Orthogonal Expression System for Eukaryotic Hosts
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Solution Overview
Problem
Current gene expression systems for eukaryotic hosts face challenges in achieving controlled and predictable expression, especially in diverse cultivation conditions, and are often limited by the need for host-specific promoters, which restricts their use across different species and requires customized systems for each host, making it difficult to establish robust and stable expression of heterologous genes.
Innovation Solution
Development of an orthogonal expression system using a universal core promoter that allows for the expression of synthetic transcription factors independently of host-specific regulatory mechanisms, enabling stable and tunable expression levels across a wide range of eukaryotic organisms by using a core promoter alone for synthetic transcription factor expression and synthetic promoters with specific binding sites for target genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If host-specific promoters are used for gene expression, then expression control is achieved in specific hosts, but the system cannot be transferred across different species and requires customized systems for each host
Solution Approach 1:
The patent employs a universal promoter that can function across multiple eukaryotic host species, eliminating the need for host-specific customization. This universal promoter drives expression of synthetic transcription factors that recognize synthetic promoter sequences, creating a transferable expression system that works in diverse eukaryotic organisms without requiring species-specific optimization
Solution Approach 2:
The patent introduces synthetic transcription factors as intermediary components that bridge the universal promoter and target genes. These synthetic transcription factors are expressed from the universal promoter and bind to synthetic promoter sequences upstream of target genes, mediating gene expression in a host-independent manner that enables cross-species transferability
2Reliability
If orthogonal expression systems with synthetic transcription factors are used, then expression can be controlled independently of host regulatory mechanisms, but the system complexity increases
Solution Approach 1:
The patent divides the expression system into distinct functional modules: a universal promoter for synthetic transcription factor expression, synthetic promoter sequences with specific binding sites for the transcription factors, and target genes. This segmentation allows each component to be optimized and transferred independently, reducing overall system complexity while maintaining expression stability across hosts
Solution Approach 2:
The patent modifies promoter sequences to create synthetic promoters with specific binding sites for synthetic transcription factors. By changing the DNA sequence parameters of the promoters to be non-host-specific, the system achieves independence from host regulatory mechanisms while maintaining controlled expression through the synthetic transcription factor-binder interaction
3Productivity
If multiple genes are expressed simultaneously in metabolic pathway engineering, then production pathways can be established, but achieving balanced expression ratios of multiple genes becomes difficult
Solution Approach 1:
The patent employs dynamic control of gene expression through synthetic transcription factors that can be regulated by small molecule inducers. This allows the expression ratios of multiple genes in a metabolic pathway to be adjusted dynamically during cultivation, enabling optimization of metabolic flux and product formation while maintaining balanced expression of pathway enzymes
Data Source
AI summary
The present invention provides an expression system for a eukaryotic host, which comprises 1) an expression cassette comprising a core promoter, the core promoter controlling the expression of a DNA sequence encoding a synthetic transcription factor (sTF), and 2) one or more expression cassettes each comprising a DNA sequence encoding a desired product operably linked to a synthetic promoter, the synthetic promoter comprising a core promoter, and sTF-specific binding sites upstream of the core promoter. The present invention also provides a method for identifying universal core promoters for eukaryotic hosts, expression systems using universal core promoters, hosts comprising the systems, and methods for producing protein products in eukaryotic hosts.