Breaking through acetone inhibition: integrated protein engineering and bioreactor design for sustainable chiral aryl alcohol synthesis

The asymmetric biosynthesis of chiral aryl alcohols at high substrate concentrations is hindered by acetone accumulation, a byproduct of isopropanol-mediated cofactor regeneration. Here, a structure-guided mutant carbonyl reductase LXCAR-S154Y/I145A/R191Q (LXCAR-Q₃) was engineered to alleviate acetone competitive inhibition by expanding an enzyme active pocket, achieving a 224% increase in catalytic efficiency (k_cat/K_m) for 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) and a 59% reduction for acetone. Furthermore, coupled with an efficient in situ acetone removal bioreactor (EIARB), a neat isopropanol system enabled the complete conversion of 1000 g L⁻¹ CFPO to (S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) within 7.5 h, yielding a record space–time yield of 3041 g L⁻¹ d⁻¹. The biocatalyst retained full activity over five consecutive cycles at 400 g L⁻¹ CFPO, achieving a peak yield of 7299 g L⁻¹ d⁻¹. This neat isopropanol system, coupled with in situ isopropanol recovery and simplified product isolation, demonstrated applicability for synthesizing various chiral aryl alcohols at 2 M substrate loading, underscoring its industrial viability for sustainable and cost-effective biocatalysis. This study presents an environmentally benign approach for chiral alcohol biosynthesis and introduces a flexible mutation strategy for the robustness of carbonyl reductases in biocatalysis.