Dynamic Protease Control for Robust Protein Expression
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Solution Overview
Problem
Recombinant protein expression in E. coli is challenging for 'hard to express' proteins, such as toxic, slow-folding, and large proteins, which often result in reduced growth and expression due to competition with cellular growth and potential cellular toxicity, necessitating the use of alternative expression hosts that are costly and time-consuming to develop expertise in.
Innovation Solution
Development of engineered E. coli strains with controlled expression of key housekeeping proteases through CRISPR-based gene silencing and controlled protein degradation, allowing for two-stage dynamic control of protease activity, particularly in the stationary phase, to improve protein expression while minimizing the negative impacts of complete protease deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If protease activity is completely deleted to prevent protein degradation, then protein expression is improved, but cellular growth and essential functions are severely impacted
Solution Approach 1:
The invention employs dynamic control of protease activity through conditional deletion strategies and inducible expression systems. Protease genes are deleted or inhibited only under specific conditions (e.g., stationary phase, presence of inducer) rather than constitutively, allowing the system to adapt between growth and production phases. This dynamic approach enables high protein expression when needed while preserving cellular functions during growth phases.
Solution Approach 2:
The invention segments the protease control strategy by targeting specific proteases (Lon, FtsH, HslUV) rather than all proteases globally. This selective approach allows degradation of non-essential proteases that interfere with protein expression while preserving essential proteases needed for cellular growth and homeostasis, thus resolving the contradiction between protein stability and cellular health.
2Productivity
If alternative expression hosts are used to express difficult proteins, then protein expression capability is improved, but development time and cost increase significantly
Solution Approach 1:
The invention modifies parameters within the E. coli system (protease activity levels, growth phase timing, induction conditions) to achieve expression capabilities previously requiring alternative hosts. By changing the protease activity parameter dynamically and optimizing growth phase parameters, the system can express difficult proteins including toxic and large proteins without switching to complex alternative expression systems.
3Productivity
If protease activity is reduced to protect slow-folding proteins, then protein maturation is improved, but cellular protein quality control may be compromised
Solution Approach 1:
The invention selectively targets specific proteases (Lon, FtsH, HslUV) for deletion or inhibition rather than globally reducing all protease activity. This segmentation allows protection of slow-folding proteins from degradation by specific proteases while preserving other proteases that maintain cellular protein quality control, thus resolving the contradiction between protein maturation and quality control.
Data Source
AI summary
Provide herein are engineered microbial strains having greatly improved expression of many challenging protein product. This system relies on controlled expression or dynamic reduction in activity of key housekeeping proteases. Dynamic control, implemented with CRISPR based gene silencing and or controlled protein degradation enables increased protein expression while minimizing the negative impact of complete deletions of these housekeeping enzymes.