Team:Paris Bettencourt/Project/TMAU

From 2014.igem.org

Revision as of 02:32, 18 October 2014 by Roussetfrancois (Talk | contribs)

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).
Introduction Results Methods


Figure 1. Trimethylamine production and degradation in the human body.
(A) In humans, choline is converted into trimethylamine and acetaldehyde by the gut bacteria Desulfovibrio desulfuricans.
(B) Trimethylamine is degraded in the liver into trimethylamine-N-oxyde by FMO3 (flavin-containing mono oxygenase 3).
(C) Mutations in the FMO3 gene can cause the loss of the function of the enzyme. In these patients, trimethylamine is excreted into sweat, urine and breath, causing a strong fish odor.

Introduction

Trimethylamine (TMA) is a volatile compound smelling strongly of spoiled fish. It is produced during human digestion when choline, a B-complex vitamin, is fermented by gut bacteria (Craciun, S. et al., 2012) (Fig. 1A). Normally it proceeds to the liver where it is oxidized by FMO3, a flavin-containing monooxygenase (Fig. 1B). The product of FMO3 is trimethylamine oxide (TMAO), which is odorless.
Trimethylaminuria (TMAU), or fish odor syndrome, is a rare genetic disorder in which patients are not able to fully oxidize TMA. TMAO is an autosomal recessive disorder, requiring two nonfunctional alleles of the FMO3 gene. More than 30 known mutations can inactivate FMO3, and inactive alleles have an estimated 0.1-1% global frequency (Mitchell, S.C et al., 2001). TMAU currently has no cure.
We propose that TMAU could be treated with a genetically modified skin bacteria. The trimethylamine monooxygenase (Tmm) of Ruegeria pomeroyi also oxdizes TMA to TMAO (Chen, Y. et al., 2011). Applying this enzyme to the skin should eliminate TMA in sweat and, therefore, unpleasant odor. Expression in a bacterium of the skin microbiome could be a cheap and stable way to deliver the therapeutic enzyme. Here we present the expression and characterization of Tmm in E. coli and Corynebacterium striatum, a skin-native bacterium.

Figure 2. The human nose can detect fish odor from a solution with a concentration of 1mM.
10 participants were asked to smell trimethylamine samples at different concentrations and asked whether they could detect any difference with a control (water). An odor threshold was found at 1 mM and trimethylamine at concentrations of 0.1 mM or below was not detected.







Figure 3. Characterisation of TMM using indigo production.
(A) TMM allows the degradation of trimethylamine into trimethylamine-N-oxide.
(B) TMM activity can be detected by the presence of indigo, a blue dye. Indole is naturally produced from tryptophan by tryptophanase in E. coli and can be converted into indigo by Tmm.
(C) Indigo can be detected in TMM-expressing E.coli (left) but not in the control (right) after 14h of culture in LB medium supplemented with 2g/l of tryptophan.


Figure 4. Average absorbances of DMSO extractions from bacterial lysates shows Tmm activity.
This graph shows the absorbance spectra of DMSO extractions from TMM-expressing E. coli (+TMM) or control (-TMM) in LB medium (-trp) or in LB supplemented with tryptophan (+trp). The grey area shows the expected absorbance peak of indigo.


Figure 5. GC/MS analysis of cultures of E. coli expressing TMM or an empty vector (control).
(A) GC/MS spectra of triplicates of cultures of E. coli expressing TMM or an empty vector (control). The peaks were analysed and compared to a database, showing the confidence rate for TMA.
RT = Retention time
MA = Measured Area
(B) Average area of the peaks corresponding to TMA in TMM-expressing E.coli and control. p-value=0.0199


Figure 6. Diffusion model of trimethylamine through air.
This animation shows the concentration of trimethylamine diffusing through air from a patient over a time period of 0 to 1000 hours. From the graph on the left of the animation, you can see that the odor threshold is reached very early in the model, given that the concentration of trimethylamine at the skin surface is kept at a constant concentration of 500-600 ppm.

Results

Odor panel of trimethylamine
We first wanted to determine which concentration of trimethylamine we had to use for our experiments. Ten volonteers were asked to smell trimethylamine samples at different concentrations and to say whether they could detect any difference with a control (water). An odor threshold was found at 1 mM and trimethylamine at concentrations of 0.1 mM or below was not detected (Fig. 2).

