Phi15 RNA Polymerase Expression in Pseudomonas With Low Toxicity
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Solution Overview
Problem
The T7 RNAP-based expression system, widely used in E. coli, is toxic and poorly regulated in Pseudomonas hosts like P. putida, leading to cell growth arrest and high mutational pressure, limiting its use for high-yield protein production and biosensor applications.
Innovation Solution
Characterization and utilization of non-toxic, orthogonally functioning RNA polymerases from Pseudomonas phages such as phi15, along with optimized phage lysozymes, to create a regulated expression system in Pseudomonas species, utilizing the XyIS/Pm system for inducible control and integrating these elements into the host genome for improved expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the T7 RNAP-based expression system is used in Pseudomonas hosts, then high-yield protein production is achieved, but cell growth arrest and high mutational pressure occur
Solution Approach 1:
The patent extracts the T7 RNAP system from its original E. coli host and adapts it to Pseudomonas hosts by removing the toxic components (T7 RNAP and T7 lysozyme) while retaining the beneficial high-yield transcriptional activity through alternative phage RNAPs from Pseudomonas phages, thus solving the contradiction between high protein yield and cell growth arrest
Solution Approach 2:
The patent changes the key parameter of RNAP origin from coliphage T7 to Pseudomonas phage RNAPs, fundamentally altering the transcriptional machinery parameters to be compatible with Pseudomonas metabolism while maintaining high transcriptional activity for protein production
2Power
If the T7 RNAP-based expression system is used in Pseudomonas hosts, then high transcriptional activity is achieved, but the system is poorly regulated
Solution Approach 1:
The patent implements feedback regulation by introducing phage lysozymes that inhibit their corresponding phage RNAPs, creating a self-regulating system where the lysozyme product feeds back to control the RNAP activity, thus achieving both high transcriptional activity and tight regulation
Solution Approach 2:
The patent uses phage lysozymes as intermediary molecules that mediate between the RNAP and the cell environment, providing controlled inhibition of RNAP activity to regulate protein expression levels while maintaining the high transcriptional potential of the phage RNAP system
3Productivity
If the LacI system is used to express T7 RNAP, then high protein yield is achieved, but extremely high levels of T7 RNAP are produced under uninduced conditions
Solution Approach 1:
The patent converts the harmful effect of basal RNAP expression into a beneficial regulatory mechanism by using phage lysozymes that are also expressed under basal conditions to inhibit the phage RNAP, thus turning the problem of basal expression into a solution for tight regulation
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 phi15 RNAP system achieves high-yield recombinant protein production with reduced toxicity and improved regulation, enabling sensitive biosensor systems with low detection limits and broad application in Pseudomonas hosts.
Implementation Method 1
the phi15 RNAP system achieves high-yield recombinant protein production
Implementation Method 2
introducing the corresponding phage lysozymes... improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition
Data Source
AI summary
The invention relates to a Pseudomonas sp. strain for use in the production of a recombinant protein characterised in that said strain comprises a nucleotide sequence encoding a phi15 RNA polymerase. The invention further relates to a plasmid, capable of integrating or replicating in Pseudomonas sp., comprising a phi15 promoter sequence operably linked to a nucleotide comprising one or more restriction sites for the insertion of a nucleotide sequence encoding a recombinant protein, or operably linked to a nucleotide sequence encoding a recombinant protein.