Inducible Expression System for Streptococcus thermophilus
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Solution Overview
Problem
There is a lack of effective systems for controlled protein expression in Streptococcus thermophilus, which hinders the production of therapeutic proteins and other molecules, as existing systems are not adequately controlled and suffer from yield losses and surface proteolysis.
Innovation Solution
A new inducible expression system based on the quorum sensing cellular communication mechanism involving a small hydrophobic peptide (SHP) and a regulator protein (Rgg), allowing for self-induction or external induction of protein expression, thereby controlling protein production and minimizing yield losses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the NisRK regulatory gene expression system is used in L. lactis, then protein expression can be induced, but the promoter remains active at a significant level in the absence of the inducer, leading to leaky expression
Solution Approach 1:
The invention extracts and eliminates the leaky expression problem by using a different regulatory system (SHP-Rgg quorum sensing) that does not suffer from the same defect as the NisRK system, thereby removing the harmful background expression while preserving the inducible expression capability
Solution Approach 2:
The invention changes the regulatory parameter from the NisRK two-component system to the SHP-Rgg quorum sensing system, which fundamentally alters the expression control mechanism to eliminate leaky expression while maintaining inducible expression capability
2Productivity
If existing expression systems are used in S. thermophilus, then protein production is possible, but the systems are not adequately controlled and suffer from yield losses and surface proteolysis
Solution Approach 1:
The invention performs preliminary action by controlling protein expression timing through quorum sensing mechanisms, ensuring that protein production occurs only when bacteria reach appropriate cell density, thereby preventing premature expression that would lead to yield losses and surface proteolysis
Solution Approach 2:
The invention implements feedback control through the quorum sensing system where the SHP peptide and Rgg regulator monitor bacterial cell density and automatically regulate promoter activity, creating a self-regulating system that optimizes protein production while minimizing losses
3Reliability
If a quorum sensing system with SHP peptide and Rgg regulator is used, then precise control of protein expression is achieved, but the system complexity increases
Solution Approach 1:
The invention applies self-service by utilizing the bacterium's own native quorum sensing components (SHP peptide and Rgg regulator) that already exist in S. thermophilus, thereby achieving precise expression control without introducing complex external regulatory systems
Solution Approach 2:
The invention leverages the universal quorum sensing mechanism that naturally regulates bacterial group behavior for multiple functions, including both native bacterial coordination and heterologous protein expression control, thereby simplifying the overall system architecture
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 system enables precise and controlled expression of proteins, reducing leaky expression and surface proteolysis, and allows for the production of therapeutic proteins like elafin with enhanced yield and stability.
Implementation Method 1
a promoter positively regulated by a protein complex formed by a small hydrophobic peptide called "SHP" and a regulator protein called "Rgg"
Implementation Method 2
They also showed that an Ami oligopeptide transporter allows the import of the mature SHP. Finally, they demonstrated an interaction between the mature SHP peptide and a transcriptional regulator of the Rgg family
Data Source
AI summary
The present invention relates to genetically modified bacteria capable of inducibly expressing a nucleic acid sequence of interest, genetic constructs, vectors and uses thereof.