Bacillus Fermentation Sulfate Precipitation for Stable Protein Yield
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Solution Overview
Problem
Existing methods struggle to stabilize and increase the yield of native or wild-type enzymes prone to (auto)hydrolysis or instability during large-scale industrial fermentation processes in Bacillus host cells, without affecting the viability of the host cells.
Innovation Solution
A method involving inoculation of a fermentation medium with Bacillus host cells containing a gene of interest under a promoter, adding sulfate to achieve at least 20 mM concentration, and optionally including an amino acid derivative, to allow protein precipitation and/or crystallization under conditions conducive to host cell growth and protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high titers of protein of interest are achieved through increased protein expression, then protein yield is improved, but protein stability deteriorates due to aggregation and inactivation
Solution Approach 1:
The invention changes the chemical composition parameters of the fermentation medium by adding sulfate (to at least 20 mM) and amino acid derivatives (0.1-30 g/L), which fundamentally alters the protein solubility and stability characteristics during high-titer production
Solution Approach 2:
Sulfate acts as an intermediary substance that mediates between high protein expression and protein stability by inducing precipitation of the protein of interest, thereby protecting it from aggregation and inactivation while maintaining high yield
2Stability of the object's composition
If protein precipitation is induced to stabilize the protein, then protein stability is improved, but host cell viability may be affected
Solution Approach 1:
The invention carefully controls the chemical parameters (sulfate concentration ≥20 mM, amino acid derivative 0.1-30 g/L, pH, temperature) to induce protein precipitation while maintaining conditions that are non-toxic to host cells
Solution Approach 2:
The invention converts the potentially harmful effect of high protein concentration (which causes aggregation and inactivation) into a beneficial precipitation process that stabilizes the protein while maintaining host cell viability through controlled chemical conditions
3Productivity
If conventional media composition is used for high protein production, then productivity is improved, but protein stability deteriorates due to lack of stabilization agents
Solution Approach 1:
The invention creates a composite fermentation medium system combining sulfate, amino acid derivatives, and conventional nutrients, which synergistically provides both high productivity and protein stability
Solution Approach 2:
Sulfate and amino acid derivatives serve as intermediary stabilization agents that are added to conventional media to enable high-titer production while maintaining protein stability through precipitation
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
Stabilizes protein production by precipitation, maintaining host cell viability and increasing yield, facilitating simple purification of the protein of interest.
Implementation Method 1
adding sulfate to the fermentation medium to reach a concentration of at least 20 mM of sulfate in the fermentation medium; and d) allowing the protein of interest to precipitate and/or crystallize during cultivation
Implementation Method 2
allowing the protein of interest to precipitate and/or crystallize during cultivation
Data Source
AI summary
The present invention relates to the field of industrial fermentation and protein production. In particular, it relates to a method for producing a protein of interest in a fermentation medium comprising the following steps a) inoculating a fermentation medium with a Bacillus host cell comprising a gene encoding a protein of interest under the control of a promoter; b) cultivating the Bacillus host cell in the fermentation medium under conditions conducive for the growth of the Bacillus host cell and the expression of the protein of interest, c) adding sulfate to the fermentation medium to reach a concentration of at least 20 mM of sulfate in the fermentation medium; and d) allowing the protein of interest to precipitate and/or crystallize during cultivation; wherein the fermentation medium comprises an amino acid derivative in an amount of 0-30 g/l of fermentation medium. Further contemplated is the use of a combination of a sulfate and an amino acid derivative for producing a protein of interest in Bacillus host cell in a fermentation medium and a crystallized protein of interest obtained by or obtainable by the method of the invention.