Flavin-containing monooxygenase (FMO) is the enzyme responsible for the conversion of indole to indoxyl using NADPH as its co-factor1. Under the presence of oxygen, indoxyl spontaneously dimerizes to indigo.
This enzyme is responsible for producing one of the intermediates of our biological dyeing process, indoxyl. Characterization of this enzyme is important because we need have information about its kinetics and conditions for activity in order to successfully implement this enzyme in our biological dyeing process to make soluble indigo (indican). After synthesizing this enzyme, we looked to verify functionality by checking indigo presence, checked for factors affecting indoxyl production through bio-indigo quantification, and calculated kinetic data for our enzyme.
In addition, we wanted to provide good characterization of this FMO part so that other iGEM teams in the future can have access to this reliable and well characterized biobrick and its data.
We first checked the functionality of FMO by checking for the presence of indigo. This is because our product, indoxyl, spontaneously dimerizes in the presence of oxygen to indigo. To do this, we prepared a standard of store bought indigo and compared its peak on the HPLC with that of bioindigo. The two graphs are shown below (bio-indigo production followed by indigo standard).
By running a standard of indirubin, a side product of indoxyl's oxidation, we were able to see what proportion of our final solution was indigo vs. indirubin by comparing the Area(%) under the curve. This data is shown below, and shows that we have a significantly larger amount of indigo being produced in comparison to indirubin, the predominant side product.
An important aspect in characterizing our FMO enzyme is to look into factors affecting indoxyl synthesis. We looked at these factors by quantifying and titrating the indigo production. To do this, we ran a series of experiments with varying levels of tryptophan, pH buffering, and differing salts.
After culturing E. coli cells in plain LB media with resistance for over 30 hours, we quantified indigo concentration to be approximately 2.06 mg/L. Once we increased the tryptophan concentration in the media, we got an appreciable increase in bio-indigo production. We also tried two different salts, KCl and NaCl due to previous research suggesting potassium and sodium ions have an effect on E. coli tryptophanase, one of the enzymes involved in converting tryptophan to indole in our pathway2. However, there was little to no difference in indigo production from the different salts. Additionally, after buffering the pH level with sodium phosphate at pH 7, we observed little to no difference in indigo production.
From this, we concluded that the main factor affecting indigo production was the level of tryptophan present in the media. This makes sense because this is the raw material for cells in order to produce indole, the substrate the FMO enzyme acts on. The maximum amount of indigo we were able to produce was approximately 224 mg/L in buffered conditions and 2.4 g/L tryptophan concentration in the media. The results of these titrations are summarized below.
Cells were cultured in 5 ml glass tubes in the appropriate media at 30o C for over 30 hours. YE stands for yeast extract. Buffering was done by the addition of sodium phosphate. Refreshment refers to the replacement of media after approximately 13 hours of growth.
All indigo quantifications were done through absorbance measurements at 620 nm with TECAN. In order to quantify our indigo production using this method, we created a standard indigo calibration curve with a stock solution of pure indigo in dimethyl sulfoxide (DMSO). The results of the calibration curve are below with a R2 value of 0.994.
Additionally, we wanted to measure how quickly FMO generated indoxyl from its substrate, indole. We used Michaelis-Menten kinetics to model the behavior of FMO with various concentrations of indole substrate to determine the Km and Vmax of the enzyme.
To generate kinetic data for FMO, we first needed to obtain purified enzyme. We used the T7 expression system and chemically competent BL21 cells to express a His6 tagged FMO and purify it using a nickel column. With this purified enzyme, we ran an assay with different concentrations of indole in a mixture containing 0.1 mM NADPH, 0.1 mM EDTA, and Bicine/KOH buffer (pH 8.5).
From this data, we generated a Michaelis-Menten kinetics graph, and found the Vmax and Km to be 1.166*10-5 mM/s and 0.8698 mM respectively. At the 95% confidence level, Vmax is in the range of [0.00000935 mM/s, 0.00001398 mM/s], while Km is in the range of [0.12 mM, 1.62 mM].
The kinetics assay was adopted from previous work with FMO on substrates such as trimethylamine3. To our knowledge, this is the first kinetic assay done with indole. All kinetic assays were carried out measuring absorbance at 620 nm with TECAN. A limitation in our kinetic assay is that we assumed that the oxidation step from indoxyl to indigo was nearly instantaneous, and subsequently measured only the indigo concentration. However, we believe that because of sufficient aeration and oxygen content during our assay, the error coming from this assumption would be very limited.
We were also interested in whether over-accumulation of indigo would be toxic to E. coli cells. This was of particular interest to us because large scale production of bio-indigo if toxicity occurred. To test the toxicity of indigo, we ran a time course experiment in which we measured the number of bacteria over a 24 hour period. With each time point over a 24 hour period, we counted cells with the use of a hemocytometer. Cells with the FMO plasmid and mutant FMO plasmid were cultured in 5 ml tubes with 0.74 g/L tryptophan at 30o C. The mutant FMO, which contains mutagenized catalytic sites of the FMO, served as a control for this experiment. From this experiment, we observed no noticeable toxicity with indigo.
2:Högberg-Raibaud, A., Raibaud, O., & Goldberg, M. E. (1975). Kinetic and equilibrium studies on the activation of Escherichia coli K12 tryptophanase by pyridoxal 5’-phosphate and monovalent cations. The Journal of Biological Chemistry, 250, 3352–3358.
3:Choi, H. S., Kim, J. K., Cho, E. H., Kim, Y. C., Kim, J. Il, & Kim, S. W. (2003). A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli. Biochemical and Biophysical Research Communications, 306, 930–936.