Bacterial Host Cell Plasmid Stability via Alanine Racemase
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Recombinant production hosts in bacterial fermentation processes face challenges with plasmid instability and metabolic burden, leading to reduced productivity due to outgrowth of wild-type cells and loss of plasmids, especially under conditions of high compound production, where antibiotic resistance markers are ineffective and auxotrophic markers can be detrimental to cell growth.
Innovation Solution
Inactivating both chromosomal genes encoding alanine racemases in a bacterial host cell and introducing a plasmid with an autonomous replication sequence, a polynucleotide encoding a polypeptide of interest, and a third alanine racemase operably linked to a promoter, to enhance plasmid stability and polypeptide expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotic resistance markers are used for plasmid selection, then plasmid maintenance is achieved, but the system becomes ineffective under long-term cultivation due to antibiotic degradation and dilution, and requires additional purification steps
Solution Approach 1:
The patent removes antibiotic resistance markers from the plasmid system and replaces them with an auxotrophic marker (purF gene). This extraction of the harmful antibiotic dependency while retaining plasmid maintenance functionality resolves the contradiction between reliable plasmid maintenance and productivity.
Solution Approach 2:
The patent uses a metabolic pathway (purine biosynthesis) that is naturally essential for cell survival but can be supplemented in the growth medium. The auxotrophic marker provides continuous selective pressure without requiring persistent antibiotic presence, effectively replacing a long-lived selection mechanism with a metabolically-based one that works throughout cultivation.
2Reliability
If auxotrophic markers are used on multi-copy plasmids for positive selection, then plasmid maintenance is achieved, but cell growth and productivity are negatively impacted due to unbalanced enzymatic function
Solution Approach 1:
The patent applies local quality by providing the purF gene product (phosphoribosylformylglycinamidine synthase) specifically where needed - in plasmid-carrying cells - to balance the purine biosynthesis pathway. This localized supplementation allows auxotrophic selection without globally disrupting cellular metabolism, resolving the contradiction between plasmid maintenance and cell growth.
Solution Approach 2:
The patent changes the selection mechanism from antibiotic-based to metabolism-based, utilizing the purine biosynthesis pathway. By modifying the metabolic state of the cell (creating a purine auxotroph) rather than imposing external chemical selection, the system achieves plasmid maintenance while minimizing impact on overall cellular physiology and productivity.
3Stability of the object's composition
If chromosomal genes are deleted to create auxotrophic markers, then plasmid stability is improved, but the system becomes less effective when cell lysis occurs due to cross-feeding
Solution Approach 1:
The patent uses D-alanine as an intermediary substance that mediates between plasmid stability and auxotrophic marker effectiveness. The dal gene on the plasmid encodes D-alanine racemase, which produces D-alanine to compensate for the deleted chromosomal dal gene. This intermediary metabolite ensures continuous selection pressure even when cell lysis occurs, maintaining both plasmid stability and marker reliability.
4Productivity
If recombinant production hosts are genetically modified for high-level compound production, then productivity is improved, but fitness decreases leading to outgrowth of wild-type cells and loss of plasmids
Solution Approach 1:
The patent applies preliminary action by pre-modifying the host cell genome to delete the chromosomal dal gene before introducing the plasmid. This creates a dependency on the plasmid-carrying dal gene, ensuring that recombinant hosts with high productivity traits are selectively maintained. The preliminary genetic modification establishes a selection system that automatically favors productive recombinant strains over wild-type cells throughout fermentation.
Data Source
AI summary
The present invention relates to a bacterial host cell in which a first chromosomal gene encoding a first alanine racemase and a second chromosomal gene encoding a second alanine racemase have been inactivated. Said bacterial host cell comprises – either on a plasmid comprising at least one autonomous replication sequence or present as multiple copies in the chromosome – a gene expression cassette comprising a polynucleotide encoding at least one polypeptide of interest, operably linked to a promoter, and a polynucleotide encoding a third alanine racemase, operably linked to a promoter. The present invention further relates to a method for producing at least one polypeptide of interest based on cultivating the bacterial host cell of the present invention.