Heterologous expression and whole-cell activity assay of human CYP8B1 in Escherichia coli

Abstract:

Background Studies have shown that inhibiting human cytochrome P450 8B1 (CYP8B1) can regulate bile acid metabolism and further influence glucose and lipid homeostasis, representing a potential therapeutic strategy for metabolic diseases such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease. However, the development of CYP8B1 inhibitors remains slow, possibly due to the inability of existing small-molecule screening systems targeting CYP8B1 activity to achieve medium-to-high-throughput screening.Objective This study constructed an Escherichia coli system co-expressing human CYP8B1 and cytochrome P450 reductase (CPR) and identified the strains with high catalytic activity of 12α-hydroxylase, aiming to establish a medium-to-high-throughput screening system for CYP8B1 activity.Methods First, on the basis of bicistronic or dual-expression vector strategies, recombinant plasmids co-expressing CYP8B1 and CPR were constructed with the medium-to-high-copy plasmid pET-17b or the high-copy plasmid pRSFDuet-1 as vectors. Subsequently, pGro7/pGro12 (capable of expressing the molecular chaperone proteins groES-groEL) and the constructed co-expression plasmids were successively transformed into different host strains C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3) to construct different recombinant expression strains. After fermentation of the recombinant strains, 7α-hydroxy-4-cholesten-3-one (7α-HCO) was used as the substrate for CYP8B1, and reverse phase-high performance liquid chromatography (RP-HPLC) was employed to measure the whole-cell catalytic activity. Finally, the optimal reaction conditions and expression system were determined by comparing the activity assay results.Results We successfully constructed a heterologous expression system of human CYP8B1 in E. coli. With the medium-to-high-copy plasmid pET-17b and the high-copy plasmid pRSFDuet-1 as vectors, along with plasmids pGro7 and pGro12 for expressing the molecular chaperone proteins groES-groEL, 12 recombinant expression strains of CYP8B1 were constructed in five host strains: C41(DE3), C43(DE3), BL21-CodonPlus(DE3)-RIPL, Rosetta(DE3), and GSsetta(DE3). By comparing their whole-cell activities, we identified the recombinant strain C43-pRSF-tCYP8B1-tCPR as the optimal expression system for CYP8B1, with the optimal reaction conditions being no polymyxin B added to the reaction system and dimethyl sulfoxide (DMSO) as the solvent for the substrate 7α-HCO.Conclusion The engineered E. coli system enables effective heterologous expression of human CYP8B1 with robust whole-cell catalytic activity.