Mutant RNase E mRNA Stabilization for Toxic Protein Expression

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Solution Overview

Problem

Traditional recombinant protein expression systems, particularly in E. coli, face challenges with impaired growth and fitness loss when producing toxic proteins, lacking clear guiding principles for gene and cell factory optimization.

Innovation Solution

A microbial host cell with a mutant RNase E enzyme, engineered to have decreased activity through specific amino acid substitutions, is co-expressed with a target gene on an auxiliary plasmid to modulate RNase activity, enhancing recombinant protein expression, especially of toxic proteins.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional E. coli expression systems are used for recombinant protein production, then high cell density and fast growth are achieved, but impaired growth and fitness loss occur when producing toxic proteins

Engineering Contradiction:

Improverecombinant protein expression levelsVSAvoidbacterial growth and fitness

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention modifies the RNase E enzyme through site-directed mutagenesis, changing specific amino acid residues (D303N, D346N, E297Q) to alter its catalytic activity. This parameter change in enzyme function allows reduced RNA degradation of target proteins while maintaining cell viability, resolving the contradiction between protein expression levels and bacterial growth health.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The mutant RNase E acts as an intermediary element that mediates between the toxic recombinant protein and the cellular RNA degradation machinery. By expressing the mutant enzyme, the system creates a buffering layer that selectively protects target mRNA from degradation without completely shutting down RNA turnover, thus improving protein expression while maintaining cellular homeostasis.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If T7 RNA polymerase activity is increased to enhance gene expression, then protein production increases, but toxicity of membrane protein production worsens

Engineering Contradiction:

Improvegene expression levelsVSAvoidmembrane protein toxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The invention converts the harmful effect of high T7 RNA polymerase activity (which causes toxic mRNA accumulation) into a beneficial outcome. By introducing mutant RNase E with reduced catalytic activity, the system allows high-level transcription to proceed while preventing the detrimental accumulation of unstable mRNA, thereby transforming the toxic effect into enhanced protein production without cellular damage.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Stability of the object's composition

If RNA degradation is enhanced to control gene expression, then mRNA turnover is improved, but recombinant protein expression levels decrease

Engineering Contradiction:

ImprovemRNA turnover controlVSAvoidrecombinant protein expression

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The mutant RNase E exhibits local quality differentiation in its substrate recognition and degradation properties. The amino acid substitutions (particularly D303N and D346N in the catalytic domain) create a enzyme variant that selectively reduces degradation of specific target mRNAs while maintaining overall RNA turnover control. This localized modification in enzymatic activity allows simultaneous achievement of mRNA stability control and enhanced protein expression.

Inventive Principle:

Principle #3Local quality

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

The mutant RNase E enzyme acts as a competitive inhibitor, reducing RNA degradation and improving the expression levels of toxic proteins, overcoming the limitations of traditional systems by stabilizing mRNA and enhancing protein production.

Implementation Method 1

The mutant RNase E enzyme acts as a competitive inhibitor, reducing RNA degradation and improving the expression levels of toxic proteins

Methodology Applied

Scientific EffectCompetitive inhibition:

Implementation Method 2

RNase E is an essential membrane-associated enzyme in E. coli involved in the maturation of both ribosomal RNA and tRNA, as well as total mRNA decay

Methodology Applied

Scientific EffectEnzyme activity: Enzyme

Data Source

PatentUS20250263679A1Mutant rnase e for enhancing recombinant protein expression

Publication Date: 2025.08.21 DANMARKS TEKNISKE UNIV

AI summary

The invention provides a microbial host cell for enhanced recombinant expression of a target protein, said host cell comprising a mutant RNase E enzyme to be coexpressed with a target gene of interest. The invention further provides a method of enhancing recombinant protein expression using said microbial host cell. The method is particularly useful for the expression of proteins that are otherwise difficult to express in traditional expression systems, such as proteins which are toxic to the host cell. The invention further provides an auxiliary plasmid comprising a rne* gene encoding a mutant RNase E enzyme and a LysS gene encoding T7 lysozyme.