Recombinant Nuclease Expression in Host Cells
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Solution Overview
Problem
Existing recombinant host cells used for producing enzymes often face challenges with contaminating DNA in the final enzyme product, which can lead to regulatory issues and reduced productivity due to high viscosity in cell lysates.
Innovation Solution
Engineering recombinant Bacillus host cells to express a recombinant nuclease using the pstS promoter, which is regulated by phosphate levels, allowing for efficient degradation of DNA during fermentation, thereby separating enzyme expression from DNA contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant host cells are used for enzyme production, then productivity is improved, but contaminating DNA is present in the final product
Solution Approach 1:
The nuclease gene is integrated into the host cell genome along with the enzyme gene, enabling DNA degradation capability to be established before fermentation begins. The promoter is designed to be activated at the end of fermentation when DNA contamination becomes a problem, allowing preliminary preparation without immediate DNA degradation that would interfere with enzyme production.
Solution Approach 2:
The system uses a phosphate-responsive promoter that dynamically changes its activity based on phosphate availability. During early fermentation when phosphate is abundant, the promoter is repressed and nuclease is not expressed. When phosphate is depleted at the end of fermentation, the promoter is activated and nuclease expression increases, automatically adapting to the fermentation stage.
2Manufacturing precision
If nuclease is expressed during fermentation, then DNA contamination is reduced, but enzyme productivity decreases due to interference
Solution Approach 1:
The nuclease expression is implemented as a periodic action rather than continuous expression. The phosphate-responsive promoter ensures nuclease is only expressed during the phosphate-depleted phase at the end of fermentation, creating a temporal separation between enzyme production (early phase) and DNA degradation (late phase), thus avoiding interference with enzyme productivity.
3Manufacturing precision
If constitutive nuclease expression is used, then DNA degradation is continuous, but phosphate resources are wasted during growth phase
Solution Approach 1:
The system implements feedback control through the phosphate-responsive promoter that senses intracellular phosphate levels. When phosphate is abundant during growth, the promoter remains repressed and nuclease is not expressed. When phosphate becomes depleted, the repression is lifted and nuclease expression is activated, creating a feedback mechanism that responds to phosphate status and prevents unnecessary phosphate consumption during the growth phase.
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 approach enables the production of enzymes free from contaminating DNA, improving fermentation efficiency and compliance with regulatory standards by controlling nuclease expression to specifically target and degrade DNA at the end of fermentation.
Implementation Method 1
The pstS promoter is regulated by the level of phosphate in the medium during fermentation in a way where the promoter is activated by low levels of phosphate and blocked by high levels of phosphate
Implementation Method 2
The gene nucB coding for this extracellular DNase (nuclease) from B. subtilis and B. licheniformis was cloned downstream of the pstS promoter
Implementation Method 3
efficient degradation of DNA
Data Source
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AI summary
The present invention relates to cells producing at least one polypeptide of interest and expressing one or more recombinant nuclease encoding gene(s) thereby producing the nuclease(s), and methods for producing a polypeptide of interest essentially free from contaminating DNA, said method comprising the steps of: (a) cultivating a cell that produces at least one polypeptide of interest and expresses one or more recombinant nuclease encoding gene(s) thereby producing the nuclease(s); and (b) isolating the polypeptide of interest.