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    Antibiotic resistance marker-free protein production

    products-servicesenGenes Biotech GmbH
    November 10th 2020

    A major advantage of enGenes Biotech’s proprietary -eXpress™ proprietary technology platform for outstanding  recombinant protein production in Escherichia coli (E.coli) is that it also facilitates the cloning and vector optimization required to ensure proteins that are free of antibiotic markers or antibiotic-resistant genes.

    The enGenes-eXpress advanced growth decoupled mechanism allows customers to achieve customized solutions for the cost-effective production of antibiotic marker-free proteins in microbial expression systems.

    Eliminating antibiotic markers through essential gene complementation

    A major problem with the use of microorganisms for enzyme production has been that many of the plasmids used for recombinant protein production in bacteria are based on the use of antibiotic resistant genes as selection markers. This has raised increasing safety concerns, particularly concerning the β-lactams associated with allergic responses in humans or animals or the horizontal gene transfer of these resistance genes.

    One solution to the problem is to complement the essential gene with an expression vector in a strain with a defect or inhibited expression of the same essential gene, using for example the dapD gene, which has a role in the Lysine biosynthetic pathway as well as cell wall assembly. While mutations in the dap pathway are lethal, a common limitation has been intrinsic difficulty in construction of a dapD – mutant strain and dependence towards defined culture media composition.

    As alternatives, other essential genes have been targeted for the same purpose, particularly infA coding for a translation initiation factor. This system is tightly regulated, with no cross-feeding effect observed since initiation factors released into the media from lyzed cells are not absorbed by plasmid-free cells.

    A further variant of the essential gen complementation strategy uses amino-acid auxotrophy complementation where a proline-auxotrophic strain obtained through chromosomal pro BA gene deletion is used to express antibody Fab fragment and plasmid-mediated complementation is used as a second selection mechanism to completely abolish plasmid loss during fermentation. On this principle, an E. coli M15-derivated glycine-auxotrophic strain has been shown to produce comparable amounts of recombinant protein as a conventional system.

    Other strategies for eliminating antibiotic markers include:

    • Post-segregational killing: Also known as the Hok/Soksystem, the toxin gene, responsible for host death, is integrated into the genome of a strain lacking the complete operon while an antitoxin gene that inactivates the toxin is located in the expression plasmid. The antibiotic resistance gene integrated along with the toxin is eliminated by the targeted recombinase to generate a final host strain free of any antibiotic resistance marker.
    • Plasmid selection using an endogenous essential gene marker: To overcome need to engineer mutant host strains and use specifically defined media, the fabl-triclosan model is based on over-expressing a host essential gene in presence of a chemical inhibitor of its product However, such systems are more suitable for DNA production rather than recombinant protein expression.
    • Poison/Antidote selection: A ‘poison’ gene like ccdB is inserted into the bacterial genome of an coli strain, encoding a stable protein and binding gyrase to impair DNA replication and induce cell death. A plasmid-born antidote gene (e.g. ccdA) then encodes an instable protein under control of the mob promoter, acting as a natural inhibitor of the poison gene.

    enGenes-eXpress advantages for antibiotic marker-free expression

    The -eXpress technology platform supports essential gene complementation by providing an engineered strain ready to accept antibiotic resistance-free plasmids. This genetically engineered host cells  modified host allows introduction of standard expression vectors (with T7 promoter) wherein the antibiotic resistance expression unit has been replaced for a particular E. coli gene. The fermentation process can be implemented with minimal medium and standard fermentation equipment and is fully scalable.

    According to the -eXpress principle reprogramming of the host cell is performed by co-expression of a bacteriophage-derived peptide that stops cell division and host mRNA production and at the same time modulates the host cell metabolism for improved soluble, high level protein production.

    The technology allows secretion of proteins targeted to the periplasmic space to the cell free supernatant, thereby allowing a cost-effective manufacturing option comparable to yeast-based expression systems. It also enables recombinant product formation to be decoupled from cell growth, enabling bioprocessing with clear separation of biomass growth and product formation. This in turn makes it possible to generate significantly higher specific and volumetric yields compared to the previous state-of-the-art (Escherichia coli BL21 (DE3).

    -eXpress growth-decoupled expression can be combined with plasmid expression vectors that allow antibiotic resistance marker-free selection. This is a particularly relevant advantage in food/feed-applications or human or veterinary applications where regulatory authorities are especially sensitive to the use of antibiotic resistance genes.

    enGenes already provides -eXpress with a ready-to-go optimized vector backbone and host strain. However, where necessary, it can also perform optimization on different levels to achieve client yield expectations. enGenes can also provide an ‘out of the box’ engineered -eXpress strain to meet demands for antibiotic resistance marker-free selection.

    Other elements of the enGenes portfolio, such as generating expression strain, host cell engineering, feasibility studies, bioprocess optimization, and final purification protocols can all be tailored into an antibiotic-free protein project, with additional assurance of greater cost-effectives and readier acceptance by regulatory authorities, due to the absence of antibiotic resistance markers.

    Furthermore, the -eXpress technology provides a more stable production process. In the case of the antibiotic resistance marker-free selection technology, the cell’s survival becomes connected to plasmid presence in the cell. Thus -eXpress cells resist loss of plasmids, thereby remaining productive.


    Antibiotic resistance marker-free protein production

    enGenes Schematic of enGenes -eXpress process

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