Constitutive Promoters for Low Metabolic Burden Bacterial Expression

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Solution Overview

Problem

Current genome editing technologies in bacteria face challenges with high metabolic burden and plasmid yield limitations, particularly when using inducible promoters, which require careful tuning of inducer dosage and timing, and often result in reduced scalability and increased procedural time.

Innovation Solution

Development of a method involving synthetic regulatory nucleic acid molecules that confer reduced constitutive expression when combined with high-copy vectors, allowing for stable and efficient protein expression without significant interference with bacterial growth, using constitutive promoters like Pveg or derivatives, and optimized replication origins to maintain vector copy numbers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of operation

If inducible promoters are used for genome editing in bacteria, then gene expression can be controlled, but metabolic burden increases and plasmid yield is limited

Engineering Contradiction:

Improvegene expression controlVSAvoidplasmid yield

Core Design Contradiction:

Ease of operationVSQuantity of substance

Solution Approach 1:

The patent changes the promoter type from inducible to constitutive, fundamentally altering the expression control mechanism. This parameter change eliminates the need for inducer molecules while maintaining controlled expression through promoter strength selection, thereby resolving the contradiction between expression control and plasmid yield

Inventive Principle:

Principle #35Parameter changes

2Productivity

If strong constitutive promoters are used, then protein expression efficiency increases, but bacterial growth is significantly interfered with

Engineering Contradiction:

Improveprotein expression efficiencyVSAvoidbacterial growth

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies local quality by using different promoter strengths in different contexts - strong constitutive promoters for applications requiring high expression, and weaker constitutive promoters for applications requiring minimal growth interference. This localized selection of promoter characteristics resolves the contradiction between expression efficiency and growth reliability

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent introduces dynamic selection of promoter variants based on experimental needs. Researchers can dynamically choose between different constitutive promoter strengths (strong or weak) depending on whether the priority is expression efficiency or growth maintenance, making the system adaptable to different application requirements

Inventive Principle:

Principle #15Dynamics

3Adaptability or versatility

If inducible promoters are used, then gene expression can be activated on demand, but procedural time increases and scalability is reduced

Engineering Contradiction:

Improveinducible expressionVSAvoidprocedural time

Core Design Contradiction:

Adaptability or versatilityVSLoss of time

Solution Approach 1:

The patent extracts the inducibility feature from the promoter system by replacing inducible promoters with constitutive promoters. This removal of the induction mechanism eliminates the time-consuming steps of inducer addition and timing optimization, while the desired gene expression is achieved directly through the constitutive promoter activity

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentUS20230212593A1Method for the production of constitutive bacterial promoters conferring low to medium expression

Publication Date: 2023.07.06 BASF SE

AI summary

Disclosed herein are methods for the production of low to medium expressing constitutive promoters in bacteria and promoters produced therewith.