Two-Stage Fermentation for Disulfide Proteins in E. coli
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Solution Overview
Problem
E. coli's reducing cytoplasmic environment hampers the soluble expression of proteins with cysteine residues, leading to misfolding and aggregation due to disulfide bond reduction, and previous strain modifications either induce toxicity or require specialized growth conditions.
Innovation Solution
A two-stage fermentation approach is employed to dynamically control the cytoplasmic redox state by maintaining a reducing environment during growth and shifting to an oxidative environment for protein production, utilizing chromosomal deletions and conditional overexpression of disulfide bond enzymes like dsbC and evr1p to facilitate proper folding of heterologous proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If constitutive oxidative stress is applied to improve disulfide bond formation, then protein folding is improved, but growth rate decreases and expression conditions are limited
Solution Approach 1:
The patent implements dynamic control of cytoplasmic redox state by transitioning from a reducing environment during growth phase to an oxidative environment during production phase. This is achieved through conditional expression of reductase enzymes (trxB, gor) using inducible promoters, allowing the system to adapt redox conditions to different physiological stages rather than maintaining constitutive oxidation throughout
Solution Approach 2:
The patent divides the fermentation process into distinct growth phase and production phase, with each phase having optimized redox conditions. The growth phase maintains reducing conditions for cell proliferation, while the production phase switches to oxidative conditions for disulfide bond formation, allowing independent optimization of each phase without mutual interference
2Reliability
If reductase deletions are made to increase oxidative potential, then disulfide bond formation is improved, but toxicity increases and growth is slowed
Solution Approach 1:
Instead of completely deleting reductase genes, the patent uses conditional expression control to dynamically adjust reductase activity levels. The reductase enzymes are expressed only when needed (during phase transition) rather than constitutively, changing the temporal parameter of enzyme availability to achieve oxidative conditions without permanent genetic damage or toxicity
3Reliability
If specialized growth conditions are applied to limit oxidative stress, then cell viability is maintained, but expression efficiency decreases
Solution Approach 1:
The patent dynamically switches redox conditions between growth and production phases rather than maintaining specialized conditions throughout. During growth, standard conditions maintain viability; during production, induced oxidative conditions maximize expression efficiency, eliminating the need for continuous specialized conditions that would limit overall productivity
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 enables robust and scalable expression of disulfide-containing proteins, improving their solubility and reducing toxicity, while maintaining exponential growth and oxidative conditions for proper folding, as demonstrated with proteins like human hyaluronidase-I and nanobodies.
Implementation Method 1
maintains a reducing cytosolic environment that is comparable to the reducing cytosolic environment of a microorganism lacking genetic modifications
Implementation Method 2
the synthetic metabolic valve(s) resulting in a shift to an oxidative cytosolic environment, and inducing expression or overexpression of the heterologous protein to result in proper folding of the heterologous protein product in an oxidative environment
Implementation Method 3
dsbC isomerizes disulfide bonds to improve correct folding when multiple disulfide bonds are present
Implementation Method 4
evr1p catalyzes cysteine oxidation and dsbC isomerizes disulfide bonds to improve correct folding when multiple disulfide bonds are present
Data Source
AI summary
Methods and microorganism for expression of a protein requiring at least one disulfide bond for proper folding of the protein are described. The biofermentation methods comprise growth of a microorganism that may conditionally expressing the protein in addition to at least one synthetic metabolic valve designed to regulate of at least one enzyme effective for altering the redox characteristics of the cytosolic environment of the genetically modified microorganism. In a product producing step of the method, an oxidative cytosolic environment is conditionally provided.