Revision as of 20:30, 13 October 2022

E. copro unknown MFS transporter

This novel MFS transporter was taken from the same operon as ismA in the genome of E. copro. ismA is involved in the metabolism of cholesterol, so we hypothesized that this MFS transporter is involved in cholesterol transport.

Introduction

The McGill iGEM team set out to develop a cholesterol lowering probiotic as a preventative for cardiovascular disease. Both endogenously synthesized cholesterol and dietary cholesterol end up in the gut, where they are absorbed and sent around the body. McGill iGEM’s project consists of developing a novel metabolic pathway to convert cholesterol, which is absorbed in the gut, into coprostanol, a molecule that cannot be absorbed and is thus excreted from the gut. The metabolic pathway consists of a three-step pathway with four metabolites: cholesterol, which is converted to cholestenone, then coprostanone and finally coprostanol. We repurposed existing enzymes to engineer a metabolic pathway to do this conversion, then packaged it in a probiotic bacterium. By converting intestinal cholesterol into coprostanol, this probiotic bacterium can prevent cholesterol absorption as a preventative for high cholesterol-induced cardiovascular disease.

Biology

To complete this project, whatever bacterial chassis we use must also be to uptake cholesterol. Given that most bacterial species do not have this capability, we need a way to create this functionality in the chassis that we want to use.

This MFS transporter was discovered in a bacterium known as Eubacterium coprostanoligenes, a species known to be able to uptake and catalyze the metabolism of cholesterol. The gene for the MFS transporter was found just downstream, and within the same operon, of a gene that metabolized cholesterol into cholestenone, the first step of the cholesterol to coprostanol conversion pathway. Thus, we hypothesize that this MFS transporter could potentially play a role in cholesterol transport.

Results

After cloning the unknown MFS transporter into Bacillus subtilis, we were ready to test if it is involved in cholesterol transport. We did so through a cholesterol uptake assay. We measured the cholesterol concentration in the media containing bacteria, allowed it to grow some time, then measured concentration again. The more the cholesterol concentration decreases, the more the bacteria uptakes cholesterol. We compared uptake in B. subtilis containing MFS transporter with wild-type B. subtilis and E. coli.

Cultures were grown of each of these in lecithin media (LB + 0.1% lecithin) then diluted to OD600=0.1. At the 0h time point, 100X (8mg/mL) cholesterol stock in ethanol was added (final concentration 80μg/mL). Using a cholesterol quantification kit, cholesterol concentration in the culture was determined through a fluorometric assay, with emission corresponding to cholesterol concentration. We took 10-plicate measurements of each culture to reduce standard deviation. Cultures were allowed to grow for 24 hours before OD600 and cholesterol concentration were measured again, then Δemission/Δ10^OD600 (since OD vs log(CFU) is linear) was determined, which corresponds directly to the relative rate at which the bacteria uptake cholesterol as they grow. Emission was normalized by subtracting the emission of a negative control containing bacteria but no cholesterol. All values were determined based on the average of all replicate measurements.

24-hour cholesterol uptake assay by B. subtilis and B. subtilis + MFS. Cholesterol concentration of cultures containing E. coli, B. subtilis, and B. subtilis with MFS transporter were measured using a fluorometric quantification kit at time points separated by 24 hours. OD600 was measured at the same time points. A bar graph was generated representing the Δemission⁵⁸⁷/Δ10^OD600 of E. coli, B. subtilis, and B. subtilis with pBS1C + Pveg + MFS.

The standard deviation was quite high for all samples, but clear trends could still be determined. The MFS transporter did not appear to make a significant difference in cholesterol uptake compared to B. subtilis, as can be seen by how the error bars for “B. subtilis” and “B. subtilis w/ Pveg + MFS” in the bar graph overlap. There could be many reasons for this: protein is not expressed in significant quantities, protein is expressed but it is not trafficking correctly to the membrane, or protein is not involved in cholesterol transport. However, B. subtilis did clearly uptake more cholesterol than E. coli. This evidence suggests that when in the presence of free cholesterol, B. subtilis will import cholesterol or integrate it into its membrane, allowing our proteins which are expressed in the interior to catalyze the conversion to coprostanol.

Sequence and Features