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. 2023 Nov 25;8(4):741-748.
doi: 10.1016/j.synbio.2023.11.008. eCollection 2023 Dec.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Chixiang Sun  1   2   3   4 , Baodong Hu  1   2   3   4 , Yanchun Li  2   5 , Zhimeng Wu  2   5 , Jingwen Zhou  1   2   3   4 , Jianghua Li  1   2   3   4 , Jian Chen  1   2   3   4 , Guocheng Du  1   2   3   4   5 , Xinrui Zhao  1   2   3   4
Affiliations

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Chixiang Sun et al. Synth Syst Biotechnol. .

Abstract

Deoxycholic acid (DCA) has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat. The hydroxylated product (6β-OH DCA) was developed to improve the solubility and pharmaceutic properties of DCA for further applications. Herein, a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst (CYP107D1, OleP) to convert DCA to 6β-OH DCA. Firstly, the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E. coli C41 (DE3) strain. Next, the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway. In addition, a new biosensor was developed to select the appropriate redox partner. Furthermore, a cost-effective whole-cell catalytic system was constructed, resulting in the highest reported conversion rate of 6β-OH DCA (from 4.8% to 99.1%). The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

Keywords: Deoxycholic acid; Hydroxylation; OleP; Redox partners; Whole-cell catalysis.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
The characteristics of expressed OleP in E. coli. (A) Full wavelength scan of purified OleP in three states. The blue, orange, and red circles mark the characteristic absorbance of the purified OleP, OleP-CO complex, and OleP–CO–sodium dithionite complex, respectively. (B) SDS-PAGE analysis of OleP. Lane 1, soluble expression of OleP; Lane 2, inclusion body of OleP; Lane 3, purified soluble OleP. M, marker (C) The analysis of the hydroxylated product of DCA catalyzed by OleP. a control reaction catalyzed by E. coli C43 (DE3) strain harboring pET28a empty plasmid; b the hydroxylation of DCA catalyzed by E. coli O1 strain harboring pET28a-oleP and pACYC-camA-camB plasmids. The black and red arrows point to the substrate (DCA) and the hydroxylated product (6β-OH DCA), respectively.
Fig. 2
Fig. 2
Three strategies to improve the heterologous expression of OleP in E. coli. (A) Selection of suitable plasmid. WT represents the E. coli O1 strain. (B) Selection of proper host. (C) The optimal induction temperature for OleP expression (°C). (D) The proper concentration of IPTG for OleP expression (mM). (E) The optimal induction time for OleP expression (h). The blue-filled triangle represents the biomass (OD600). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.
Fig. 3
Fig. 3
Strategies to improve heme supply in E. coli for the efficient catalysis of OleP. (A) The heme-binding ratio of wild-type OleP, MBP-OleP, and TF-OleP. (B) Heme biosynthetic pathways in E. coli. The purple arrow represents the C5 pathway and the pink arrow represents the downstream biosynthetic pathway of heme. The pCDFDuet-hemA-hemL plasmid was constructed to enhance the C5 pathway; the pETDuet-hemH-hemB-hemD-hemC plasmid was constructed to enhance the downstream biosynthetic pathway. (C) The effect of supplements on the OleP catalysis. (D) The color of engineered strains and pure enzyme. 1: E. coli O1 strain; 2: E. coli O2 strain; 3: E. coli O2 strain cultivated with ALA and FeCl3; 4: E. coli AL strain; 5: OleP enzyme purified from E. coli AL strain. The lower values represent the content of intracellular heme in different engineered strains. (E) The effect of enhancing heme biosynthesis on the OleP catalysis. The blue-filled triangle represents the heme-binding ratio (%). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.
Fig. 4
Fig. 4
Strategies to construct sfGFP sensor to screen redox partners. (A) The scheme of constructing the sfGFP sensor. (B) The self-assembly of OleP and Fdx was based on the three-dimensional structure of sfGFP (PDB: 5BT0). (C) Screening proper redox partners for OleP from different sources. The G1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The fluorescent intensities were calculated and the color of cells and fluorescent images were presented for G2-G5 strains that express different redox partners-sfGFP-1-10 and sfGFP-11-OleP, respectively. (D) The conversion rates were calculated for R2-R5 strains that express different redox partners and OleP, respectively. The R1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The blue-filled triangle represents the fluorescent intensity/OD600. The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.
Fig. 5
Fig. 5
The simulated analysis and design of redox partners for OleP. (A) The structures and interactions between OleP and Fdxs are presented. The key interacting residues in OleP-Fdx complexes are depicted as sticks and highlighted in yellow. Heme and substrates are displayed as sticks, colored in red and wheat, respectively. The Fe2S2 cluster is visualized as spheres. The distances (Å) between the iron-sulfur cluster and heme-iron are measured and indicated by dashed red lines. The interaction areas of OleP-Fdx are calculated by NovoPro (https://www.novopro.cn/). The numbers of hydrogen bonds and salt bridges are predicted by PDBePISA (https://www.ebi.ac.uk/pdbe/). (B) The fusion expression and the different expressional ratios between OleP and PetH/PetF. (C) The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.
Fig. 6
Fig. 6
The optimal whole-cell catalytic system for OleP catalysis. (A) Effect of catalytic form. (B) Effect of different biomass of resting whole-cell (OD600). (C) Effect of concentration of DCA (mg/mL). (D) Effect of catalytic time (h). (E) Effect of NADPH addition and intracellular NADPH regeneration system. The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.
Fig. 7
Fig. 7
LCMS analysis of the hydroxylation of LCA by OleP. (A) Biocatalytic reaction of LCA. a control reaction catalyzed by E. coli C41 (DE3) strain harboring pRSFDuet-1 empty plasmid; b the hydroxylation of LCA catalyzed by E. coli O1 strain harboring pET28a-oleP and pACYCDuet-camA-camB plasmids; c the hydroxylation of LCA catalyzed by E. coli C4 strain harboring pRSFDuet-petH-petF-oleP, pCDFDuet-hemA-hemL, and pACYCDuet-pntAB-nadK plasmids. The black and red arrows point to the substrate (LCA) and the hydroxylated product (MDCA), respectively. (B) MS analysis of substrate LCA. (C) MS analysis of hydroxylated product MDCA.
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References

    1. Hofmann A.F., Hagey L.R., Krasowski M.D. Bile salts of vertebrates: structural variation and possible evolutionary significance. J Lipid Res. 2010;51(2):226–246. doi: 10.1194/jlr.R000042. - DOI - PMC - PubMed
    1. Ajouz H., Mukherji D., Shamseddine A. Secondary bile acids: an underrecognized cause of colon cancer. World J Surg Oncol. 2014;12:1–5. doi: 10.1186/1477-7819-12-164. Artn 164. - DOI - PMC - PubMed
    1. Sykes J.M., Allak A., Klink B. Future applications of deoxycholic acid in body contouring. J Drugs Dermatol. 2017;16(1):43–46. doi: 10.1007/s40257-016-0231-3. - DOI - PubMed
    1. Hofmann A.F., Herdt T., Ames N.K., Chen Z., Hagey L.R. Bile acids and the microbiome in the cow: lack of deoxycholic acid hydroxylation. Lipids. 2018;53(3):269–270. doi: 10.1002/lipd.12036. - DOI - PubMed
    1. Wietholtz H., Marschall H.U., Sjovall J., Matern S. Stimulation of bile acid 6 alpha-hydroxylation by rifampin. J Hepatol. 1996;24(6):713–718. doi: 10.1016/S0168-8278(96)80268-6. - DOI - PubMed