound smelling strongly of spoiled fish. It is produced by bacteria in the human gut and oxidized into a non-volatile compound in the liver by a flavin-containing monooxygenase 3 (FMO3). Trimethylaminuria (TMAU), or Fish Odor Syndrome, is a rare genetic disease caused by inactivating mutations in the FMO3 gene. Consequently, TMA accumulates in sweat, saliva, and urine, causing a strong fish odor. Patients suffer no other serious symptoms, except a difficult social condition.

AIMS

TMA is also processed by the trimethylamine monooxygenase (TMM) of Ruegeria pomeroyi, an enzyme similar to human FMO3. Expressing this enzyme in human skin bacteria should remove trimethylamine from sweat and reduce its unpleasant odor. TMM-expressing bacteria in a cream or spray could be a cheap and stable way to deliver the therapeutic enzyme to TMAU patients.

RESULTS

  • Cloned tmm into E.coli using pSB1C3 and pSEVA315 vectors, creating a new Biobrick: BBa_K1403015.
  • Characterized and quantified the activity of TMM by a colorimetric assay.
  • Confirmed the degradation of trimethylamine by gas chromatography - mass spectrometry (GC/MS).
Aims and Achievement Introduction Results Methods References

Results

After cloning tmm into a Biobrick vector (pSB1C3), the construct was successfully expressed in E. coli. TMM activity was found in TMM-expressing E. coli but not in empty vector-expressing E. coli. TMM does not only degrade trimethylamine into trimethylamine-N-oxide, but also converts indole into indigo. To measure the activity of TMM, the growth medium was supplemented with tryptophan, a precursor of indole, which is the substrate of TMM. After 14h of culture, cells were pelleted, washed twice with sterile water, resuspended in DMSO and sonicated. TMM activity was determined by measuring the absorbance spectrum of bacterial extractions. Peaks at 620 nm were found in TMM-expressing E.coli cultures supplemented with tryptophan, which was identified as indigo according to absorbance spectrum analysis. Gas chromatography-mass spectrometry (GC/MS) confirmed the activity of TMM by showing a significant decrease (p-value<0.05) of the concentration of TMA by TMM-expressing E.coli.

References

- ref1
- ref2

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
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75014 Paris, France
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