Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β‐Hydroxylation of Lithocholic Acid

Abstract

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo-and regioselective 7b-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7b-position but LCA is exclusively hydroxylated at the 6b-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificitydetermining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regioand stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA. Ursodeoxycholic acid (UDCA) is a valuable bile acid frequently prescribed for the treatment of cholecystitis as it can solubilize cholesterol gallstones with fewer side effects than chenodeoxycholic acid (CDCA). [1] UDCA also has anti-inflammatory properties [2] and is applied in the therapy of cystic fibrosis [3] and liver diseases like primary biliary cholangitis. [4] The major natural source of UDCA is bear bile, [5] a popular traditional medicine obtained by biliary catheterization of farmed bears. Alternatively, semi-synthetic UDCA can be produced from cholic acid (CA) [6] or CDCA. [7, 8] The synthesis route starting from CA forms CDCA within 5 steps, including a Wolff-Kishner reduction, and an epimerization at C7 to produce UDCA (Scheme 1 a, Scheme S1). [9] The yields of this pathway do not exceed 30 %. To overcome these limitations, a shorter synthesis route based on the biocatalytic epimerization of CDCA to UDCA (Scheme 1 a) has been developed. [7, 10] LCA is an abundant and inexpensive waste product of meat production [11] as this bile acid is found in farmed animals like sheep, [12] cattle, [12] and pigs. [13] Currently, no biotechnological process [14] utilizing LCA originating from these sources is known, making it a desirable starting material for the synthesis of UDCA. A few microbial organisms have been reported to form UDCA from LCA. [15] For example, the fungus Fusarium equiseti converts LCA to a number of products, including UDCA at 35 % yield. [16] However, there is currently no enzyme known to selectively hydroxylate LCA at the 7b-position to form UDCA and its synthesis pathway in microbial organisms, starting from LCA, remains enigmatic. An enzyme for direct 7b-hydroxylation would be a valuable tool for direct conversion of LCA to UDCA without involving the complex metabolism of fungi that invariably [16] produce multiple undesired side products, [15] complicating downstream processing. A major challenge in the enzymatic conversion of LCA to UDCA is the hydrophobicity and thus, extremely low water solubility of LCA co...

Enabling highly (R)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases

“…Similar approaches have been applied to construct small and smart libraries, which efficiently decrease the effort involved in developing stereoselective isozymes or multi-functional enzyme variants by directed evolution. 24 Therefore, a library containing approximately 60 combinations of these ten mutations in AI was created and screened for styrene epoxidation activity and enantioselectivity (Table S2 and Fig. S5 † ).…”

Recent trends in biocatalysis

“…This can be sufficient if single site-saturation or small combinatorial libraries have to be screened. 29,30 While these methods are still applicable to larger random and combinatorial libraries, they are not capable of screening a significant fraction of the library diversity. 31 One solution to this problem is to make the wells smaller, going through 1536-well plates to microcapillary arrays.…”

“…Limitations are directly caused by the intrinsic properties of steroidal compounds such as low solubility in aqueous media and cellular toxicity. 29 Some of these issues have been addressed by the emulsification of substrates with surfactants or the utilization of two-phase systems with organic solvents. 333,339,346 Furthermore, steroid transformations in well-characterised recombinant host cells (e.g., E. coli, S. cerevisiae) regularly yield low product titres simply because steroid-modifying enzymes only poorly express or function outside their native hosts, combined with insufficient substrate uptake and unintended metabolism.…”

“…Binding of a ligand causes the spin shift of the heme iron that can be detected as a signal spectrophotometrically. [402][403][404] Although these selected examples certainly highlight the power of directed evolution to engineer CYPs to execute highly desired steroid modifications, they are typically far from industrial applications due to low yields (2% isolated yield after LCA to UDCA transformation by the best OleP mutant in E. coli co-expressing putidaredoxin and putidaredoxin reductase as redox partner proteins) 29 and/or low substrate loads (1 mM testosterone for different hydroxylations in E. coli by selfsufficient P450BM3 variants). 383,385 None of these studies addressed the optimisation of CYP enzyme production in vivo apart from precursor supplementation for heme production.…”

“…383,385 None of these studies addressed the optimisation of CYP enzyme production in vivo apart from precursor supplementation for heme production. 29 Khatari et al adjusted the stoichiometry of CYP260A1 from Sorangium cellulosum and the redox partner proteins adenoredoxin reductase (AdR) and adenoredoxin (AdX) and showed that CYP260A1 hydroxylates 11-deoxycorticosterone (11-DOC) at high ratios of the redox partners (e.g., CYP260A1 : AdR : AdX = 1 : 3 : 10) mainly at the C1a-position. 405 At lower ratios (CYP260A1 : AdR : AdX = 1 : 3 : 5), also C1-C2-ene-11-DOC was produced in vitro.…”