Polyphosphate Kinase Mutant for ATP Regeneration
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Solution Overview
Problem
Existing ATP regeneration systems are inefficient and costly due to the need for multiple enzymes and unstable phosphate donors, particularly for biosynthesis processes requiring ATP generation from AMP, which complicates biotransformation processes.
Innovation Solution
A polyphosphate kinase mutant with specific amino acid mutations at positions 79, 106, 108, 111, and 285 is developed, enhancing enzyme activity and enabling an efficient ATP regeneration system using polyphosphoric acid as a phosphate donor, reducing ATP consumption in biotransformation reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple enzymes and phosphate donors are used to regenerate ATP from AMP, then ATP regeneration is achieved, but the biotransformation process becomes complicated
Solution Approach 1:
The invention combines the functions of multiple enzymes (AMP kinase and ADP kinase) into a single polyphosphate kinase enzyme that can directly convert AMP to ATP using polyphosphoric acid as phosphate donor, thereby simplifying the biotransformation process while maintaining ATP regeneration capability
Solution Approach 2:
The polyphosphate kinase enzyme exhibits multi-functionality by catalyzing both the phosphorylation of AMP to ADP and ADP to ATP in a single enzymatic step, replacing the need for separate enzymes and reducing process complexity
2Productivity
If long-chain polyP is used as phosphate donor for PPK2-III, then ATP regeneration efficiency is improved, but the cost increases
Solution Approach 1:
The invention changes the parameter of phosphate donor from long-chain polyP to polyphosphoric acid (PPA), which is a more cost-effective material commonly used in industrial applications, while maintaining the ability to support efficient ATP regeneration through the engineered polyphosphate kinase
Solution Approach 2:
The invention employs polyphosphoric acid, a cheaper and more readily available phosphate donor compared to long-chain polyP, making the ATP regeneration system more economically viable for industrial biotransformation processes
3Quantity of substance
If polyphosphoric acid is used as phosphate donor, then cost is reduced, but enzyme activity decreases
Solution Approach 1:
The invention introduces specific amino acid mutations at positions 79, 106, 108, 111, and 285 of the polyphosphate kinase to optimize its catalytic activity toward polyphosphoric acid, thereby compensating for the lower inherent activity observed with this phosphate donor while maintaining cost advantages
Solution Approach 2:
The engineered polyphosphate kinase acts as an intermediary that facilitates the transfer of phosphate groups from polyphosphoric acid to AMP and ADP, enabling efficient ATP regeneration despite the use of a less optimal phosphate donor compared to long-chain polyP
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 mutant polyphosphate kinase system achieves enzyme activity 2.7-17.9 times higher than the parent enzyme, reducing ATP usage by over 70% in synthesizing NMN and G6P without affecting final yields, offering broad industrial application prospects.
Implementation Method 1
a phosphotransferase that catalyzes the interaction between ADP and the phosphate donor
Implementation Method 2
polyphosphate kinase mutant acts as biocatalyst
Data Source
AI summary
The present invention discloses a polyphosphate kinase mutant, engineered strain and application thereof, wherein the polyphosphate kinase mutant is obtained by single- or multi-site mutations of the amino acid at position 79, 106, 108, 111 or 285 of the amino acid sequence shown in SEQ ID No. 2. The present invention provides a variety of polyphosphate kinase mutants derived from Cytophaga hutchinsonii, and the specific enzyme activity of these mutants is 2.7-17.9 times higher than that of the parent polyphosphate kinase, more than 70% of the amount of adenosine triphosphate (ATP) consumption in ATP-dependent biocatalytic synthesis reactions may be reduced by the ATP regeneration system constituted by the mutants, which has broad industrial application prospects.