Part:BBa_K4348000

AKR1D1_his

A human 5-beta reductase used in bile acid synthesis. Used in the 2022 McGill project to catalyze the second step of the cholesterol -> coprostanol pathway, which is 4-cholesten-3-one to coprostanone. Coprostanol cannot be absorbed by the gut, which is a unique property.

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

AKR1D1 falls under the 5 beta reductase class of enzymes, and is expressed in the livers of humans. It is responsible for reducing the double bond between the fourth and fifth carbons of the sterol backbone, mimicking the second step (conversion of cholestenone to coprostanone) of the cholesterol to coprostanol pathway. In humans, AKR1D1 is able to recognize a wide variety of sterol substrates such as cortisone, aldosterone, and in particular 7α-hydroxycholestenone, and process them into their double bond reduced products. Since the only difference between 7α-hydroxycholestenone (one of AKR1D1’s endogenous substrates) and cholestenone (the substrate we want it to accept) is a hydroxyl group on the 7th carbon, we hypothesized that AKR1D1 could process our substrate cholestenone as well.

Results

AKR1D1 was optimized for IPTG induction concentration, temperature and incubation time for best protein expression yield. 30mL cultures of AKR1D1 were induced and grown at 37˚C for 4 hours, 30˚C for 6 hours, 24˚C for 12 hours and 16˚C for 24 hours with ampicillin and 4mM IPTG. The conditions that yielded the darkest bands were then used for protein purification using his-tag cobalt resin beads.

The flowthrough and dialysis samples AKR1D1 were run on SDS-PAGE. The gel was subsequently stained with coomassie blue stain.

Figure 1. 12% SDS-Page gel of AKR1D1 IPTG induction optimization. SDS-Page was performed on AKR1D1 at various IPTG incubation times and temperatures. The expected length is 37kDa.

We began by assembling our reactions following our tested reaction mixture to confirm the activity of our proteins. We started off by testing our proteins individually to see if they would be able to perform the hypothesized substrate to product conversion. To achieve this we incubated AKR1D1 with cholestenone and then performed an ethyl acetate extraction, derivatization, and resuspension before measuring enzyme activity on GC-MS.

Figure 2. GC-MS chromatogram for the optimized derivatization of AKR1D1. The GC-MS chromatogram shows the 0.01% coprostanone standard, which is the hypothesized product of AKR1D1’s conversion from cholestenone, along with the product of a reaction containing AKR1D1, 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.0), 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37 ̊C.

We then ran a pop assay on AKR1D1, which is a protocol we developed to test proteins without protein purification, which builds off of a pre-existing protocol for protein expression. Liquid cultures of AKR1D1 protein-expressing E. coli are pelleted, washed with PBS, frozen at -80°C, resuspended in 2mL PBS. These samples are sonicated on ice, 3 times for 30 second bursts at an amplitude of 20kHz, then spun at 12000 rpm and 4°C for 20 minutes to release proteins into the supernatant. The protein-containing supernatant (potassium phosphate buffer) is extracted and has other reaction components added to it before being run on the GCMS.

This allowed us to test AKR1D1 and confirm whether it still maintains activity following our incubation of extract with the appropriate sterol, and cofactor, which not having to protein purify the enzyme.

Figure 3. GC-MS chromatogram of three AKR1D1 pop assays in E. coli mixed with cholestenone substrate. The GC-MS chromatogram shows the 0.01% coprostanone standard along with the product of a three AKR1D1 reactions containing, 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.0), 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37°C. AKR1D1 was added to the reaction using the pop assay protocol. The AKR1D1 reaction chromatogram shows a peak directly underneath the coprostanone standard, indicating product formation using the pop assay protocol.

Instead of incubating our reactions for 16h, we incubated the reactions for 1h to see if they would still have catalytic activity that would be detectable on the GC-MS.

This allowed us to test AKR1D1 and confirm whether it still maintains activity following our reduced incubation time.

Figure 4. GC-MS chromatogram of two in vitro assays containing AKR1D1 mixed with cholestenone incubated for 1 hour. A GC-MS chromatogram showing the 0.01% coprostanone standard, along with the product of two AKR1D1 in vitro reaction containing AKR1D1, 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.0), 0.2% Triton X-100, and 5% ethanol, incubated for 1 hour at 37°C, a significantly shorter incubation time than the previously-run 16 hours. The asterisk denotes that the concentration of protein, and substrate was doubled for this particular reaction. On both AKR1D1 in vitro RIT reaction product chromatograms, there is a peak directly underneath the coprostanone standard.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]