Part:BBa_K1403015
Trimethylamine moonoxygenase (tmm) expression cassette This Biobrick encodes trimethylamine mono-oxygenase (tmm) which transforms trimethylamine into trimethylamin-N-oxide. This enzyme is originally expressed by Ruegeria pomeroyi and is similar to human FMO3.
Trimethylamine + NADPH + H+ + O2 = Trimethylamine-N-oxide + NADP+ + H2O
- Constitutive promoter BBa_J23108
- Synthetic RBS
- Histidine tag
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 438
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 262
Illegal AgeI site found at 485 - 1000COMPATIBLE WITH RFC[1000]
Characterisation
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. 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 with absorbance properties matching indigo (Fig. 1). Without TMM expression or without tryptophan, indigo production was minimal or absent.
GC/MS confirmed the activity of TMM by proving the degradation of trimethylamine (Fig. 2). 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. 5D). This confirms the efficiency of degradation of the fish odor by TMM.
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