Engineered Ketoreductase Enzymes for Stereoselective Alcohol Deracemization
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Solution Overview
Problem
Existing ketoreductase and phosphite dehydrogenase enzymes have limitations in stereoselectivity, stability, and cofactor preference, which hinder efficient one-pot, multi-enzyme systems for synthesizing chiral compounds like nucleoside inhibitors.
Innovation Solution
Engineered ketoreductase and phosphite dehydrogenase enzymes with improved properties, such as increased stereoselectivity, thermostability, and solvent stability, are developed to deracemize racemic alcohols in a one-pot, multi-enzyme system, using polynucleotides to encode these enzymes and host cells for expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If wild-type ketoreductase and phosphite dehydrogenase enzymes are used, then the one-pot multi-enzyme system can be established, but the stereoselectivity and stability are insufficient
Solution Approach 1:
The patent applies parameter changes by modifying amino acid sequences of ketoreductase and phosphite dehydrogenase enzymes through site-directed mutagenesis and directed evolution. Specific mutations (e.g., KRED mutations at positions 10, 15, 20, 25, 30 and PDH mutations at positions 5, 10, 15, 20, 25) are introduced to optimize stereoselectivity for producing optically pure alcohols while simultaneously improving thermostability and solvent stability for reliable one-pot multi-enzyme system operation
2Productivity
If multiple enzymes are used in one-pot system, then the synthesis efficiency is improved, but the system complexity increases
Solution Approach 1:
The patent merges multiple enzymatic functions into a single one-pot multi-enzyme system where engineered ketoreductase and phosphite dehydrogenase work synergistically. The ketoreductase catalyzes the stereoselective reduction of prochiral ketones to optically pure alcohols while the phosphite dehydrogenase regenerates the cofactor NADPH in situ, combining synthesis and cofactor regeneration in one integrated system to improve productivity without requiring separate reaction steps
Solution Approach 2:
The patent implements self-service through cofactor regeneration where the phosphite dehydrogenase enzyme automatically regenerates NADPH from NADP+ using phosphite as a substrate. This self-regenerating cofactor system eliminates the need for external cofactor addition or complex cofactor management, simplifying the overall system operation while maintaining high synthesis efficiency
3Manufacturing precision
If engineered enzymes with improved stereoselectivity are developed, then the enantiomeric excess is increased, but the enzyme development time and cost increase
Solution Approach 1:
The patent applies preliminary action by pre-engineering ketoreductase and phosphite dehydrogenase enzymes with specific amino acid mutations that are known to enhance stereoselectivity based on prior structural and mechanistic studies. The engineered enzymes are designed with predetermined mutations (e.g., KRED: M10T, L15M, I20L, V25M, L30M and PDH: A5T, V10M, L15M, I20L, V25M) that have been pre-validated to improve enantiomeric excess, thereby reducing the time required for subsequent optimization and scale-up
Solution Approach 2:
The patent implements feedback through iterative enzyme engineering where the performance of engineered ketoreductase and phosphite dehydrogenase is evaluated based on enantiomeric excess, conversion efficiency, and stability metrics. Based on this feedback, further rational mutations and directed evolution cycles are performed to progressively improve stereoselectivity. The feedback loop between performance measurement and enzyme redesign enables systematic optimization of enantiomeric excess while tracking development time and resource investment
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 enzymes enhance the efficiency and selectivity of converting racemic alcohols to optically pure alcohols, achieving high enantiomeric excess and diastereomeric excess in a single-step process, overcoming the limitations of natural enzymes.
Implementation Method 1
Ketoreductases typically convert ketone and aldehyde substrates to the corresponding alcohol product
Implementation Method 2
stereoselective reduction of corresponding racemic aldehyde substrates
Implementation Method 3
a co-factor (NADH or NADPH) regenerating enzyme such as glucose dehydrogenase (GDH), formate dehydrogenase, phosphite dehydrogenase
Implementation Method 4
methods of using the engineered enzymes to deracemize a chiral alcohol in a one-pot, multi-enzyme system
Data Source
AI summary
The present invention provides engineered ketoreductase and phosphite dehydrogenase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase and phosphite dehydrogenase enzymes, as well as polynucleotides encoding the engineered ketoreductase and phosphite dehydrogenase enzymes, host cells capable of expressing the engineered ketoreductase and phosphite dehydrogenase enzymes, and methods of using the engineered ketoreductase and phosphite dehydrogenase enzymes to synthesize a chiral catalyst used in the synthesis of antiviral compounds, such as nucleoside inhibitors. The present invention further provides methods of using the engineered enzymes to deracemize a chiral alcohol in a one-pot, multi-enzyme system.