Cognate Foldase Co-Expression for Gram-Positive Protein Secretion
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Solution Overview
Problem
Existing methods for improving protein production yield in industrial biotechnology face challenges in overcoming bottlenecks in the secretory machinery of production host cells, particularly when expressing heterologous polypeptides.
Innovation Solution
Co-expression of a heterologous polypeptide of interest with a cognate foldase in Gram-positive host cells, such as PrsA, enhances protein folding and reduces secretion stress, leading to improved expression and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If gene over-expression is employed to improve production yield, then the expression level of the target protein increases, but bottlenecks in the secretory machinery cause reduced secretion efficiency
Solution Approach 1:
The patent introduces foldases (PrsA, PpiA, PpiB) as intermediary proteins that mediate between the over-expressed target protein and the secretory machinery. These foldases assist in proper protein folding and facilitate efficient secretion, resolving the bottleneck caused by high expression levels overwhelming the secretory system.
Solution Approach 2:
The patent modifies the folding parameters of the secretory pathway by introducing exogenous foldases with specific folding capabilities. This changes the folding environment and kinetics, allowing properly folded proteins to be efficiently secreted even when produced at high levels through gene over-expression.
2Reliability
If a non-cognate foldase is co-expressed with the heterologous polypeptide, then protein folding assistance is provided, but secretion stress remains high and expression is not optimized
Solution Approach 1:
The patent applies the principle of local quality by matching specific foldases to specific heterologous polypeptides based on their origin species. This creates an optimized local folding environment where the cognate foldase's specific folding mechanisms are tailored to the structural requirements of its native polypeptide, maximizing folding efficiency and secretion yield.
Solution Approach 2:
The patent implements a feedback mechanism where the cognate relationship between foldase and polypeptide ensures optimal interaction. The foldase from the same species as the polypeptide provides feedback-compatible folding assistance, recognizing and processing the polypeptide's specific structural features, thereby reducing secretion stress and improving overall expression yield.
3Quantity of substance
If gene over-expression is used to increase target protein levels, then production capacity increases, but the secretory machinery becomes overwhelmed
Solution Approach 1:
The patent introduces foldases as intermediary proteins that buffer the interaction between high levels of target protein and the secretory machinery. These intermediaries facilitate proper folding and smooth transit through the secretion pathway, preventing overwhelming of the secretory system even when target protein levels are dramatically increased through gene over-expression.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The co-expression strategy results in on par or improved expression and reduced secretion stress compared to non-cognate foldases, optimizing polypeptide production in Gram-positive host cells.
Implementation Method 1
Foldases are proteins that assist in folding of other proteins
Implementation Method 2
PrsA exists as a dimeric lipoprotein anchored in the outer leaflet of the cell membrane, where it aids folding of proteins secreted via the conserved SecA-YEG pathway
Data Source
AI summary
The present invention relates to means and methods for optimizing expression of a heterologous polypeptide of interest in Gram-positive host cells by co-expression with a foldase that is cognate to the heterologous polypeptide of interest.