Bidirectional Promoter Library for Coexpression Optimization
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Solution Overview
Problem
Current bidirectional expression vectors in Pichia pastoris and Saccharomyces cerevisiae lack a diverse range of promoters to optimize coexpression of multiple genes, limiting expression levels, ratios, and regulatory profiles, which are crucial for maximizing protein yields and metabolic pathway efficiency.
Innovation Solution
A library of bidirectional expression cassettes with a repertoire of 50-1000 different bidirectional promoter sequences, allowing for the coexpression of genes with varying expression levels, ratios, and regulatory profiles, including constitutive, inducible, and derepressed expression, by using type IIS restriction enzymes for cloning and TA cloning strategies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a single bidirectional promoter is used in expression vectors, then cloning is simplified, but the ability to optimize expression levels and ratios of coexpressed genes is limited
Solution Approach 1:
The patent divides the promoter selection into modular segments by creating a library of bidirectional promoters with different expression strengths (strong, medium, weak) and regulatory profiles (constitutive, inducible, derepressed). Each promoter segment can be independently selected and combined with gene pairs to achieve optimal coexpression, replacing the need for a single universal promoter with a segmented library of specialized promoters.
Solution Approach 2:
The patent changes the parameter of promoter diversity by introducing a library of 50-1000 different bidirectional promoter sequences with varying expression levels, ratios, and regulatory characteristics. This parameter change enables fine-tuning of coexpression optimization for different gene pairs while maintaining the bidirectional expression capability.
2Adaptability or versatility
If multiple separate vectors are used for coexpression of two genes, then each gene can have its own optimized promoter, but the system complexity increases and requires multiple resistance markers
Solution Approach 1:
The patent merges two separate monodirectional expression systems into a single bidirectional expression vector. The bidirectional promoter simultaneously drives expression of two genes in opposite orientations within one vector, eliminating the need for two separate vectors, multiple resistance markers, and complex cotransformation or mating procedures while still allowing individual promoter optimization through library selection.
Solution Approach 2:
The bidirectional expression vector achieves multi-functionality by enabling coexpression of any pair of genes with optimized expression levels and ratios through the promoter library. A single vector design with bidirectional promoters can replace multiple specialized vectors, providing universal applicability for coexpression of heterodimeric proteins, metabolic pathways, and other multi-gene systems.
3Device complexity
If the same monodirectional promoter is cloned in front of two genes on the same vector, then multiple resistance markers are avoided, but recombination events may occur due to identical promoter sequences
Solution Approach 1:
The patent applies asymmetry by using bidirectional promoters where the two promoter sequences face opposite directions (head-to-head orientation). Even when similar promoter sequences are used, their opposite orientations prevent homologous recombination between them, as the asymmetric arrangement disrupts the symmetry required for recombination events while maintaining comparable expression capabilities.
4Productivity
If strong promoters are used for high expression, then protein yields increase, but cellular machinery may be overburdened when protein folding or post translational modifications are limiting
Solution Approach 1:
The patent introduces dynamics into the expression system by providing a library of bidirectional promoters with varying strengths (strong, medium, weak) and regulatory profiles (constitutive, inducible, derepressed). This dynamic range allows selection of appropriate promoter strength based on the specific requirements of each gene pair, enabling adjustment of expression levels to match cellular processing capacity and avoid overburdening the folding and modification machinery while maintaining high productivity.
Data Source
AI summary
The invention refers to a library of bidirectional expression cassettes or expression vectors comprising a repertoire of bidirectional promoter sequences, each expression cassette comprising a promoter sequence operably linked to a first gene in one direction, and operably linked to an oppositely oriented second gene in the other direction which is different from the first gene, and bidirectional Pichia pastoris or CHO cells promoter sequences. The invention further refers to a method of screening or selecting a bidirectional promoter suitable for expressing at least two GOI in a host cell and a kit comprising a) an expression cassette consisting of the first and second genes and a stuffer sequence separating them, which stuffer sequence comprises a recognition site for a type IIS restriction enzyme at both ends; b) the type IIS restriction enzyme; c) and a repertoire of promoter, preferably a promoter library including bidirectional promoters.