Team:Paris Bettencourt/Project/TMAU

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 produced in the intestine by Desulfovibrio desulfuricans by fermentation of choline (Craciun, S. et al., 2012) (Fig. 1A). In healthy patients, the fmo3 gene allows the degradation of TMA in the liver into a non-volatile compound, trimethylamine-N-oxide (Fig.1B). But a mutation in the FMO3 sequence is most of the time the cause of trimethylaminuria: TMA is not degraded and is then excreted in sweat, saliva and urine leading to a strong fish odor (Fig. 1C). This autosomal recessive disorder requires two nonfunctional alleles of the FMO3 gene. More than 40 known mutations can inactivate FMO3, and inactive alleles have an estimated 0.1-1% global frequency (Mitchell, S.C et al., 2001). The patients are otherwise healthy but the disease affect their social relationships and can lead to depression. There is currently no cure for this metabolic disorder. Some treatments, often focused on reducing the choline absorption from food, tend to lower the symptoms (RJ Mackay et al., 2011).

Figure 2. 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 3. Average absorbances of DMSO extractions from bacterial lysates.
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 4. 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
RT = Retention time
MA = Measured Area

Results

Cloning
tmm was cloned into a Biobrick vector (pSB1C3) using XbaI and PstI restriction sites. We also cloned it into pSEVA315, an universal vector with a high-copy replication origin (Standard European Vector Architecture) using XbaI and HindIII restriction sites in order to express it into C. striatum. Both of the constructs were expressed in E. coli.

Characterisation of TMM activity in E.coli
The characterisation was performed on E.coli expressing tmm cloned into pSB1C3. 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 (Fig. 2A), but also converts indole into indigo (Fig. 2B). 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 (Fig. 2C). TMM activity was determined by measuring the absorbance spectra of the bacterial extractions. Peaks at 620 nm were found in TMM-expressing E.coli cultures supplemented with tryptophan, which was identified as indigo. Without tmm expression or without tryptophan, indigo production was minimal or absent (Fig. 3).

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. The results show a significant decrease (p-value<0.05) of the concentration of TMA in TMM-expressing E.coli (Fig. 4B).

Methods

Cloning
The tmm gene was produced by gene synthesis (IDT). The final construct was codon-optimized for E. coli expresion 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 using XbaI and PstI restriction sites. For expression in C. striatum we used the vector pSEVA351, an universal vector with a high-copy replication origin (Standard European Vector Architecture)using XbaI and HindIII restriction sites.

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