Mutant Ldc2 Polypeptides for High-Yield Cadaverine Biosynthesis
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Solution Overview
Problem
Current methods for cadaverine production, such as fermentative production and in vitro enzyme catalysis, result in low yields, necessitating a more effective process to enhance cadaverine production.
Innovation Solution
The use of a transformant comprising an expression plasmid vector in Escherichia coli (E. coli) or Hafnia alvei cells with specific mutants of the Ldc2 polypeptide, such as S111C, N262T, K265N, S111C/N262T, S111C/K265N, N262T/K265N, and S111C/N262T/K265N, to increase cadaverine yields through improved lysine decarboxylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional lysine decarboxylases (CadA or LdcC) are used in fermentative production, then the lysine biosynthesis pathway can be extended to cadaverine biosynthesis, but the cadaverine production yield remains low
Solution Approach 1:
The patent applies parameter changes by introducing specific mutations at positions 111, 262, and 265 of the Ldc2 enzyme sequence. These amino acid substitutions (S111C, N262T, K265N) modify the enzyme's catalytic parameters to enhance lysine decarboxylation activity, directly increasing cadaverine production yield from low levels to unexpectedly high yields
Solution Approach 2:
The patent uses copying by creating recombinant expression constructs of the mutated ldc2 gene and introducing them into host bacteria. The mutant Ldc2 polypeptide sequences are copied and expressed in E. coli or Hafnia alvei cells, replacing or supplementing the traditional CadA or LdcC enzymes to achieve improved cadaverine production
2Quantity of substance
If in vitro enzyme catalysis is used with traditional lysine decarboxylases, then lysine can be converted to cadaverine, but the conversion yield is limited
Solution Approach 1:
The patent modifies the enzymatic conversion parameters by introducing specific amino acid mutations (S111C, N262T, K265N) in the Ldc2 enzyme. These parameter changes enhance the decarboxylation efficiency and substrate affinity, enabling higher cadaverine conversion yields while maintaining the simplicity of the in vitro enzyme catalysis approach
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
This approach leads to unexpectedly high cadaverine production yields, exceeding those achieved with traditional E. coli lysine decarboxylases like CadA, demonstrating the effectiveness of the mutant Ldc2 polypeptides in enhancing cadaverine biosynthesis.
Implementation Method 1
Lysine decarboxylases are the enzymes that catalyze production of cadaverine by removing the carboxyl group from lysine
Data Source
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AI summary
The expression plasmid vectors comprise a polynucleotide sequence encoding Ldc2 polypeptide, a fragment, and/or a mutant. A backbone plasmid is capable of autonomous replication in a host cell. The host cell is not a P. aeruginosa cell. Transformants are transformed with expression plasmid vector. The transformants are not P. aeruginosa.Mutant host cells comprise a polynucleotide sequence encoding Ldc2 polypeptide, a fragment and/or a mutant that has been integrated into the host cell chromosome. A polypeptide, a fragment and/or a mutant comprise Ldc2. A non-naturally occurring polynucleotide, and/or a mutant encodes polypeptide comprising Ldc2. Biobased cadaverine is produced using the transformants and the biobased cadaverine is prepared by the method. Polyamides are formed using the biobased cadaverine and compositions.