TKT Gene Mutants for Higher L-Lysine Fermentation Yield
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing L-lysine through fermentation using Corynebacterium glutamicum strains are limited in efficiency and yield, necessitating improvements in fermentation techniques, nutrient media composition, and microorganism performance.
Innovation Solution
Introduction of a TKT gene mutant protein with specific amino acid substitutions or additions, and fusion proteins with purification tags, expressed in recombinant microorganisms like Corynebacterium glutamicum, to enhance L-lysine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional fermentation methods using Corynebacterium glutamicum are used, then L-lysine production is achieved, but production efficiency and yield are limited
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (positions 327, 328, 329) in the TKT enzyme protein sequence to create variants with improved catalytic activity. These mutations alter the enzyme's kinetic parameters, enabling higher L-lysine production efficiency and yield while maintaining stability
Solution Approach 2:
The patent creates composite protein structures by fusing purification tags (such as His-tag, GST-tag, or FLAG-tag) with the TKT enzyme protein. These fusion proteins combine the enzymatic function with purification capabilities, allowing for easier recovery and higher production efficiency of L-lysine
2Productivity
If TKT gene mutant protein is introduced to enhance L-lysine production, then production capacity improves, but protein purification becomes more complex
Solution Approach 1:
The patent uses purification tags as intermediary elements that facilitate protein recovery. These tags (His-tag, GST-tag, FLAG-tag) serve as mediators between the TKT enzyme and purification systems, enabling simple affinity chromatography or other purification methods without complicating the overall process
Solution Approach 2:
The patent extracts and removes the purification tag from the final protein product through proteolytic cleavage or other methods, separating the functional TKT enzyme from the purification auxiliary. This allows the enzyme to be used in L-lysine production while the tag is discarded, simplifying the purification process
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
Enhances the production capacity and efficiency of L-lysine synthesis in recombinant microorganisms, improving yield and productivity.
Implementation Method 1
a TKT protein, and the TKT protein being A1) or A2) or A3) as follows: A1) a protein comprising (or being) SEQ ID No. 2, or a mutant protein which is obtained by mutating an alanine residue at position 327 in SEQ ID No. 2 into a threonine residue, a serine residue, a cysteine residue, a proline residue, an asparagine residue, a glutamine residue, a phenylalanine residue, a leucine residue, a valine residue, an isoleucine residue, an aspartic acid residue, a methionine residue, an arginine residue, a glutamic acid residue, a glycine residue, a histidine residue, a lysine residue, a tryptophan residue, or a tyrosine residue
Implementation Method 2
L-lysine has physiological effects such as promoting development, enhancing immunity and improving central nervous system functions. It is one of the eight essential amino acids that humans and animals cannot synthesize on their own and are necessary for growth. At present, L-lysine, as the second largest amino acid variety in the world, is mainly produced by a fermentation method
Data Source
AI summary
Provided are a TKT gene mutant and an application thereof in the preparation of L-lysine. The TKT gene mutant is a DNA molecule as represented by SEQ ID NO: 3, 5, 7, 9, 11, or 13 in the sequence listing, which encodes a protein as represented by SEQ ID NO: 4, 6, 8, 10, 12, or 14, and a wild type TKT gene is a DNA molecule as represented by SEQ ID NO: 1, which encodes a protein represented by SEQ ID NO: 2. It has been experimentally verified that the TKT gene and the mutant thereof can improve the yield of L-lysine and can be used for the production of L-lysine, and same have good application prospects.