Part:BBa_K4348001

ismA_his

An enzyme that converts cholesterol to 4-cholesten-3-one. It was discovered in Eubacterium coprostanoligenes by a team at the Broad Institute of MIT in 2021. This conversion is the first of the three-step pathway of converting cholesterol to coprostanol, a sterol that does not get absorbed by the gut. A 6x his-tag is attached to the N-terminal end, allowing for easy purification and analysis through nickel/cobalt columns and western blotting.

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

ismA is a 3β hydroxysteroid dehydrogenase that is known to convert cholesterol to cholestenone. It was discovered in the bacterium E. coprostanoligenes by Kenny et al, 2020 Cell Host and Microbe, which is able to convert cholesterol to coprostanol, although the other two enzymes of the pathway are not known.

Results

In order to solubilize ismA, a SUMO tag was attached on the N-terminus using Gibson assembly. The primers used were psmt_ifit_F, psmt_ifit_R, pPROEX_ismA_F and pPROEX_ismA_R. The ligations were verified via NotI and NcoI double digestion. A successful ligation was transformed into BL21, then incubated with 0.5mM IPTG at 16˚C for 24 hours to favor slower production of proteins to prevent misfolding. The proteins were extracted with B-per reagent and the soluble fraction was purified. A western blot with anti-his antibodies was performed, showing successful purification of ismA.

Figure 1. Anti-his Western Blot of SUMO-ismA and ismA (no SUMO) protein purification. ismA-SUMO was detected in the elution of the ismA-SUMO sample group, indicating that it was successfully purified out from the inclusion bodies. However, the only detectable ismA in the ismA (no SUMO) sample was found in the cell lysate, indicating that all the ismA was stuck completely in the inclusion bodies.

We tested our purified, uncleaved SUMO ismA by combining it in our reaction buffer with NADP+ (ismA’s cofactor) and cholesterol, ismA’s substrate. We looked for the formation of cholestenone on the GCMS, its product.

Figure 2. GC-MS chromatogram of two in vitro assays containing SUMO ismA mixed with cholesterol, both incubated for 16h. A GC-MS chromatogram showing the 0.01% cholestenone standard, along with the product of an ismA in vitro reaction containing ismA, 100µM cholesterol, 500µM NADP+, 100mM potassium phosphate buffer (pH 6.5), 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37°C.

For both SUMO-ismA in vitro reactions there is a small cholestenone product that can be observed under the peak of the standard. This indicates that ismA does in fact work in vitro.

Contribution

Group：iGEM23_LZU-CHINA

This year we used the IsmA gene to assemble a new composite cholesterol treatment part. After characterization testing, as shown in Figure 3, we found that the addition of BCoAT gene and BSH gene seems to improve the cholesterol degradation ability.

Figure3: a) Cholesterol residue in the culture medium after cultivation of engineering bacteria on basic cholesterol medium b) Percentage of cholesterol degradation in the culture medium after cultivation of engineering bacteria on basic cholesterol medium

Sequence and Features

Assembly Compatibility:

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• 12
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• 21
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• 23
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• 25
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  Illegal AgeI site found at 58
  
• 1000
  COMPATIBLE WITH RFC[1000]