enGenes technologies deliver cost-effective, high-yield bioprocesses over broad range of for specific Applications

enGenes Biotech has developed a suite of patented technology platforms for the customized production of recombinant proteins, enzymes, bioconjugates, metabolites, small molecules, and plasmid DNA (pDNA) or RNA.

Based on enGenes-e^Xpress™ technology that enables the growth-decoupled production of biomolecules in Escherichia coli at radically increased yields, platforms including -e^Xtra™, -e^Xpand™, -excite™, -e^Xcess™, and -e^Xchange™ also allow enhanced expression for desired characteristics. These can include extracellular secretion, expanded genetic code with incorporated non-canonical amino acids (ncAAs), continuous production, pDNA upstream and downstream processing and antibiotic resistance-free biomolecules production.

enGenes-e^Xpress™ adaptable platform

The patented enGenes-e^Xpress™ proprietary technology platform uses an advanced growth decoupled mechanism that allows customers to achieve customized solutions for high-yield recombinant protein production in E. coli. The technology is based on a genetically modified host cell based on BL21 (DE3) that allows the introduction of standard expression vectors (with T7 promoter) with no need for cumbersome adaptations to a two-stage process (accumulation of biomass, followed by production of recombinant protein) that can be implemented on standard fermentation equipment.

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. This enables the production of difficult-to-express proteins.

The technology allows the 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 up to 80% higher specific and volumetric yields compared to standard E. coli BL21 (DE3) systems with improved scalability and controllability, robustness, fewer optimization steps and shorter development cycles across the whole pharmaceutical contract manufacturing process.

Related technologies

The -e^Xpress™ core technology is a versatile one that can be intelligently applied in many ways in conjunction with other enGenes products to produce proteins, enzymes, bioconjugates, metabolites, small molecules, and plasmid DNA (pDNA) or RNA.

enGenes has filed patents for these related technology platforms, which can be used in combination to produce very specifically optimized bioconjugates, proteins and plasmid DNA.

These five related technologies comprise:

• enGenes-eXtra™: -e^Xtra™ technology enables the extracellular production of recombinant proteins by secretion, utilizing the periplasmic space of coli and a leaky outer membrane to release the protein into the extracellular space. This approach allows for simplified purification processes and potentially higher yields of the desired protein. enGenes-X-tra technology is a novel and efficient way for extracellular production of recombinant proteins by secretion that also offers cost-effectiveness through reduced downstream processing needs, making it an attractive option for high-quality protein production. Volumetric yields beyond the 10 g/L scale have been achieved regularly.
• enGenes-e^Xpand™: -e^Xpand™ technology allows site-specific incorporation of non-canonical amino acids (NCAAs) into the bioconjugate, with diverse applications in fields like protein engineering, drug discovery, and biotechnology, by enabling the synthesis of modified proteins with tailored properties, as well as cost-effective incorporation of NCAAs into proteins for bioconjugation through click-chemistry.
• enGenes-e^Xcite™: -e^Xcite™ is a cutting-edge technology that enables the continuous manufacturing of biomolecules in coli, providing high productivity from a very small footprint. With its high yield and scalability, enGenes-excite™ represents a promising solution for the cost-effective and sustainable production of biomolecules.
• enGenes-e^Xcess™: -e^Xcess™ technology provides an efficient and robust platform for the manufacturing of high-quality plasmid DNA. Its upstream and downstream processes have been optimized to enable significant yield improvements and facilitate large-scale production of plasmid DNA for gene therapy and vaccine development. This technology allows for very cost-effective production of high-quality plasmid DNA in a process that is also highly precise and reproducible. Volumetric yields up to 3 g/L supercoiled plasmid DNA have been achieved using optimized upstream processes.
• enGenes-eXchange™: The -e^Xchange™ platform offers a sustainable solution for antibiotic resistance marker-free recombinant protein production, using modified plasmids and coli strains that allow for antibiotic-free manufacturing of recombinant proteins and plasmid DNA. The technology offers a viable alternative to traditional methods that rely on the use of antibiotics, resulting in safer and more environmentally friendly production.

