Corynebacterium Constitutive Promoter Screening via Transcriptome Sequencing

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Solution Overview

Problem

Current methods for screening constitutive promoters in Corynebacterium are complex and lead to plasmid loss and adverse effects on host cell growth and metabolism, particularly during the stationary phase of bacterial growth.

Innovation Solution

A method using transcriptome sequencing to identify promoters with low activity in the logarithmic phase and high activity in the stationary phase, incorporating green fluorescent protein as a marker to evaluate promoter activity and plasmid stability, resulting in a Corynebacterium constitutive expression vector with enhanced stability and expression efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If constitutive promoters with strong activity are used to continuously express foreign genes, then expression efficiency is improved, but plasmid loss increases and host cell growth is adversely affected

Engineering Contradiction:

Improveexpression efficiencyVSAvoidplasmid stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies dynamics by creating a phase-dependent promoter system that automatically adjusts its activity based on bacterial growth phase. The promoter is designed to be weak during logarithmic growth (supporting host growth) and strong during stationary phase (maximizing expression), allowing the system to adapt its expression strength dynamically rather than maintaining constant strong expression.

Inventive Principle:

Principle #15Dynamics

Solution Approach 2:

The patent changes the operational parameter of promoter activity based on growth phase. By identifying promoter sequences that naturally exhibit low activity in logarithmic phase and high activity in stationary phase through transcriptome sequencing, the system transitions from a static strong promoter to a dynamically regulated promoter whose activity parameter changes with physiological conditions.

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If constitutive promoters are used to simplify the operation process, then ease of operation is improved, but host cell metabolism and growth are adversely affected

Engineering Contradiction:

Improveoperation simplicityVSAvoidhost cell growth inhibition

Core Design Contradiction:

Ease of operationVSObject-affected harmful factors

Solution Approach 1:

The system maintains ease of operation by using a constitutive promoter structure that requires no external induction, while internally regulating activity based on growth phase. The promoter automatically transitions from low to high activity as bacteria enter stationary phase, eliminating the need for complex induction protocols while avoiding growth inhibition during the logarithmic phase.

Inventive Principle:

Principle #15Dynamics

Solution Approach 2:

The promoter exhibits periodic action by being weak during the logarithmic growth phase (allowing host growth) and strong during the stationary phase (maximizing product expression). This temporal separation of growth and production phases simplifies operation compared to inducible systems while protecting host cell health.

Inventive Principle:

Principle #19Periodic action

3Quantity of substance

If strong promoter activity is maintained throughout bacterial growth, then expression level is improved, but plasmid loss rate increases

Engineering Contradiction:

Improvetarget protein expression levelVSAvoidplasmid loss rate

Core Design Contradiction:

Quantity of substanceVSLoss of substance

Solution Approach 1:

The patent uses dynamics by designing a promoter that is weak during logarithmic growth (when plasmid loss is problematic) and strong during stationary phase (when expression is needed). This temporal differentiation allows high expression levels to be achieved without maintaining strong promoter activity throughout the entire growth cycle, thereby reducing plasmid loss.

Inventive Principle:

Principle #15Dynamics

Solution Approach 2:

The system performs preliminary action by allowing bacteria to grow and stabilize plasmids during the logarithmic phase with weak promoter activity, before switching to strong expression in the stationary phase. This sequencing of events prevents plasmid loss that would occur if strong expression were attempted during active growth.

Inventive Principle:

Principle #10Preliminary action

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 method ensures low target protein expression in the logarithmic phase and high expression in the stationary phase, reducing plasmid loss and adverse effects on host bacteria, making it suitable for amino acid fermentation and increasing acid yield and conversion rates.

Implementation Method 1

incorporating green fluorescent protein as a marker to evaluate promoter activity and plasmid stability

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS11639512B2<i>Corynebacterium </i>constitutive expression vector promoter screened on the basis of transcriptome sequencing, screening method thereof, and applications thereof

Publication Date: 2023.05.02 WUHAN GRAND HOYO CO LTD

AI summary

Provided is a method for screening a Corynebacterium constitutive expression vector promoter on the basis of transcriptome sequencing; and further provided are the Corynebacterium constitutive expression vector promoter screened on the basis of transcriptome sequencing, an expression vector comprising the promoter, a recombination strain obtained by transforming a host cell Corynebacterium glutamicum using the expression vector, and applications thereof.