Engineering an Epoxide Hydrolase for Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (S)- and (R)-Flutriafol.

Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase (RpEH), obtaining mutant Escherichia coli/RpehH336W/L360F with a 6.4-fold enhanced enantiomeric ratio (E) from 5.5 to 35.4. This enabled a gram-scale resolution of rac-1a by E. coli/RpehH336W/L360F, producing (S)-1a (98.2% ees) and (R)-1b (75.0% eep) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral (S)-flutriafol (98.2% ee) and (R)-flutriafol (75.0% ee) were easily synthesized by a one-step chemocatalytic process from (S)-1a and a two-step chemocatalytic process from (R)-1b, respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of RpEH toward bulky 2,2-bisaryl-substituted oxirane 1a.

Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (R)-Tebuconazole in High Optical Purity Mediated by an Epoxide Hydrolase from Rhodotorula paludigensis.

Tebuconazole is a chiral triazole fungicide used globally in agriculture as a racemic mixture, but its enantiomers exhibit significant enantioselective dissimilarities in bioactivity and environmental behaviors. The steric hindrance caused by the tert-butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of (R)-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2-tert-butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, rac-1a) by Escherichia coli/Rpeh whole cells expressed epoxide hydrolase from Rhodotorula paludigensis (RpEH), followed by a one-step chemocatalytic synthesis of (R)-tebuconazole. It was observed that (S)-1a was preferentially hydrolyzed by E. coli/Rpeh, whereas (R)-1a was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio (E value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM rac-1a was performed using 150 mg/mL E. coli/Rpeh wet cells, resulting in the retention of (R)-1a in a 97.0% ees, a 42.5% yields, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity (R)-tebuconazole (>99% ee) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor (R)-1a with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of RpEH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a.

Improving Biotransformation Efficiency of Arthrobacter simplex by Enhancement of Cell Stress Tolerance and Enzyme Activity.

Arthrobacter simplex exhibits excellent Δ1-dehydrogenation ability, but the acquisition of the desirable strain is limited due to lacking of comprehensive genetic manipulation. Herein, a promoter collection for fine-tuning gene expression was achieved. Next, the expression level was enhanced and directed evolution of the global transcriptional factor (IrrE) was applied to enhance cell viability in systems containing more substrate and ethanol, thus contributing to higher production. IrrE promotes a stronger antioxidant defense system, more energy generation, and changed signal transduction. Using a stronger promoter, the enzyme activities were boosted but their positive effects on biotransformation performance were inferior to cell stress tolerance when exposed to challenging systems. Finally, an optimal strain was created by collectively reinforcing cell stress tolerance and catalytic enzyme activity, achieving a yield 261.8% higher relative to the initial situation. Our study provided effective methods for steroid-transforming strains with high efficiency.