E. coli Strain Attenuation for Heterologous Protein Secretion
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Solution Overview
Problem
Current methods for producing recombinant proteins in E. coli strains are limited by inefficient secretion of heterologous proteins into the fermentation medium, leading to high production costs and reduced yield due to stringent control mechanisms mediated by (p)ppGpp synthetase II activity.
Innovation Solution
Attenuating the (p)ppGpp-synthetase II activity in E. coli strains by introducing mutations or deletions in the spoT gene, combined with mutations in the relA gene to achieve a 'relaxed' phenotype and 'leaky' mutations, allowing for increased secretion of heterologous proteins into the fermentation medium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If (p)ppGpp synthetase II activity is active in E. coli strains, then cell metabolism is regulated under stringent control, but secretion of heterologous proteins into fermentation medium is inhibited
Solution Approach 1:
The invention extracts or removes the harmful function of (p)ppGpp synthetase II by introducing mutations or deletions in the spoT gene. This eliminates the stringent control mechanism that prevents protein secretion, thereby resolving the contradiction between metabolic regulation and protein secretion efficiency
Solution Approach 2:
The invention changes the parameter of (p)ppGpp synthetase II activity from active to attenuated or eliminated state through genetic modifications. This parameter change transforms the metabolic regulation system from a stringent control mode to a relaxed mode that permits efficient protein secretion
2Quantity of substance
If standard E. coli strains are used for recombinant protein production, then production costs are high, but protein yield is reduced due to inefficient secretion
Solution Approach 1:
The invention changes the genetic parameters of E. coli strains by introducing mutations in spoT and relA genes. This creates a new strain type with altered metabolic control that enables efficient protein secretion, thereby simultaneously improving protein yield and reducing production costs
Solution Approach 2:
The invention creates modified E. coli strains that copy the desirable traits of relaxed phenotype strains while maintaining the ability to produce recombinant proteins. These modified strains serve as improved copies that overcome the secretion limitations of standard strains
Data Source
AI summary
A process for producing a heterologous protein is provided. The process comprises culturing an E. coli strain in a fermentation medium, the E. coli strain harboring a gene which codes for the heterologous protein and is functionally linked to a signal sequence coding for a signal peptide, and releasing the heterologous protein into the fermentation medium, and the heterologous protein being removed from the fermentation medium, wherein the E. coli strain has an attenuated (p)ppGpp-synthetase II activity (PSII activity).