Part:BBa_K1131000

Flavin-containing monooxygenase (FMO); M. aminisulfidivorans

This Flavin-containing monooxygenase (FMO) from M. aminisulfidivorans can be expressed in many strains of E. Coli to produce indigo dye. In the presence of indole and oxygen, FMO can catalyze the addition of a hydroxyl group to indole generating the intermediate indoxyl. Indoxyl then naturally oxidizes to generate indigo which, due to its hydrophobicity, crashes out of solution. The part submitted is the ORF of FMO only.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1369
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

SZU-China 2020 iGEM TEAM

1.Species Source
In order to better characterize the bacterial flavin-containing monooxygenase (bFMO) documented by BBa_1131000, we found many literature about FMO that are also from M. aminisulfidivorans. The species’current name is Methylophaga aminisulfidivorans MP, and in some early literature it may be named as Methylophaga aminisulfidivorans SK1. Its taxonomy ID in NCBI is 1026882.

2.Experimental Characterization
2.1 Bio-indingo Absorption Spectrum
We used DMSO for resolubilization, and bio-indigo had an obvious absorption peak at 617nm, which was the same as that recorded in the literature. And the pigment is clearly blue in daylight.

2.2 Indigo Standard Curve
Prepare indigo standard samples with sample concentrations of 0.8mg/ml, 0.72mg/ml, 0.64mg/ml, 0.48mg/ml, 0.4mg/ml 0.32mg/ml, 0.24mg/ml, 0.16mg/ml, 0.08mg /ml, 0mg/ml, measure the absorbance at 620 nm with an ultraviolet-visible spectrophotometer, and draw a standard curve.

2.3 Determination of Bio-indigo
ProductionThe cells were harvested by centrifugation at 110,000 rpm for 5 mins, after which the supernatant was removed and the precipitation was rinsed by distilled water twice.
Then, the cell pellet was resuspended by dimethyl sulfoxide (DMSO), and tested by ultraviolet-visible spectrophotometer UV-2250 for optical density at 620 nm.
Compare the data obtained in the experiment with the standard curve of the indigo sample to determine the indigo production.

In addition, we got the unexpected orange-red dye in the fermentation product. After we contacted with SHANGHAI_SFLS_SPBS, we found that this phenomenon also happened in their experiments. After consulting the literature, we found that this may be due to the production of isatin, which is obtained by oxidation of indigo. It is preliminarily speculated that this is due to the overexpression of FMO, which caused the indigo to be further oxidized into isatin.

3.Documentary Characterization
3.1 Background
FMO
Flavin-containing monoocygenases (FMOs) are first discovered in rat liver microsomes, and are usually found in eukaryotes. The amount of enzyme present in different tissues varies with species and sex, but the highest concentration is usually found in the liver. FMOs are involved in a wide range of oxidative biological processes, including drug detoxification, xenobiotic metabolism and bio-catalytic synthesis by catalyzation of the oxygenation of many nitrogen-, sulfur-, phosphorous-, selenium-, and other nucleophilic heteroatom.
In the literature in 2003, Choi, H.S.et alcloned the first discovered bacterial FMO from a methylotrophic bacterium they isolated from the seawater at Mokpo, Korea [1]. And they named the new species Methylophaga aminisulfidivorans SK1. It grows on methanol, methylated amines, and dimethylsulfide but not on methane. Fructose and glucose are the only multicarbon compounds tested that can be used as growth substrates.
They isolated and cloned a gene of Methylophaga aminisulfidivorans SK1whose presence in E.coli produced blue dye indigo. The deduced amino acid sequence from the gene showed approximately 30% identities with FMOs of human (FMO1-FMO5). Its biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, Hack Sun Choi et al assigned the enzyme to be a bacterial FMO. And the nucleotide sequence of the bacterial fmo gene has been deposited in the GenBank database under Accession No. AF494423.
We used Clustal X to compare the nucleotide and amino acid sequence of FMOfrom the reference with that from BBa_1131000. We found that they have the same amino acid sequence, except for the redundant two amino acids, GS, on the C terminus of bFMO from BBa_1131000. But they have different DNA sequence with 77% identities.