Coryneform Protein Secretion via Mdh Reduction and Tat Export
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Solution Overview
Problem
The existing methods for secretory production of heterologous proteins in coryneform bacteria do not effectively utilize the relationship between malate dehydrogenase (Mdh) activity and protein secretion, limiting the efficiency of heterologous protein production.
Innovation Solution
Modify coryneform bacteria to reduce Mdh protein activity by decreasing mdh gene expression or disrupting the mdh gene, combined with enhancing the expression of Tat secretion system genes and using Tat-dependent signal peptides for heterologous protein secretion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Mdh protein activity is reduced in coryneform bacteria, then secretory production of heterologous proteins is enhanced, but cellular metabolic function may be affected
Solution Approach 1:
The patent applies parameter changes by precisely controlling the degree of Mdh activity reduction through various methods (gene deletion, promoter mutation, RNA interference) to achieve optimal balance between protein secretion enhancement and metabolic function maintenance. Different mutation strengths are tested to find the parameter sweet spot.
Solution Approach 2:
The patent introduces intermediary elements such as Tat-dependent signal peptides and fusion proteins as mediators between the reduced Mdh activity state and the heterologous protein secretion process, enabling efficient protein export while preserving cellular homeostasis.
2Productivity
If Tat secretion system genes are enhanced and Tat-dependent signal peptides are used, then protein secretion efficiency is improved, but system complexity increases
Solution Approach 1:
The patent exploits the universality of the Tat secretion system by using Tat-dependent signal peptides that can direct the secretion of various heterologous proteins through a single, well-characterized pathway, avoiding the need to develop multiple different secretion mechanisms.
Solution Approach 2:
The patent employs preliminary action by pre-fusing Tat-dependent signal peptides to heterologous proteins before secretion, and pre-enhancing Tat system gene expression, so that the secretion machinery is ready and optimized before the actual protein export process begins.
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
Enhances the secretory production of heterologous proteins by improving the bacteria's ability to secrete and accumulate proteins in the culture medium and cell surface layer, simplifying protein purification processes.
Implementation Method 1
a protein secretion pathway completely different from the Sec system has been found in chloroplast thylakoid membranes in plant cells (Non-Patent document 8). This novel secretion pathway has been named the 'Tat system' (Twin-Arginine Translocation system) because of the common presence of arginine-arginine sequences in the signal sequence of a protein secreted by it
Implementation Method 2
Malate dehydrogenase is a redox enzyme that catalyzes the conversion of malic acid to oxaloacetic acid and/or the reverse reaction
Data Source
AI summary
A novel technique for improving secretory production of a heterologous protein by coryneform bacteria is provided, and thereby a method for secretory production of a heterologous protein is provided. A coryneform bacterium having an ability of secretory producing a heterologous protein and having been modified so that an activity of a Mdh protein is reduced is cultured to produce the heterologous protein by secretory production.