Used in combination, this suite of technologies also has the potential to increase yields up to five-fold with overall production costs reduced by as much as 80%. The effectiveness of these platforms has been confirmed in numerous research studies, cited below.

​References

1. Stargardt, Patrick, Lukas Feuchtenhofer, Monika Cserjan-Puschmann, Gerald Striedner, and Juergen Mairhofer. “Bacteriophage Inspired Growth-Decoupled Recombinant Protein Production in Escherichia Coli.” ACS Synthetic Biology9 (6): 1336–48. https://doi.org/10.1021/acssynbio.0c00028.
2. Stargardt, P., Striedner, G. and Mairhofer, J. (2021). Tunable expression rate control of a growth-decoupled T7 expression system by l-arabinose only. Microbial Cell Factories, 20(1). doi: https://doi.org/10.1186/s12934-021-01512-7.
3. ‌ Lemmerer, M., Juergen Mairhofer, Lepak, A., Longus, K., Hahn, R. and Bernd Nidetzky (2019). Decoupling of recombinant protein production from Escherichia colicell growth enhances functional expression of plant Leloir glycosyltransferases. Biotechnology and Bioengineering, [online] 116(6), pp.1259–1268. doi: https://doi.org/10.1002/bit.26934.
4. ‌ Danielewicz, N., Dai, W., Rosato, F., Webb, M.E., Striedner, G., Römer, W., Turnbull, W.B. and Mairhofer, J. (2022). In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli. Toxins, [online] 14(6), p.396. doi: https://doi.org/10.3390/toxins14060396.
5. ‌ De Vos, J., Pereira Aguilar, P., Köppl, C., Fischer, A., Grünwald-Gruber, C., Dürkop, M., Klausberger, M., Mairhofer, J., Striedner, G., Cserjan-Puschmann, M., Jungbauer, A. and Lingg, N. (2021). Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection. Talanta, [online] 235, p.122691. doi: https://doi.org/10.1016/j.talanta.2021.122691.
6. ‌ Klausberger, M., Duerkop, M., Haslacher, H., Wozniak-Knopp, G., Cserjan-Puschmann, M., Perkmann, T., Lingg, N., Aguilar, P.P., Laurent, E., De Vos, J., Hofner, M., Holzer, B., Stadler, M., Manhart, G., Vierlinger, K., Egger, M., Milchram, L., Gludovacz, E., Marx, N. and Köppl, C. (2021). A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests. EBioMedicine, [online] 67, p.103348. doi: https://doi.org/10.1016/j.ebiom.2021.103348.
7. ‌ Jens Kastenhofer, Lukas Rettenbacher, Lukas Feuchtenhofer, Juergen Mairhofer and Spadiut, O. (2020). Inhibition of coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness. 16(3), pp.2000274–2000274. doi: https://doi.org/10.1002/biot.202000274.
8. ‌ Zanker, A.A., Stargardt, P., Kurzbach, S.C., Turrina, C., Mairhofer, J., Schwaminger, S.P. and Berensmeier, S. (2022). Direct capture and selective elution of a secreted polyglutamate-tagged nanobody using bare magnetic nanoparticles. Biotechnology Journal, [online] 17(5), p.e2100577. doi: https://doi.org/10.1002/biot.202100577.
9. ‌ Galindo Casas, M., Stargardt, P., Mairhofer, J. and Wiltschi, B. (2020). Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in coli. ACS synthetic biology, [online] 9(11), pp.3052–3066. doi: https://doi.org/10.1021/acssynbio.0c00298.
10. ‌ Danielewicz, N., Rosato, F., Tomisch, J., Gräber, J., Wiltschi, B., Striedner, G., Römer, W. and Mairhofer, J. (2023). Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy. ACS omega, [online] 8(17), pp.15406–15421. doi: https://doi.org/10.1021/acsomega.3c00667.
11. ‌ Gotsmy, M., Strobl, F., Weiß, F., Gruber, P., Kraus, B., Mairhofer, J. and Zanghellini, J. (2023). Sulfate limitation increases specific plasmid DNA yield and productivity in coli fed-batch processes. Microbial Cell Factories, [online] 22(1), p.242. doi: https://doi.org/10.1186/s12934-023-02248-2.

Resources

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