Antibiotic-Free Plasmid Selection via Metabolic Pathway Genes
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Solution Overview
Problem
The bioprocess industry faces challenges with the use of antibiotic resistance markers, including increased production costs, environmental pollution, and difficulties in maintaining plasmids due to antibiotic degradation, leading to decreased productivity and the generation of secondary products during long-term fermentation.
Innovation Solution
A marker composition and method that utilize a plasmid with genes encoding enzymes in the isopentenyl diphosphate or dimethylallyl diphosphate synthetic pathway, allowing for the selection and transformation of living modified organisms without antibiotics, using pathways like the MEP or MVA pathway, and introducing complementary genes to ensure survival and productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotic resistance genes are used as selection markers, then plasmids can be stably maintained in host cells during culture, but production costs increase and environmental pollution occurs
Solution Approach 1:
The invention extracts and removes the antibiotic resistance gene from the plasmid structure, replacing it with a non-antibiotic-based selection mechanism. The plasmid is redesigned to lack any antibiotic resistance marker, fundamentally eliminating the source of antibiotic pollution while maintaining plasmid stability through alternative means.
Solution Approach 2:
The invention introduces an intermediary mechanism - a selectable phenotype based on essential metabolic pathway genes (such as folate synthesis genes) that mediates plasmid selection without requiring antibiotics. This intermediary selection system allows stable plasmid maintenance through metabolic dependency rather than antibiotic pressure.
2Reliability
If antibiotics are added to culture liquid to maintain plasmids, then plasmid stability is improved, but productivity decreases in the second half of culture due to antibiotic degradation
Solution Approach 1:
The invention performs preliminary action by designing the plasmid structure beforehand to eliminate antibiotic resistance genes, thereby preventing the need for antibiotic addition throughout the culture process. This preliminary design choice avoids the subsequent problem of antibiotic degradation and its impact on productivity.
Solution Approach 2:
The invention enables continuous useful action by maintaining plasmid stability throughout the entire culture period without antibiotic degradation. The alternative selection mechanism remains effective throughout culture, allowing continuous productivity without the decline observed in antibiotic-based systems during the second half of culture.
3Ease of operation
If antibiotic resistance markers are used, then selection of transformed organisms is achieved, but additional separation and purification processes are required
Solution Approach 1:
The invention extracts the antibiotic resistance marker from the selection process, replacing it with a selection mechanism based on essential metabolic genes. This extraction eliminates the need for antibiotic-related separation and purification processes, simplifying the overall workflow while maintaining selection efficiency.
4Object-generated harmful factors
If foreign genes are inserted into chromosomes instead of using plasmids, then antibiotic marker-free organisms can be created, but expression amount of proteins decreases
Solution Approach 1:
The invention creates a universal system that combines the benefits of both plasmid and chromosomal approaches. The plasmid carries multiple functions including the foreign genes for target product production and the essential metabolic pathway genes for selection, eliminating the need for antibiotic markers while maintaining high protein expression levels through plasmid-based amplification.
Data Source
AI summary
A marker composition for selecting a living modified organism allows transformation and the production of a target product without antibiotics or antibiotic resistance genes. The marker composition for selecting a living modified organism may basically prevent problems caused by the use of antibiotics and antibiotic resistance genes and produce a target product at a high yield.