Enantioselective synthesis of enantiopure chiral alcohols using carbonyl reductases screened from Yarrowia lipolytica

Abstract

Aims:We aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis. Methods and Results: Yarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2-hydroxyacetophenone (2-HAP) to optically pure (S)-1-phenyl-1,2-ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6Á96 U mg À1 (99Á8% e.e.) and 7Á85 U mg À1 (99Á9% e.e.), respectively, and showed moderate heat resistance at 40-50°C and acid tolerance at pH 5Á0-6Á0. An efficient whole-cell two-phase system was established using reductase-expressing recombinant Escherichia coli. The conversion of 2-HAP (20Á0 g l À1 ) conversion with the solvent of dibutyl phthalate was approximately 70-fold higher than in water. Furthermore, the two recombinant E. coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E. coli BL21 (DE3)/pET-28a-yacrII showed a broad substrate spectrum. Conclusions: A new whole-cell recombinant E. coli-based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed. Significance and Impact of the Study: We proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.

Green and scalable synthesis of chiral aromatic alcohols through an efficient biocatalytic system

“…The solubility of substrate in aqueous solution was very small, and most of the substrate was suspended in water, which made the enzyme unable to conduct effective catalytic conversion. By using water/organic solvent two‐phase system, the reaction concentration of the substrate was raised and the catalytic reaction proceeded between the two phases (Zhang et al ., 2019). In order to select and evaluate suitable organic solvents, log P value was used as the evaluation index of solvent polarity, i.e.…”

“…Recombinant protein RhADH was purified using a purifier system (ÄKTA pure; GE Healthcare) equipped with GSTrap HP (GE Healthcare, Piscataway, NJ, USA). The detailed operation was referenced to the protocol (Zhang et al ., 2019).…”

“…One unit (U) of enzyme activity was defined as the amount of enzyme that catalysed the formation and oxidation of 1 μmol of NADH (NADPH)/min. Effects of pH, temperature and various metal ions on enzymatic activity were investigated as in a previous study (Zhang et al ., 2019). Each experiment was performed in triplicate.…”

One-Pot Enzymatic–Chemical Cascade Route for Synthesizing Aromatic α-Hydroxy Ketones

“…2,4−6 Additionally, 2-HAP is used to prepare optically pure chemicals, such as (R)-and (S)-1-phenyl-1,2-ethanediol, which can be used as medicine intermediates and liquid crystal materials, respectively. 7,8 2-HAP can be produced using chemical and biocatalytic methods. The chemical synthesis methods mainly include photocatalytic oxidation of phenethyl alcohol, photocatalytic decarboxylative carbonylation of carboxylic acid, 9,10 oxidation of phenylglycol or terminal olefin by metal and nanometal catalysis, 11,12 and/or dehalogenation of bromoacetophenone catalyzed by microwave radiation.…”

“…α-Hydroxy ketones are important compounds used to synthesize a series of natural products and pharmaceuticals − in the field of organic chemistry. Among these compounds, 2-hydroxyacetophenone (2-HAP) is a promising and cost-effective synthetic intermediate widely used as a building block for the synthesis of valuable larger molecules, such as α-ketoesters, syn -amino alcohols, and antitumor Schiff bases. ,− Additionally, 2-HAP is used to prepare optically pure chemicals, such as ( R )- and ( S )-1-phenyl-1,2-ethanediol, which can be used as medicine intermediates and liquid crystal materials, respectively. , …”