Part:BBa_K4788007

IsmA-BSH-BCoAT

Transforming cholesterol into 4-cholesten-3-one while producing bile salt lyase and short chain fatty acids.

Usage and Biology

This is a complex complex coding part that can directly degrade cholesterol while producing short chain fatty acids and bile salt lyases. Short chain fatty acids and bile salt lyases play a crucial role in the indirect regulation of cholesterol. This composite gene can achieve multifaceted and multifunctional cholesterol regulation functions.

Characterization

In order to characterize the corresponding components, we need to develop different characterization schemes for different genes. For the BCoAT gene, We use gas chromatography to examine the production of short chain fatty acids in strains containing the BCoAT gene to characterize the gene, and for the IsmA gene, we used the OPA method to detect cholesterol residue after cholesterol culture medium cultivation to characterize it. And for the BSH gene, we used MRS bile salt qualitative plate inoculation experiments to characterize it. As shown in Figure 1, the triad gene strain showed good performance in IsmA gene characterization, with high significance compared to the control group.

Figure1: 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

As shown in Figure 2, the triad gene strain also showed good performance in characterizing the BSH gene, with a very clear precipitation circle.

Figure2:Qualitative Plate Control Experiment Results of Tandem Body Strain BSH under Aerobic Conditions

At the same time, in order to verify the performance of BSH in the triad in anaerobic environments, we also conducted experiments to culture strains containing BSH genes in anaerobic environments. As shown in Figure 3, the characterization of BSH in the triad under anaerobic conditions is still excellent.

Figure3:Qualitative Plate Control Experiment Results of Tandem Body Strain BSH under Anaerobic Conditions

However, for the characterization of BCoAT, the performance of the triad as shown in Figure 4 is not ideal.

Figure1: GC detection of short chain fatty acid production by BCoAT containing strains

Reference

Jia B, Zou Y, Han X, Bae JW, Jeon CO. Gut microbiome-mediated mechanisms for reducing cholesterol levels: implications for ameliorating cardiovascular disease. Trends Microbiol. 2023 Jan;31(1):76-91. doi: 10.1016/j.tim.2022.08.003. Epub 2022 Aug 22. PMID: 36008191. Kenny DJ, Plichta DR, Shungin D, Koppel N, Hall AB, Fu B, Vasan RS, Shaw SY, Vlamakis H, Balskus EP, Xavier RJ. Cholesterol Metabolism by Uncultured Human Gut Bacteria Influences Host Cholesterol Level. Cell Host Microbe. 2020 Aug 12;28(2):245-257.e6. doi: 10.1016/j.chom.2020.05.013. Epub 2020 Jun 15. PMID: 32544460; PMCID: PMC7435688.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1772
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2841
  Illegal AgeI site found at 58
  
• 1000
  COMPATIBLE WITH RFC[1000]