Ketoreductase polypeptides for the reduction of acetophenones
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Solution Overview
Problem
Existing ketoreductases from L. kefir, L. brevis, and L. minor exhibit insignificant activity towards substituted acetophenones and lack stereoselectivity for reducing (S)-alcohols, limiting their effectiveness in converting compounds like 2′,6′-dichloro-3′-fluoroacetophenone.
Innovation Solution
Engineered ketoreductases with a mutated residue at position 190, preferably to proline, exhibit reversed enantioselectivity and improved properties such as increased enzymatic activity, thermostability, and solvent stability, enabling efficient conversion of substituted acetophenones to (S)-alcohols.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If wild-type ketoreductases from L. kefir, L. brevis, and L. minor are used, then the enzymes can reduce simple ketone substrates, but they exhibit insignificant activity towards substituted acetophenones and lack stereoselectivity for reducing (S)-alcohols
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the ketoreductase sequence (particularly at positions 190, 196, and 205) to alter the enzyme's stereoselectivity and substrate scope. This transforms the enzyme from reducing simple ketones to stereoselectively reducing substituted acetophenones with (S)-selectivity, resolving the contradiction between maintaining broad substrate adaptability and achieving high stereoselectivity.
Solution Approach 2:
The patent applies local quality by making targeted mutations at specific positions (190, 196, 205) within the enzyme sequence rather than global modifications. These localized changes at critical residues in the active site or substrate binding region confer (S)-selectivity and improved activity towards substituted acetophenones while preserving overall enzyme function and substrate scope.
2Productivity
If wild-type ketoreductases are used, then the enzymes maintain native stability, but they exhibit insignificant enzymatic activity towards substituted acetophenones
Solution Approach 1:
The patent applies parameter changes through site-directed mutagenesis at positions 190, 196, and 205 to enhance enzymatic activity towards substituted acetophenones. The mutations (e.g., Y190P, V196P, A205P) are designed to improve substrate binding and catalytic efficiency while the patent subsequently evaluates and maintains enzyme stability through sequence optimization and expression system selection.
3Productivity
If conventional chemical synthesis methods are used, then various chemical compounds can be produced, but multiple synthetic procedures are required and stereoselectivity is difficult to achieve
Solution Approach 1:
The patent replaces conventional chemical synthesis methods with enzymatic catalysis using engineered ketoreductases. This substitution provides inherent stereoselectivity through the chiral enzyme active site, achieving greater than 99% stereometric excess in a single step, thereby eliminating the need for multiple chemical synthesis procedures and complex separation techniques.
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 ketoreductases achieve stereoselective reduction of 2′,6′-dichloro-3′-fluoroacetophenone to (S)-1-[2,6-dichloro-3-fluorophenyl]-ethanol with greater than 99% stereometric excess and enhanced reaction rates, surpassing wild-type enzymes by up to 1500%.
Implementation Method 1
Engineered ketoreductases with a mutated residue at position 190, preferably to proline, exhibit reversed enantioselectivity and improved properties such as increased enzymatic activity
Implementation Method 2
The engineered ketoreductases achieve stereoselective reduction of 2′,6′-dichloro-3′-fluoroacetophenone to (S)-1-[2,6-dichloro-3-fluorophenyl]-ethanol with greater than 99% stereometric excess
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 a variety of chiral compounds.