Psychrotolerant Yeast Expression System for Thermolabile Protein Stability
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Solution Overview
Problem
Current yeast expression systems, particularly those using mesophilic or thermotolerant organisms, are not optimal for producing thermolabile proteins due to high thermal lability and potential inactivation at cultivation temperatures, leading to incorrect protein folding and degradation.
Innovation Solution
A psychrotolerant strain of Debaryomyces macquariensis yeast, specifically strain D50, is used in conjunction with a plasmid expression vector containing an expression cassette with specific promoters and terminators, enabling constitutive expression and stable production of heterologous proteins, including thermolabile ones, at lower temperatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mesophilic or thermotolerant yeast expression systems are used, then high biomass production and fast growth are achieved, but thermolabile proteins are inactivated and incorrectly folded due to high cultivation temperatures
Solution Approach 1:
The patent changes the fundamental parameter of cultivation temperature by selecting a psychrotolerant yeast strain (Debaryomyces macquariensis) that can grow at low temperatures (15-25°C). This allows the expression system to maintain high productivity while producing thermolabile proteins that would be inactivated at higher temperatures, thus resolving the contradiction between fast growth and protein stability.
2Speed
If high cultivation temperatures are used, then fast cell metabolism and protein production speed are achieved, but protein aggregation and degradation increase
Solution Approach 1:
The patent changes the temperature parameter to low cultivation conditions (15-25°C) using psychrotolerant yeast, which maintains adequate metabolic activity while significantly reducing protein aggregation and degradation. This resolves the contradiction between fast metabolism and reduced protein aggregation by operating in a temperature regime where both can coexist.
3Productivity
If thermolabile proteins are produced in mesophilic yeast, then heterologous gene expression is achieved, but correct protein folding is compromised due to thermal inactivation
Solution Approach 1:
The patent changes the cultivation temperature parameter to low temperatures (15-25°C) using psychrotolerant yeast, which prevents thermal inactivation of thermolabile proteins during expression. This ensures correct protein folding and structural integrity while maintaining high heterologous protein production, thus resolving the contradiction between productivity and folding accuracy.
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AI summary
Expression system for production of heterologous protein, wherein the yeast cells are used as expression host for a heterologous gene, wherein the system comprises a psychrotolerant yeast strain of the Debaryomyces macquariensis species, preferably Debaryomyces macquariensis strain D50, accession number KKP 2066p and a plasmid expression vector comprising an expression cassette for expression of a gene coding a heterologous protein that contains a GAP promoter isolated from Debaryomyces macquariensis and a CYC1 terminator isolated from Debaryomyces macquariensis. In addition, the plasmid expression vector comprises an expression cassette for expression of an antibiotic resistance gene.