Host Cell Poison-Antidote System for Plasmid Stability
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Solution Overview
Problem
Current recombinant protein production systems face challenges with plasmid instability and reduced growth rates in microorganisms, particularly due to the burden of plasmid replication and transcription, and the use of antibiotic-resistance genes is undesirable due to contamination risks and reduced protein yield.
Innovation Solution
A host cell system is developed with at least two copies of a nucleic acid sequence encoding a poison protein, such as CcdB, and one copy of the corresponding antidote protein, CcdA, to enhance plasmid stability and protein production, while avoiding antibiotic-resistance genes by using a poison-antidote system where the antidote is carried by a plasmid and the poison is chromosomally integrated.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotic-resistance genes are used as selectable markers, then plasmid maintenance is improved, but product contamination and reduced protein yield occur
Solution Approach 1:
The invention extracts and removes the harmful antibiotic-resistance gene from the plasmid system while retaining the essential plasmid maintenance function through alternative mechanisms such as toxin-antitoxin systems or metabolic dependencies, thereby eliminating contamination risk while preserving plasmid stability
Solution Approach 2:
The invention introduces an intermediary selectable marker system that does not involve antibiotic resistance, such as auxotrophic markers or conditionally essential genes, which mediate plasmid selection through metabolic pathways rather than antibiotic resistance mechanisms, thus avoiding product contamination
2Productivity
If plasmid replication and transcription are maintained, then protein production capability is improved, but cellular metabolic burden increases and growth rate decreases
Solution Approach 1:
The invention employs dynamic control of plasmid replication and gene expression through inducible promoters and regulated replication origins, allowing the system to adapt between low-level basal expression during growth phase and high-level expression during production phase, thereby balancing growth rate with protein production capability
Solution Approach 2:
The invention implements periodic control strategies where plasmid replication and strong gene expression are activated in periodic cycles rather than continuously, allowing cells to recover and grow during low-expression periods while achieving high production during induced periods, thus resolving the conflict between growth rate and productivity
Data Source
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AI summary
The present invention relates to a host cell for producing a recombinant peptide, polypeptide or protein of interest, wherein said host cell comprises at least 2 copies of a nucleic acid sequence encoding a poison protein; and to the use thereof for producing peptides, polypeptides or proteins of interest.