Artificial Positive Feedback Loop for Stable Gene Expression
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Solution Overview
Problem
Current methods for over-expressing metabolic pathways in fungi rely on high copy plasmids and strong promoters, which are unstable and limited in expression patterns, making it difficult to achieve stable and targeted production of desired compounds.
Innovation Solution
The implementation of an artificial positive feedback loop (APFL) system that uses engineered transcription factors to induce specific promoters, allowing for the diversion of compounds from one metabolic pathway to another, eliminating the need for plasmids and enhancing the stability and yield of target molecule production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high copy plasmids and strong promoters are used to over-express metabolic pathways, then gene expression level is improved, but stability of the system deteriorates
Solution Approach 1:
The patent implements an artificial positive feedback loop where a transcription factor activates its own promoter, creating self-sustaining high-level expression. The transcription factor binds to the APFL promoter to drive its own expression and the expression of pathway genes, maintaining stable high productivity without requiring high copy plasmids
Solution Approach 2:
The system is designed to be self-regulating and self-sustaining. The transcription factor automatically activates the APFL promoter when present, creating a self-reinforcing loop that maintains high expression levels without external intervention or unstable high copy plasmid maintenance
2Productivity
If strong/constitutive promoters are used to express pathway genes, then productivity is improved, but adaptability to different fungi deteriorates
Solution Approach 1:
The APFL system uses a transcription factor that can recognize and bind to the engineered promoter sequence across different fungal species. This universal mechanism allows the same construct to achieve high-level expression in multiple fungi types, combining high productivity with broad adaptability
Solution Approach 2:
The system changes the regulatory parameter from species-specific constitutive promoters to an inducible APFL mechanism that responds to transcription factor binding. This parameter change enables the system to achieve high expression levels while being transferable across different fungal hosts
3Quantity of substance
If high copy plasmids are used for gene expression, then gene dosage is improved, but reliability of stable expression deteriorates
Solution Approach 1:
The patent extracts the high expression function from the unstable high copy plasmid system and relocates it to the genomic integration site with the APFL promoter. This separates the gene dosage effect from the plasmid maintenance requirement, achieving high reliable expression through genomic integration
Solution Approach 2:
The APFL creates a feedback mechanism where the transcription factor activates its own expression from the integrated promoter, ensuring consistent high-level expression that is reliably maintained through genomic inheritance rather than plasmid segregation
Data Source
AI summary
The present invention provides for a system for increasing the production of a compound using an artificial positive feedback loop (APFL). In some embodiments, the system diverts a compound produced in a first metabolic pathway into a second metabolic pathway in order to produce a compound of interest.