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.

Thinker-Shanghai-2023

Description

In view of the fact that the cholesterol-degrading protein will be expressed within the E. coli (Escherichia coli) strains, an attainable approach that may transfer cholesterol from guts into the probiotic strains needs to be sought, by implementing which cholesterol can be converted into coprostanol, thereby opening up a pathway that can efficaciously eliminate LDL-C (Low-density Lipoprotein Cholesterol) inside human bodies. We are currently faced with two options: utilizing the non-specific passages located on cell membranes to transfer cholesterol or using certain CETP (Cholesteryl Ester Transfer Protein). It is noticeable that the former approach is not likely to function as the passages can disrupt the homeostasis of E. coli. Therefore, seeking proper transfer proteins becomes a reliable solution.

As cholesterol transport protein allows cholesterol to enter probiotics from the external environment, so that probiotics can degrade cholesterol in food and achieve the purpose of producing low-cholesterol food, we decided to use the cholesterol transport protein.

Usage and Biology

After determining that certain bacteria like E. coprostanoligenes (AT51222, which is the only publicly available bacterial strain that is able to produce fecosterol) have evolved some CETP, we used a specific kind of CETP--the MFS CETP, which was already structured and characterized by McGill iGEM 2022【1】. Using the T7 promoter derived from the T7 bacteriophage to initiate the transcription of T7 bacteriophage, we introduced our constructed plasmids (E. coli Rosetta/E. coli Rosetta with pT7-MFS, which is the used CETP.)

Characterization

We use cholesterol transport protein（MFS） and transform the plasmids into E.coli Rosetta.

We measured the OD600 values (the optical density of the obtained solution at the optical wavelength of 600nm) and the actual concentrations of cholesterol before and after the trial. As mentioned earlier, plasmids were cultured in LB medium in each trial and the cholesterol concentration was diluted to OD600 = 0.1. Initially, ethanol cholesterol solution was added into LB medium at a concentration of 8 mg/mL to let the cholesterol concentration reach 80 μg/mL. The cholesterol concentration in each group's medium was detected with a quantitative cholesterol kit (mlbio, ml094955). OD600 and cholesterol concentrations were detected again after 12 h of culture. For reasons of standardizing the data, we calculated Δcholesterol/ΔOD600, which represents cholesterol uptake by bacteria during their growth. All data aforementioned were determined based on the mean of all repeated measurements.

Figure 1: Determination of MFS.

While maintaining identical experimental conditions, we establish trials to measure cholesterol concentrations in untreated plasmid E. coli Rosetta and plasmid E. coli Rosetta treated with protein pT7-MFS after twelve hours of cultivation conducted in LB medium. As illustrated by Fig. B and Fig. C, with identical initial cholesterol concentrations of 80 µg/mL, the untreated plasmid displayed an ultimate cholesterol concentration of roughly 72.16 µg/mL, while the cholesterol concentration plasmid treated with protein pT7-MFS dropped to approximately 55.83 µg/mL. Comparatively, the cholesterol concentrations reduced were approximately 7.84 µg/mL and 24.17 µg/mL, respectively for untreated and protein-treated plasmid samples, as shown in Fig. E. In this case, precise calculations reveal that the concentration of cholesterol is reduced by roughly 9.8% in untreated plasmid E. coli Rosetta, while in the protein-treated plasmid E. coli Rosetta, the concentration is reduced by approximately 30.21%.

Based on the experiments and outcomes aforementioned, we conclude that MFS can serve as an indispensable element in the construction of an effectual pathway to control cholesterol levels in human bodies by transporting cholesterol from human intestines into E. coli to help facilitate the degrading process of cholesterol. In this case, MFS can be used to promote the production of low-cholesterol foods, which aim to efficaciously control cholesterol levels in human bodies to mitigate risks of cardiovascular diseases. Therefore, MFS can provide a robust basis for novel insights into clinical treatments for high cholesterol and its causal diseases.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1063
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 847
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 233
1000
COMPATIBLE WITH RFC[1000]