Cutinase Mutant Promotes Extracellular Protein Secretion in Bacillus subtilis
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Solution Overview
Problem
Current methods for extracellular expression of intracellular proteins in Bacillus subtilis are inefficient, as they do not effectively promote the secretion of proteins outside the cells, unlike in E. coli systems, due to differences in cell membrane composition, limiting their application in industrial protein production.
Innovation Solution
A cutinase mutant with specific amino acid mutations at positions 175, 177, 178, 207, 209, 213, and 214 is co-expressed with intracellular proteins in Bacillus subtilis to enhance membrane permeability, facilitating extracellular secretion without compromising cell integrity, using vectors like pHY300PLK and plasmids such as pHY300PLK-xylA-cut, pHY300PLK-L175A/T177A, and pHY300PLK-I213A/P214A.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If cutinase is co-expressed with intracellular target protein in B. subtilis, then membrane permeability is enhanced, but extracellular secretion efficiency is insufficient
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (Phe175, Thr177, Ile178, Thr207, Phe209, Ile213, Pro214) in the cutinase sequence to optimize its function. These parameter changes at the molecular level transform the cutinase from having limited effect on B. subtilis membrane permeability to effectively promoting extracellular secretion of intracellular proteins while maintaining cell integrity.
Solution Approach 2:
The mutated cutinase acts as an intermediary substance that mediates between the intracellular environment and extracellular space. It facilitates the transfer of intracellular proteins to the extracellular environment by temporarily increasing membrane permeability, then restoring membrane integrity, thus enabling efficient secretion without direct cell lysis.
2Object-affected harmful factors
If cutinase hydrolyzes phospholipid component of cell membrane, then membrane permeability increases, but cell membrane integrity may be compromised
Solution Approach 1:
The mutated cutinase applies partial action by selectively and temporarily hydrolyzing only sufficient phospholipid components to achieve the desired level of membrane permeability for protein secretion. It avoids excessive hydrolysis that would compromise cell membrane integrity, thus achieving the optimal balance between permeability enhancement and cell viability maintenance.
Solution Approach 2:
The patent converts the potentially harmful effect of membrane hydrolysis into a beneficial outcome. The controlled phospholipid hydrolysis by mutated cutinase, which could otherwise damage cell membranes, is transformed into a useful mechanism for enhancing membrane permeability to facilitate protein secretion, with the mutated enzyme subsequently restoring membrane integrity.
3Productivity
If intracellular proteins are expressed inside cells, then protein production is achieved, but downstream purification becomes cumbersome and costly
Solution Approach 1:
The patent applies the extraction principle by removing intracellular proteins from the cell interior to the extracellular environment using mutated cutinase. This extraction process simplifies downstream purification because proteins are already secreted into the fermentation broth, eliminating the need for cell disruption and complex extraction steps, thereby reducing manufacturing costs and complexity.
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
This method successfully achieves extracellular expression of enzymes like xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, and branching enzyme, simplifying downstream purification and reducing production costs, with enhanced enzyme activity levels in fermentation supernatants.
Implementation Method 1
cutinase can hydrolyze the phospholipid component of the cell membrane to a certain extent and increase the permeability of the cell membrane
Data Source
AI summary
Disclosed is a method for promoting extracellular expression of proteins in B. subtilis using cutinase, which belongs to the technical fields of genetic engineering, enzyme engineering and microbial engineering. It teaches co-expressing a cutinase mutant and a target protein in B. subtilis to promote extracellular expression of the target protein which is naturally located inside cells. The target protein includes xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, branching enzyme and the like. The invention can achieve extracellular expression of intracellularly localized target protein, improve the production efficiency, reduce the production cost and simplify the subsequent extraction process.