Engineered Ketoreductase Variants for Stable R-3-Hydroxythiolane Synthesis
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Solution Overview
Problem
Existing ketoreductase enzymes face challenges in achieving high stereoselectivity, enzymatic activity, thermostability, and solvent stability when converting keto substrates to chiral alcohol products, particularly in the production of (R)-3-hydroxythiolane.
Innovation Solution
Engineered ketoreductase polypeptides with specific amino acid modifications, such as replacing residue 145 with serine, exhibit improved stereoselectivity, enzymatic activity, and stability, allowing for efficient conversion of 3-ketothiolane to (R)-3-hydroxythiolane with high stereomeric excess and increased reaction rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If wild-type ketoreductase enzymes are used, then the conversion of keto substrates to chiral alcohol products occurs, but the stereoselectivity and enzymatic activity are insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the ketoreductase enzyme sequence. Mutations at positions 145, 157, and other sites alter the enzyme's catalytic properties, improving both stereoselectivity for (R)-3-hydroxythiolane production and enzymatic activity. The engineered enzymes show up to 100-fold increase in activity compared to wild-type while maintaining >98% stereomeric excess.
2Manufacturing precision
If ketoreductase enzymes are engineered for improved stereoselectivity, then stereomeric excess increases, but enzyme stability may be compromised
Solution Approach 1:
The patent simultaneously optimizes multiple amino acid positions to achieve both high stereoselectivity and stability. Specific mutations at positions 145 (e.g., to serine), 157 (e.g., to serine or threonine), and other sites create engineered enzymes that maintain structural integrity while achieving >98% stereomeric excess. The coordinated mutations ensure both selectivity and thermostability are improved.
3Productivity
If multiple amino acid mutations are introduced to improve enzyme performance, then catalytic activity increases, but the complexity of enzyme engineering increases
Solution Approach 1:
The patent focuses mutations on specific local regions of the enzyme that are critical for catalysis and substrate binding. By targeting key positions such as 145, 157, and other structurally important sites, the engineering effort is concentrated where it has maximum impact. This localized approach improves catalytic activity without requiring comprehensive mutation of the entire enzyme sequence, thereby managing engineering complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered ketoreductase polypeptides achieve stereoselective reduction of 3-ketothiolane to (R)-3-hydroxythiolane with greater than 98% stereomeric excess and enhanced enzymatic activity, outperforming wild-type enzymes by up to 100 times, and maintain stability under various conditions.
Implementation Method 1
Ketoreductase enzymes can be found in a wide range of bacteria and yeasts... KRED enzymes can be found in a wide range of bacteria and yeasts... engineered ketoreductase polypeptides... capable of stereoselectively reducing or converting 3-ketothiolane to the corresponding (R)-3-hydroxythiolane product
Implementation Method 2
The reduction of ketones and aldehydes, and the oxidation of alcohols by enzymes such as KRED requires a co-factor, most commonly reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) for the oxidation reaction
Data Source
AI summary
The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize chiral compounds.