Variant RpoC Plasmid Copy Number Control
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Solution Overview
Problem
There is a need for mutants of RNA polymerase β' subunit that can modify the copy number of plasmids without causing a corresponding change in chromosomal copy number, as existing methods are empirical and lack predictability.
Innovation Solution
A nucleic acid molecule comprising a variant rpoC RNA-polymerase β' subunit protein coding sequence, which encodes a variant RpoC with an R47C substitution, is used to regulate the copy number of plasmids by decreasing the plasmid copy number without affecting the chromosomal copy number.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If plasmid replication is increased to achieve high target gene expression, then productivity is improved, but plasmid stability deteriorates due to metabolic burden
Solution Approach 1:
The invention changes the biochemical parameters of RNA polymerase by introducing specific mutations (e.g., R47C substitution) to create a variant that selectively affects plasmid replication. This parameter change in the enzyme's properties allows differential control of replication rates for plasmids versus chromosomes, resolving the contradiction between high expression and stability.
Solution Approach 2:
The variant RNA polymerase acts as an intermediary that mediates between the conflicting requirements of high plasmid replication and stability. By introducing this modified enzyme, the system achieves controlled plasmid copy number increase without the severe metabolic burden that would otherwise cause plasmid loss, thus mediating the trade-off between productivity and reliability.
2Adaptability or versatility
If empirical methods are used to balance plasmid copy number and stability, then adaptability is improved, but manufacturing precision deteriorates due to trial and error process
Solution Approach 1:
The invention performs preliminary action by pre-designing specific RNA polymerase mutations based on structural and functional analysis. Instead of empirical trial and error, the mutations are predicted and constructed in advance to achieve desired plasmid copy number effects, thereby improving manufacturing precision while maintaining adaptability.
Solution Approach 2:
By changing specific parameters of the RNA polymerase enzyme through defined mutations, the invention transforms the empirical balancing process into a predictable, precision-controlled approach. The specific amino acid substitutions are designed to produce predetermined effects on plasmid replication, eliminating the need for trial and error while maintaining system adaptability.
3Productivity
If plasmid size is increased to include more target genes, then productivity is improved, but plasmid copy number deteriorates due to increased metabolic burden
Solution Approach 1:
The invention changes the parameter of RNA polymerase efficiency and specificity through mutation, allowing the system to support larger plasmid sizes without the usual copy number reduction. The variant enzyme can handle the increased metabolic burden of larger plasmids, thereby maintaining higher copy numbers despite increased plasmid size and target gene capacity.
Data Source
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AI summary
A nucleic acid molecule comprising a variant rpoC coding sequence is disclosed. The variant rpoC coding sequence encodes a variant RpoC which regulates copy number of a plasmid. Also disclosed are a recombinant microorganism comprising the nucleic acid molecule, a method for regulating copy number of a subject vector in the recombinant microorganism, and a method for making a target product by use of the recombinant microorganism.