Cloning
We synthesized Tmm from R. pomeroyi and expressed it in E. coli. The construct (BBa_K1403015) included a constitutive promoter and was cloned in the standard high-copy BioBrick vector pSB1C3. Following 24 hours of incubation at 37 C, a dark pigment was visible in the transformed colonies.

Characterisation of TMM activity in E.coli
The Tmm enzyme is not specific to TMA as a substrate. It is also known to oxidize indole to indoxyl, which dimerizes into the well known blue pigment indigo (Fig 3B). Indole is a natural product of tryptophan metabolism in E. coli.
We took advangage of this indole production activity to characterize the Tmm enzyme. E. coli that were cultured in LB supplemented with tryptophan (2 g/L) produced a deep blue pigment (Fig. 3C) with absorbance properties matching indigo (Fig. 4). Without Tmm expression or without tryptophan, indigo production was minimal or absent.

Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC/MS) confirmed the activity of TMM by proving the degradation of trimethylamine(Fig. 4). It was performed on extractions from cultures of TMM-expressing E. coli (TMM) and control expressing an empty vector in a LB medium supplemented with trimethylamine (1 mM). The results show a significant decrease (p-value = 0,0199) of the concentration of TMA in TMM-expressing E.coli (Fig. 4B). This confirms the efficiency of degradation of the fish odor by TMM.

Modeling of TMA diffusion
A simple model was created for the diffusion of trimethylamine into the air using COMSOL Multiphysics (a physics-based interface to solve partial differential equations). The model is shown in Fig.6. From literature, it was found that the odor threshold of trimethylamine in air is approximately 3.2E-5 ppm air ( Nagata, 1993), using the triangle odor bag method. This odor threshold was reached approximately 10 hours in the model, at a distance of 5 cm from the skin surface.



Methods

Cloning
The Tmm gene was produced by gene synthesis (IDT). The final construct was codon-optimized for E. coli expression and included a strong constitutive promoter and ribosome binding site.
For E. coli expression, we cloned the Tmm construct into the standard high-copy BioBrick vector pSB1C3. For expression in C. striatum we used the vector pSEVA351, a universal vector with a high-copy replication origin (Standard European Vector Architecture).

Indigo absorbance
TMM does not only degrade trimethylamine into trimethylamine-N-oxide, but also converts indole into indigo. Measurement of TMM activity was performed on TMM-expressing E.coli using the isolation of indigo protocol inspired of Choi H.S., Kim J.K et al. (2003). The control is E. coli expressing pSB1C3.

Gas chromatography / Mass spectrometry
For the GC-MS analysis, 1µl of the sample was injected into the instrument in split mode. A Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df were used. The GC-MS run uses the following standardized parameters:
- Injection temperature: 300°C
- Interface temperature: 300°C
- Ion Source: 250°C
- Carrier Gas: Helium (flow rate of 1 ml min-1)

The temperature program used was 3 min of isothermal heating at 50⁰C followed by heating at 350⁰C for 10 min. Mass spectra were recorded at 2 scan sec-1 with a scanning range of 40 to 850 m/z.
X Caliber software is used to process and analyze the data from a GC-MS run. Quantify each component based on peak areas and normalization based on the internal standard.


Modeling
The model is based on a 2D axisymmetric geometry, with the air modeled as the space 5 cm around the skin surface. Here, the skin surface is kept at a constant concentration, C0 = 1.39 +/- 0.483 uM. This value refers to the approximate amount of trimethylamine in sweat (Bain, 2005). The diffusivity of trimethylamine through the air was calculated using the Chapman-Enskog theory of gas diffusivity given by the following equation:

D = (1.858E-3 * T(3/2) * (1/M1 + 1/M2)(1/2)) / (p * s122 * w)

where D is the diffusivity of the trimethylamine
T is the room temperature, 298 K
p is the air pressure, 1 atm
M1 is the mass of the trimethylamine, 59.11 g/mol
M2 is the mass of the air, 29 g/mol
s12 is the average collision diameter, which is around 340 Angstroms for gas molecules in air, and w is the dimensionless temperature-dependent collision integral, usually on the order of 1.

Thus, the diffusion coefficient of trimethylamine was tabulated to be 1.9*10-9 m2/s.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
+33 1 44 41 25 22/25
paris-bettencourt-igem@googlegroups.com
Copyright (c) 2014 igem.org. All rights reserved.