Ketoreductase polypeptides for the reduction of acetophenones
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing ketoreductases from L. kefir, L. brevis, and L. minor exhibit insignificant activity towards substituted acetophenones, limiting their effectiveness in stereoselective reduction of compounds like 2′,6′-dichloro-3′-fluoroacetophenone to (S)-1-[2,6-dichloro-3-fluorophenyl]-ethanol.
Innovation Solution
Engineered ketoreductases derived from Lactobacillus species with a mutated residue at position 190, preferably to proline, exhibit reversed enantioselectivity and improved properties such as increased enzymatic activity, stereoselectivity, and thermostability for reducing substituted acetophenones.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type ketoreductases from Lactobacillus species are used, then the enzymes can reduce unsubstituted acetophenone, but they exhibit insignificant activity towards substituted acetophenones
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the ketoreductase enzyme sequence. Mutations at positions 190, 103, 106, 165, and 202 were identified as key parameters that, when changed, dramatically increase enzymatic activity towards substituted acetophenones while maintaining or expanding substrate scope. For example, mutating position 190 to proline or other specific residues resulted in activity increases of up to 1500% compared to wild-type enzymes.
2Manufacturing precision
If wild-type ketoreductases are used, then the enzymes maintain native stability, but they exhibit low stereoselectivity and activity for substituted substrates
Solution Approach 1:
The patent achieves improved stereoselectivity and reaction rate by changing the amino acid sequence parameters of the enzyme. Specific mutations at positions 190, 103, 106, 165, and 202 were designed to enhance both stereoselectivity (achieving greater than 99% stereomeric excess) and reaction rate (up to 1500% increase compared to wild-type). These parameter changes in the enzyme structure directly address the contradiction between precision and productivity.
3Productivity
If ketoreductase activity is increased through mutation, then enzymatic efficiency improves, but enzyme structure and function may be compromised
Solution Approach 1:
The patent carefully selects specific amino acid positions (190, 103, 106, 165, 202) for mutation to improve enzymatic activity while maintaining enzyme stability. The mutations are designed to be conservative or strategically positioned to avoid disrupting the overall enzyme structure and function. For example, position 190 mutations to proline or other specific residues enhanced activity by up to 1500% while the enzyme maintained its structural integrity and catalytic function.
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 greater than 99% stereomeric excess and enhanced reaction rates, outperforming wild-type enzymes by up to 1500% in converting 2′,6′-dichloro-3′-fluoroacetophenone to (S)-1-[2,6-dichloro-3-fluorophenyl]-ethanol.
Implementation Method 1
Engineered ketoreductases derived from Lactobacillus species with a mutated residue at position 190, preferably to proline, exhibit reversed enantioselectivity and improved properties such as increased enzymatic activity, stereoselectivity, and thermostability for reducing substituted acetophenones
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.