Chromosomal Nucleic Acid Construct for Stable Recombinant Protein Expression

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Solution Overview

Problem

Current bacterial expression systems, such as plasmid-based and genome-based systems, face limitations in yield and controllability for industrial-scale production of recombinant proteins due to high metabolic burden, segregational and structural instability, and complex regulation, leading to low quality and yield of recombinant proteins.

Innovation Solution

A nucleic acid construct comprising a synthetic non-coding DNA sequence derived from the 5'-untranslated region of Escherichia coli glp genes, specifically the glpF, glpA, or glpD genes, combined with a promoter DNA sequence and a coding DNA sequence, which enhances gene expression stability and independence from promoter strength, allowing for high-yield production of biological molecules.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If plasmid-based expression systems are used to achieve high gene dosage, then gene expression level is improved, but metabolic burden on host cells increases and stability deteriorates

Engineering Contradiction:

Improvegene expression levelVSAvoidsystem stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The invention separates the high gene dosage function from the plasmid replication mechanism by integrating multiple copies of the gene of interest directly into the bacterial chromosome, eliminating the need for plasmid replication while maintaining high expression levels through chromosomal integration

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention merges the gene of interest with the bacterial chromosome by integrating it at a specific attB site, combining the stability of chromosomal inheritance with the high expression capability through multiple integrated copies, thereby achieving both reliability and productivity

Inventive Principle:

Principle #5Merging (Combining)

2Productivity

If strong promoters are used in combination with high gene dosage to increase protein formation rate, then productivity is improved, but host cell metabolism breaks down

Engineering Contradiction:

Improveprotein formation rateVSAvoidmetabolic burden

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The invention changes the parameter of gene copy number by integrating multiple copies of the gene into the chromosome, achieving high productivity through increased gene dosage without requiring strong promoters that would overload cell metabolism

Inventive Principle:

Principle #35Parameter changes

3Productivity

If plasmid copy number is increased to improve gene dosage, then expression level is improved, but controllability of the process deteriorates

Engineering Contradiction:

Improveexpression levelVSAvoidprocess controllability

Core Design Contradiction:

ProductivityVSEase of operation

Solution Approach 1:

Instead of increasing plasmid copy number to achieve high gene dosage, the invention inverts the approach by integrating multiple gene copies directly into the chromosome, where copy number is controlled by the integration process itself rather than plasmid replication dynamics

Inventive Principle:

Principle #13The other way round (Inversion)

Data Source

PatentUS11608504B2Nucleic acid construct for in vitro and in vivo gene expression

Publication Date: 2023.03.21 GLYCOM AS

AI summary

The present invention relates to the field of recombining production of biological molecules in host cells. The invention provides nucleic acid constructs that allow to modify expression of a desired gene using both in vitro and in vivo gene expression systems. The constructs can advantageously be used to produce a variety of biological molecules recombinantly in industrial scales, e.g. human milk oligosaccharides (HMO).