E. coli Recombinant Strain Mutations for Stable L-Threonine Production
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Solution Overview
Problem
Conventional methods for producing L-threonine using E. coli strains face challenges in achieving high yield and stability due to random mutations leading to slow growth and increased byproducts, necessitating a more efficient genetic modification approach.
Innovation Solution
Site-directed mutagenesis is applied to the deoB gene at the 1049th base and the rhtA gene promoter at the −67th base to create recombinant strains, enhancing L-threonine production efficiency and stability by introducing specific mutations in the deoB(G1049A) and PrhtA(A−67)G sequences, respectively.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional random mutation breeding is used to create E. coli strains for L-threonine production, then strain diversity is increased, but the strain grows slowly and generates more byproducts
Solution Approach 1:
The patent applies site-directed mutagenesis to specifically change the deoB gene sequence, introducing precise nucleotide substitutions (such as G to A transitions) at defined positions. This targeted parameter change in the gene sequence achieves improved L-threonine production without the negative effects of random mutation, resolving the contradiction between strain diversity and productivity.
Solution Approach 2:
The patent focuses on specific segments of the deoB gene rather than subjecting the entire genome to random mutation. By targeting particular nucleotide positions within the deoB gene, the method segments the mutation process to achieve desired traits while avoiding unwanted variations that cause slow growth and byproduct formation.
2Quantity of substance
If random mutation is applied to create production strains, then genetic variation is generated, but L-threonine yield remains low due to slow growth and increased byproducts
Solution Approach 1:
The patent introduces specific parameter changes in the deoB gene through site-directed mutagenesis, such as substituting particular nucleotides at defined positions. These precise changes generate the necessary genetic variation for improved L-threonine production while avoiding the random variations that lead to low yield, thereby resolving the contradiction between genetic variation and productivity.
3Productivity
If metabolic engineering is used to construct recombinant E. coli, then L-threonine production is improved, but production cost remains high
Solution Approach 1:
The patent achieves improved L-threonine production through simple point mutations in the deoB gene rather than complex recombinant construction. By changing specific nucleotide parameters in the native gene, the method avoids the high costs associated with plasmid construction, transformation, and maintenance, thereby resolving the contradiction between production improvement and manufacturing cost.
Data Source
AI summary
The present disclosure discloses an Escherichia coli-based genetically-modified recombinant strain, a construction method therefor and use thereof. A mutant gene obtained by subjecting a wild-type deoB gene (ORF sequence is shown in a sequence 3902352-3903575 in GenBank accession No. CP032667.1) and a wild-type rhtA gene promoter sequence PrhtA (shown in a sequence 850520-850871 in GenBank accession No. AP009048.1) of an E. coli K12 strain and a derivative strain thereof (such as MG1655 and W3110) to site-directed mutagenesis, and a recombinant strain obtained therefrom can be used for the production of L-threonine, and compared with an unmutated wild-type strain, the obtained strain can produce L-threonine with a higher concentration and has good strain stability, and also has lower production cost as an L-threonine production strain.