Yeast Strain Engineering for High-Level Protein Secretion
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Solution Overview
Problem
Current methods for high-level secretory production of proteins in yeast, especially those with complex structures, face challenges such as protein degradation and the need for high methanol concentrations, which are hazardous and costly, and do not efficiently combine chaperone co-expression for improved productivity.
Innovation Solution
A transformed yeast strain is developed by introducing chaperone genes and disrupting the aoxl gene and protease genes, specifically proteinase B, to enable high-level secretory production of proteins with reduced methanol usage, thereby enhancing safety and industrial viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high concentrations of methanol are used to induce protein expression in yeast, then protein productivity is improved, but safety hazards and operational complexity increase
Solution Approach 1:
The patent changes the chemical parameter by substituting methanol with glucose as the carbon source and inducer. This parameter change maintains protein expression induction capability while eliminating the safety hazards associated with high concentrations of methanol, thus resolving the contradiction between productivity and safety
Solution Approach 2:
The patent employs glucose, a cheap and readily available carbon source, to replace methanol for inducing protein expression. This substitution uses a safe, inexpensive substance that can be easily handled and disposed of, resolving the safety and cost issues while maintaining induction efficiency
2Productivity
If chaperone genes are co-expressed to improve protein folding, then protein secretion efficiency is improved, but system complexity increases
Solution Approach 1:
The patent merges the expression of multiple chaperone genes (PDI1, ERO1, Kar2) into a coordinated expression system under glucose induction. This combining approach enhances protein folding and secretion efficiency while managing system complexity through unified regulatory control
Solution Approach 2:
The patent introduces chaperone genes into the yeast system in advance before protein expression induction. This preliminary action ensures that the molecular chaperones are present and ready to assist protein folding as soon as the target protein is expressed, improving secretion efficiency without requiring complex real-time control mechanisms
3Stability of the object's composition
If protease genes are disrupted to reduce protein degradation, then protein stability is improved, but genetic manipulation complexity increases
Solution Approach 1:
The patent extracts or removes the function of proteases by disrupting their genes (such as PRB1). This extraction of the harmful degradation function prevents protein breakdown, improving protein stability in the culture system while the genetic manipulation is performed once during strain construction
Solution Approach 2:
The patent applies preliminary anti-action by disrupting protease genes before protein expression and secretion. This preemptive measure prevents protein degradation from occurring in the first place, ensuring protein stability throughout the culture process without requiring additional protective measures during production
4Productivity
If multiple gene modifications are made to improve protein production, then productivity is improved, but manufacturing complexity increases
Solution Approach 1:
The patent performs all necessary genetic modifications (chaperone gene introduction, protease gene disruption, aox1 gene disruption) in advance during strain construction. This preliminary action creates a optimized host strain that requires no further complex manipulations during production, simplifying the manufacturing process while maintaining high productivity
Solution Approach 2:
The patent combines multiple gene modifications into a single integrated strain construction process. By merging the introduction of chaperone genes, disruption of protease genes, and disruption of aox1 gene into one comprehensive genetic engineering approach, the patent achieves high protein production while managing manufacturing complexity through unified strain development
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 allows for high-level production of correctly folded proteins with complex structures, significantly reducing methanol requirements and inhibiting protease activity, making the process safer and more suitable for industrial-scale production.
Implementation Method 1
When secretory proteins pass through the translocon, the higher-order structures thereof are loosened, and the proteins are folded in the endoplasmic reticulum. While secretory protein folding is able to spontaneously occur, various molecular chaperones assist such folding.
Implementation Method 2
covalent bonds between sulfur atoms that are formed upon two-electron oxidation of two cysteines (which are referred to as 'disulfide bonds') play very important roles in stabilizing protein steric structure
Implementation Method 3
a protease existing in a vacuole that is known as a protein-degrading organelle of yeast is reported to be associated with secretory protein degradation
Data Source
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AI summary
The object of the present invention is to provide a production system that is capable of high-level secretory production of a protein (and in particular, a protein with a complicated structure such as a structure with S-S bonds) in a host cell such as yeast and is suitable for industrial production with high safety that does not require explosion-proof facilities. The present invention provides a transformed yeast into which a chaperone gene has been introduced and in which the aox1 gene and/or the protease gene have been disrupted and a method for producing a protein involving the use of such transformed yeast.