Plasmid Vector Par System for Stable Retention in Ralstonia
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Solution Overview
Problem
Recombinant plasmids used in bacteria of the genus Ralstonia, Cupriavidus, or Wautersia for producing polyhydroxyalkanoates are unstable, leading to challenges in commercial production due to plasmid elimination during bacterial proliferation, and existing stabilization methods rely on antibiotic selective pressure, which is undesirable for environmental and cost reasons.
Innovation Solution
Development of a novel recombinant vector that includes an origin of replication specific to these bacteria and the par system for stable plasmid retention without antibiotic selective pressure, eliminating the need for conjugation transferability and antibiotic resistance markers, allowing for stable production of polyhydroxyalkanoates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If pJRD215-derived vectors or pBBR-derived vectors are used for gene transfer into Ralstonia, Wautersia, or Cupriavidus bacteria, then broad-host-range capability is achieved, but plasmid stability deteriorates when polyhydroxyalkanoates accumulate in cells
Solution Approach 1:
The invention extracts and utilizes the par system (partitioning system) from native plasmids of Ralstonia, Wautersia, or Cupriavidus bacteria. By incorporating this native partitioning mechanism into the vector design, the plasmid stability is dramatically improved without compromising the broad-host-range capability provided by the replication origin and selectable marker.
2Reliability
If antibiotic-due selective pressure is applied to stabilize plasmids, then plasmid retention is improved, but production costs increase and environmental safety deteriorates
Solution Approach 1:
The invention enables the plasmid to stabilize itself through the native par system without requiring external antibiotic selection pressure. The partitioning system automatically ensures plasmid retention during cell division, making the system self-regulating and eliminating the need for continuous antibiotic addition, thereby reducing production costs and environmental concerns.
3Reliability
If antibiotic-due selective pressure is applied to stabilize plasmids, then plasmid retention is improved, but environmental safety and applicability to human/animal treatment substances deteriorates
Solution Approach 1:
The plasmid stabilization is achieved through the self-sufficient native par system that automatically partitions plasmids to daughter cells during division. This eliminates the need for antibiotic selection pressure, removing the harmful environmental factors associated with antibiotic use while maintaining high plasmid retention rates throughout the culture process.
4Adaptability or versatility
If multicopy plasmids are used to secure stable transmission, then gene introduction capability is improved, but plasmid elimination during bacterial proliferation increases
Solution Approach 1:
The invention extracts and implements the native par partitioning system from Ralstonia, Wautersia, or Cupriavidus plasmids to ensure stable plasmid transmission during bacterial proliferation. This active partitioning mechanism counteracts plasmid elimination and maintains plasmid stability throughout the culture process, enabling reliable commercial production without antibiotic selection.
Data Source
AI summary
The object of the present invention is to develop a novel vector. Preferably, the object is to develop a novel vector which can be stably retained in bacteria of the genus Ralstonia, Cupriavidus or Wautersia without any antibiotic-due selective pressure and has no transferability by conjugation. Another object is to provide a strain which can stably produce polyhydroxyalkanoate using the vector, and a method for producing a polyhydroxyalkanoate using the strain. The present invention provides a novel recombinant vector which contains an origin of DNA replication functioning in bacteria of the genus Ralstonia, Cupriavidus or Wautersia. Particularly, the transformant, which is obtained by using a recombinant vector which contains the origin of DNA replication functioning in bacteria of the genus Ralstonia, Cupriavidus or Wautersia and contains a region for a recombinant vector stabilization (par region) can make the vector to be stably retained in bacteria, and can efficiently produce a polyhydroxyalkanoate.