Mutant Nampt Enzyme Engineering for NMN Yield
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Solution Overview
Problem
The natural enzyme Nampt has relatively low enzyme activity, leading to high costs, harsh reaction conditions, unstable production, and low productivity in the traditional enzymatic synthesis of nicotinamide mononucleotide (NMN), which restricts large-scale industrial production and application.
Innovation Solution
A mutant protein of Nampt is designed with specific point mutations (N67K, N164L, R166W, A208G, A245T, S248A, V365L, or S382M) to enhance enzyme activity, and a recombinant expression vector encoding this mutant protein is used to express the enzyme in recombinant bacteria, such as E. coli, to achieve improved NMN yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If natural Nampt enzyme is used for NMN synthesis, then the enzymatic reaction can proceed under mild conditions, but the enzyme activity is low resulting in high costs and low productivity
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the Nampt enzyme through site-directed mutagenesis. Specific residues at positions 67, 164, 166, 208, 245, 248, 365, and 382 are mutated to improve enzyme activity while maintaining catalytic function under mild conditions. This allows the enzyme to achieve higher productivity without requiring harsh reaction conditions
2Device complexity
If natural Nampt enzyme is used for NMN synthesis, then the process can be simplified, but the production is unstable with great difference in index among batches
Solution Approach 1:
The patent improves production stability by changing the enzymatic parameters through amino acid substitution. The mutant Nampt enzymes with modified residues demonstrate consistent high activity across multiple batches, eliminating the variability observed with natural enzyme while keeping the overall process simple and straightforward
3Adaptability or versatility
If natural Nampt enzyme is used for NMN synthesis, then fewer enzymes are needed in the system, but the reaction efficiency is low resulting in harsh conditions and high costs
Solution Approach 1:
The patent resolves this contradiction by changing the catalytic parameters of the Nampt enzyme through rational design and mutagenesis. The mutant enzymes achieve high reaction efficiency with improved turnover numbers, allowing the system to maintain simplicity with fewer enzyme components while dramatically improving productivity and eliminating the need for harsh conditions
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 Nampt enzymes exhibit significantly improved enzyme activity, with some mutants showing up to a 62% increase in NMN yield compared to the wild-type enzyme, facilitating large-scale industrial production of NMN.
Implementation Method 1
The present disclosure provides a mutant protein of a nicotinamide phosphoribosyltransferase Nampt, which is configured to perform a point mutation on an amino acid sequence of the nicotinamide phosphoribosyltransferase Nampt
Data Source
AI summary
The present disclosure provides an innovative method for improving the enzyme activity of an NMN biosynthetic enzyme Nampt, and relates to the technical field of genetic engineering. A mutant protein of the present disclosure is obtained by firstly analyzing a target protein Nampt using two softwares FoldX and DeepDDG, and then predicting multiple key sites influencing the enzyme functions and finally performing the semi-rational design of the enzyme. In the examples of the present disclosure, 10 mutant strains are constructed using the designed primers according to the principle of point mutation, and 8 of the mutants have higher activity than a wild-type strain, in which the NMN yield of the mutant Nampt-V365L is increased by 62%, and the NMN yields of the mutants Nampt-S248A, Nampt-N164L, Nampt-S382M, Nampt-A245T and Nampt-A208G are increased by 34%, 27%, 27%, 22% and 17% respectively.