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Coupled (R)-carbonyl reductase and glucose dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent stereochemical selectivity

Paper ID Volume ID Publish Year Pages File Format Full-Text
34280 45016 2015 7 PDF Available
Title
Coupled (R)-carbonyl reductase and glucose dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent stereochemical selectivity
Abstract

•The construction of coupling system contain RCR and GDH.•The introduction of GDH has little effects on cell-growth.•The functions of RCR and GDH were rebalanced through protein-expression optimization.•The coupling system produced (R)-PED with excellent stereochemical selectivity.

The biotransformation of 2-hydroxyacetophenone to (R)-1-phenyl-1, 2-ethanediol (PED) by NADH-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis is slow and gives low yields, probably as a result of insufficient cofactors. To improve the biotransformation efficiency of (R)-PED from 2-hydroxyacetophenon, an enzyme-coupling system containing RCR and glucose dehydrogenase (GDH) was constructed to strengthen NADH-recycling pathway in Escherichia coli, in which the Shine-Dalgarno sequence and the aligned spacing sequence were used as linkers between them. The introduction of glucose dehydrogenase had little affects on the cell-growth. The co-expression conditions of RCR and glucose dehydrogenase was optimized to rebalance their catalytic functions. The ratio of kcat/KM for enzyme-coupling system catalyzing 2-HAP and glucose was about 1.0, suggesting the good balance between the functions of RCR and GDH. The rebalanced system gave excellent performance in (R)-PED biotransformation: an optical purity of 99.9% and a yield of 99.9% at optimal conditions: 35 °C and pH 7.0. The introduction of glucose dehydrogenase stimulated increases of 23.8% and 63.8%, in optical purity and yield of (R)-PED, and simultaneously reduced the reaction time two-fold. This work provided a valuable method for efficient chiral alcohol production through protein-expression and biotransformation optimization to rebalance cofactor pathways.

Graphical abstractThe coupled system based on RCR and GDH was rebalanced through protein-expression optimization. It gave excellent performance on (R)-PED biotransformation without exogenous cofactors.Figure optionsDownload full-size imageDownload as PowerPoint slide

Keywords
GDH, glucose dehydrogenase; 2-HAP, 2-hydroxyacetophenone; RCR, (R)-carbonyl reductase; (R)-PED, (R)-1-phenyl-1, 2-ethanediol; (S)-PED, (S)-1-phenyl-1, 2-ethanediol(R)-1-Phenyl-1, 2-ethanediol; (R)-Carbonyl reductase; NADH regeneration; Glucose dehydrogena
First Page Preview
Coupled (R)-carbonyl reductase and glucose dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol biosynthesis with excellent stereochemical selectivity
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 11, November 2015, Pages 1807–1813
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering