Engineered Imine Reductases for Stereoselective Reductive Amination
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Solution Overview
Problem
There is a need for efficient biocatalytic routes to synthesize chiral secondary and tertiary amines under industrially applicable conditions, as existing methods rely on chiral boron reagents or multi-step synthesis, and there is a lack of identified enzymes that can efficiently catalyze these reactions.
Innovation Solution
Engineered polypeptides derived from opine dehydrogenases, specifically through directed evolution of enzymes from Arthrobacter sp. strain 1C, exhibit imine reductase activity, enabling the conversion of unactivated ketones and amines to secondary or tertiary amines, with enhanced stereoselectivity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing chemical methods are used to synthesize chiral secondary and tertiary amines, then the products can be obtained, but the process requires chiral boron reagents or multi-step synthesis, increasing process complexity and cost
Solution Approach 1:
The patent replaces complex chemical synthesis mechanisms with a biocatalytic mechanism. Engineered opine dehydrogenase enzymes catalyze the reductive amination reaction, substituting the need for chiral boron reagents and multiple synthetic steps with a single enzymatic step that inherently provides chirality through the enzyme's stereoselective active site.
Solution Approach 2:
The patent changes the reaction parameters by using engineered enzymes with modified amino acid sequences that alter substrate specificity and catalytic efficiency. The directed evolution process optimizes parameters such as stereoselectivity, reaction rate, and substrate scope, transforming the enzyme's properties to match industrial requirements for chiral amine production.
2Productivity
If native opine dehydrogenases are used for reductive amination, then the natural reaction can be performed, but the catalytic activity and stereoselectivity are insufficient for industrial applications
Solution Approach 1:
The patent applies directed evolution to dynamically optimize the enzyme's properties. Through iterative cycles of mutagenesis and screening, the enzyme's amino acid sequence is dynamically adjusted to enhance both catalytic activity and stereoselectivity, transforming a static native enzyme into a dynamically optimized biocatalyst suited for industrial production.
Solution Approach 2:
The patent creates multiple copies of the opine dehydrogenase gene with introduced mutations, then screens these copies for improved performance. The directed evolution process generates a library of enzyme variants, and the best-performing variant is selected and copied for further optimization or industrial use, effectively copying and improving upon the native enzyme's function.
3Adaptability or versatility
If whole cells of Acetobacterium woodii are used for biocatalysis, then imine reductase activity is present, but the substrate scope is limited to specific imines and aldehydes with activated groups
Solution Approach 1:
The patent modifies the local quality of the enzyme's active site through site-directed mutagenesis. Specific amino acid residues in the substrate binding pocket are mutated to alter steric and electronic properties, enabling the enzyme to accommodate a broader range of substrates including unactivated ketones and amines, thereby expanding substrate scope without sacrificing catalytic efficiency.
Solution Approach 2:
The engineered opine dehydrogenase becomes a universal biocatalyst capable of performing reductive amination with diverse substrate types. The enzyme maintains its natural activity with activated substrates while gaining the ability to process unactivated ketones and amines, achieving multi-functionality that covers a broad spectrum of industrial amine synthesis applications.
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 imine reductases demonstrate increased catalytic activity and stereoselectivity, allowing for the efficient synthesis of chiral amines from a wide range of substrates, including unactivated ketones and amines, under industrially relevant conditions.
Implementation Method 1
The engineered polypeptides are capable of catalyzing the conversion of a ketone (including unactivated ketone substrates such as cyclohexanone and 2-pentanone) or aldehyde substrate, and a primary or secondary amine substrate
Implementation Method 2
Opine dehydrogenases are a class of oxidoreductase that act on CH—NH bonds using NADH or NADPH as co-factor. The enzymatic activity of these engineered polypeptides derived from opine dehydrogenases is referred to as 'imine reductase activity'
Data Source
AI summary
The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.