Engineered Adenosine Kinase pH Optima Shift
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Solution Overview
Problem
Existing enzyme cascades for ATP regeneration or production of synthetic nucleotides face incompatibility issues between individual enzymatic reactions, limiting their efficiency and effectiveness.
Innovation Solution
Engineered adenosine kinases with specific amino acid sequences, exhibiting at least 70% to 99% sequence identity to reference sequences, are developed to enhance the transformation of nucleoside substrates into corresponding nucleoside monophosphates, thereby improving the compatibility and efficiency of enzyme cascades.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If enzyme cascades are used for ATP regeneration or production of synthetic nucleotides, then the opportunity to deal with unstable or toxic intermediates and shift equilibrium is improved, but incompatibility issues between individual enzymatic reactions limit efficiency
Solution Approach 1:
The patent applies parameter changes by modifying the pH optima of adenosine kinase through engineered variants (e.g., Ak2p with pH optimum shifted to 7.0-7.5). This allows the enzyme to function effectively in the neutral pH range required by other cascade enzymes like nucleoside phosphorylase and nucleotide kinases, thereby resolving incompatibility issues while maintaining high reaction efficiency and productivity
2Productivity
If adenosine kinase is used in enzyme cascades, then ATP regeneration or production of synthetic nucleotides is achieved, but incompatibility issues between individual enzymatic reactions occur
Solution Approach 1:
The patent engineers adenosine kinase variants with modified pH optima (e.g., Ak2p variant with pH optimum at 7.0-7.5) to match the operational conditions of other enzymes in the cascade. This parameter modification enables compatibility with neutral pH-requiring enzymes while maintaining high ATP regeneration efficiency
Solution Approach 2:
The engineered adenosine kinase is designed to function universally across different enzyme cascade configurations. The variant demonstrates broad adaptability by working effectively with multiple different nucleoside substrates and compatible with various downstream enzymes in the cascade, achieving multi-functional versatility
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 adenosine kinases demonstrate improved properties such as increased activity, thermostability, and compatibility with other enzymatic reactions, leading to enhanced ATP regeneration and nucleotide production efficiencies.
Implementation Method 1
Adenosine kinase catalyzes the phosphorylation of adenosine (Ado) to nucleoside monophosphate (5'-AMP) using ATP as a phosphate donor
Data Source
AI summary
The present disclosure provides engineered adenosine kinase polypeptides, and recombinant polynucleotides encoding the engineered adenosine kinases. The present disclosure further provides method of using the engineered adenosine kinases.