Mutated ArgB and ArgR Proteins for Amino Acid Production
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Solution Overview
Problem
Current methods lack an efficient process for enhancing the productivity of L-arginine, L-ornithine, and L-citrulline production in microorganisms, particularly in Corynebacterium species, as there is no reported mutation strategy for both ArgB and ArgR DNA that effectively increases these amino acid productivities.
Innovation Solution
Development of polypeptides with modified amino acid sequences in the N-terminal regions of N-acetylglutamate kinase (ArgB) and arginine repressor (ArgR) proteins, including specific substitutions and deletions, which are incorporated into recombinant DNA to reduce feedback inhibition and enhance enzyme activity, thereby increasing the production of L-arginine, L-ornithine, and L-citrulline.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type ArgB and ArgR are used, then normal cellular regulation is maintained, but L-arginine productivity is limited due to feedback inhibition and transcriptional repression
Solution Approach 1:
The patent extracts and eliminates the harmful feedback inhibition mechanism by introducing mutated ArgB genes that produce kinase enzymes insensitive to L-arginine feedback. Similarly, mutated ArgR genes are introduced to eliminate transcriptional repression. This removes the regulatory constraints that limit productivity while maintaining the essential biosynthetic function.
Solution Approach 2:
The patent applies parameter changes by mutating specific amino acid residues in ArgB (positions 26 and/or 31) and ArgR proteins to alter their regulatory properties. These parameter changes make the enzymes insensitive to L-arginine feedback inhibition and transcriptional repression, thereby enhancing productivity without compromising cellular viability.
2Productivity
If mutation strategies are applied to enhance productivity, then L-arginine production increases, but the complexity of strain development and optimization increases
Solution Approach 1:
The patent segments the complex productivity enhancement problem into two distinct genetic targets: ArgB for feedback inhibition control and ArgR for transcriptional repression control. By addressing these separately through targeted mutations, the patent simplifies the overall strain development process compared to random mutagenesis or simultaneous multi-gene modification.
Solution Approach 2:
The patent performs preliminary action by identifying and mutating specific critical amino acid positions (26 and/or 31 for ArgB, and corresponding positions for ArgR) before industrial strain development. This pre-optimization reduces subsequent complexity in strain screening and optimization by establishing a foundation of insensitivity to feedback repression mechanisms.
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 modified polypeptides and DNA sequences lead to increased production of L-arginine, L-ornithine, and L-citrulline by reducing feedback inhibition and transcriptional repression, resulting in enhanced productivity in microorganisms like Corynebacterium glutamicum.
Implementation Method 1
it is known that in coryneform bacteria N-acetylglutamate kinase, which is the second enzyme on the biosynthetic pathway from L-glutamic acid to L-arginine (EC: 2.7.2.8, hereinafter sometimes abbreviated as ArgB), is subject to feedback inhibition by L-arginine
Implementation Method 2
transcription of an operon composed of genes encoding enzymes responsible for L-arginine biosynthesis (hereinafter abbreviated as arginine operon) is repressed by the arginine repressor (hereinafter referred to as ArgR)
Data Source
AI summary
The present invention provides a polypeptide which has: (i) an amino acid sequence wherein one or more amino acid residues are substituted in the region at positions 20 to 38 from the N terminus of the amino acid sequence shown in SEQ ID NO: 1; or (ii) an amino acid sequence wherein one or more amino acid residues are substituted in the region at positions 20 to 38 from the N terminus of the amino acid sequence shown in SEQ ID NO: 1 and one or more amino acid residues are deleted, substituted or added in the region at positions 1 to 19 or 39 to 294; and which has N-acetylglutamate kinase activity.