Engineered Dehydrogenases for Unnatural Cofactor Recycling
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Solution Overview
Problem
Existing enzymatic biotransformation processes face challenges with the high cost and difficulty in controlling redox cofactor regeneration, particularly due to the expense and inefficiency of using native enzymes with unnatural cofactors.
Innovation Solution
Development of computationally designed engineered polypeptides with increased catalytic efficiency for unnatural cofactors, such as nicotinamide-based cofactors, to support diverse redox chemistries and enhance cofactor recycling in whole-cell biomanufacturing and cell-free applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If native enzymes are used with unnatural redox cofactors, then the system is simple and easy to implement, but the catalytic activity is low and inefficient
Solution Approach 1:
The patent combines computational protein design with directed evolution to create engineered polypeptides that merge the simplicity of using unnatural cofactors with high catalytic activity. The computational design phase creates initial variants with improved electrostatic complementarity, which are then refined through experimental evolution to achieve both ease of implementation and high productivity.
Solution Approach 2:
The patent systematically changes electrostatic parameters in the cofactor binding site through amino acid substitutions. By modifying charge distribution and electrostatic complementarity in the binding pocket, the engineered polypeptides achieve high catalytic activity with unnatural cofactors while maintaining system simplicity.
2Productivity
If amino acid substitutions are introduced to increase electrostatic complementarity, then catalytic efficiency for unnatural cofactors increases, but protein sequence complexity increases
Solution Approach 1:
The patent applies local quality by introducing amino acid substitutions specifically in the cofactor binding site rather than throughout the entire protein. This localized modification approach increases catalytic efficiency for unnatural cofactors while minimizing overall sequence complexity and maintaining the rest of the protein structure unchanged.
Solution Approach 2:
The patent changes electrostatic parameters locally in the binding pocket through targeted amino acid substitutions. By modifying only the electrostatic properties of residues lining the cofactor binding site, the patent achieves high catalytic efficiency without requiring comprehensive sequence redesign.
3Reliability
If conventional redox cofactor regeneration systems are used, then the system is well-established and reliable, but the cost is high due to stoichiometric consumption of expensive cofactors
Solution Approach 1:
The patent implements cofactor recovery by designing engineered polypeptides that enable regeneration of unnatural redox cofactors through catalytic cycles. The system recovers and recycles cofactors multiple times rather than consuming them stoichiometrically, reducing overall cofactor quantity requirements while maintaining reliable operation through the engineered catalytic efficiency.
4Quantity of substance
If simple unnatural cofactor analogs are used, then the cost is reduced and availability is improved, but native enzyme activity towards these cofactors is low
Solution Approach 1:
The patent applies inversion by reversing the traditional approach: instead of modifying unnatural cofactors to fit native enzymes, the patent engineers polypeptides to recognize and catalyze reactions with simple, inexpensive unnatural cofactors. This inverted strategy enables high enzyme activity towards readily available, low-cost cofactor analogs.
Solution Approach 2:
The patent changes the electrostatic parameters of the enzyme's cofactor binding site to match the properties of simple unnatural cofactors. By adjusting the charge distribution and electrostatic complementarity in the binding pocket, the engineered polypeptides achieve high activity with inexpensive cofactor analogs that have different physicochemical properties from natural cofactors.
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 polypeptides demonstrate high total turnover numbers and metabolic flux, enabling efficient redox chemistry support for chiral compound synthesis and pharmaceutical intermediate production, with enhanced catalytic activity for unnatural cofactors compared to wild-type polypeptides.
Implementation Method 1
the one or more amino acid substitutions increases electrostatic complementarity between the engineered polypeptide and the unnatural cofactor
Implementation Method 2
the engineered polypeptide reduces the unnatural cofactor by extracting electrons from an inexpensive feedstock
Data Source
AI summary
Provided are engineered polypeptides having dehydrogenase activity that exhibit increased catalytic efficiency for nicotinamide-based unnatural cofactors relative to wild-type or parent polypeptides that encode a dehydrogenase that utilizes nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. Also provided are cell-free and whole cell biotransformation systems for converting a substrate into a product using a redox reaction with a re-cycled unnatural cofactor that utilizes an engineered polypeptide that has dehydrogenase activity and which exhibits increased catalytic efficiency for nicotinamide-based unnatural cofactor.