Antibiotic-Free Polypeptide Expression via Toxin-Antitoxin Plasmid Stability

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current biotechnological methods for producing polypeptides rely heavily on antibiotic-resistance genes for selection, which are costly and contribute to antibiotic resistance in pathogenic strains, and existing systems struggle to maintain high copy numbers of genes in industrially complex media without selection pressure.

Innovation Solution

An expression system using extrachromosomal DNA with a glycerine-3-phosphate dehydrogenase gene as a selection marker, eliminating antibiotic-resistance genes and maintaining high plasmid copy numbers through episomal complementation in auxotrophic host cells, allowing polypeptide production without antibiotics.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If antibiotic-resistance genes are used for selection of host cells, then plasmid stability and high copy numbers are maintained, but ecological harm increases and production costs rise

Engineering Contradiction:

Improveplasmid stabilityVSAvoidecological harm from antibiotic resistance

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and removes the antibiotic-resistance gene from the plasmid, replacing it with a toxin-antitoxin selection system. The plasmid contains a toxin gene (relE) and an antitoxin gene (relA) where the plasmid itself provides the protective function, eliminating the need for separate antibiotic-resistance markers while maintaining plasmid stability through the toxin-antitoxin mechanism.

Inventive Principle:

Principle #2Taking out (Extraction)

2Reliability

If antibiotic-resistance genes are used for selection, then plasmid copy numbers are maintained, but production costs increase due to antibiotic addition

Engineering Contradiction:

Improveplasmid copy number maintenanceVSAvoidproduction cost

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The plasmid becomes self-regulating through the toxin-antitoxin system. When plasmid copy number decreases, the antitoxin protein degrades faster than the toxin, allowing the toxin to accumulate and kill the cell. This self-service mechanism maintains plasmid copy number without requiring external antibiotics or resistance genes, reducing production costs.

Inventive Principle:

Principle #25Self-service

3Productivity

If extrachromosomal DNA is used for polypeptide production, then high yield is achieved, but plasmid loss occurs without antibiotic selection pressure

Engineering Contradiction:

Improvepolypeptide yieldVSAvoidplasmid retention

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The invention introduces a toxin-antitoxin system as an intermediary mechanism between the plasmid and the host cell. The toxin (relE) and antitoxin (relA) proteins act as mediators that regulate plasmid maintenance: the antitoxin protects the cell from the toxin's lethal effect, and this protection is contingent on plasmid presence, thereby ensuring plasmid retention without antibiotic selection.

Inventive Principle:

Principle #24Intermediary (Mediator)

4Measurement precision

If antibiotic markers are used for selection, then successful transformation is detected, but the system contributes to antibiotic resistance in pathogenic strains

Engineering Contradiction:

Improvetransformation detectionVSAvoidantibiotic resistance spread

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

Instead of using antibiotic resistance to detect successful transformation, the invention inverts the approach by using a toxin-antitoxin system where the plasmid provides protection against cell death. Successful transformation is detected by the cell's survival in the presence of the toxin, rather than by resistance to external antibiotics, thereby eliminating the contribution to antibiotic resistance spread.

Inventive Principle:

Principle #13The other way round (Inversion)

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

Enables high-yield polypeptide production without antibiotic markers, maintaining plasmid stability in industrial media, and reducing ecological concerns associated with antibiotic use.

Implementation Method 1

genes that code glycerine-3-phosphate dehydrogenase (also named NAD(P)H-dependent dihydroxyacetone phosphate reductase...), and, thus, the production of the desired polypeptide does not need the addition of antibiotics

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

NAD(P)H-dependent dihydroxyacetone phosphate reductase... L-glycerine-3-phosphate: NAD(P) oxidoredutase

Methodology Applied

Scientific EffectRedox reaction: Redox Reactions

Data Source

PatentUS8465946B2Expression system for the antibiotic-free production of polypeptides

Publication Date: 2013.06.18 AB ENZYMES GMBH

AI summary

The present invention relates to a microbial expression system for the production of polypeptides based on the use of extrachromosomal DNA, whereby no antibiotic marker genes for the selection of the host cell but DNA sequences that encode glycerine-3-phosphate dehydrogenase are used, and, thus, the production of the desired polypeptide, e.g., xylanase, does not need the addition of antibiotics. The expression system is free from antibiotic-resistance genes. The invention further relates to a DNA sequence that encodes a polypeptide with glycerine-3-phosphate dehydrogenase activity as well as a polypeptide with glycerine-3-phosphate dehydrogenase activity.