Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr;121(4):1216-1230.
doi: 10.1002/bit.28645. Epub 2024 Jan 4.

Recombinant protein expression in proteome-reduced cells under aerobic and oxygen-limited regimes

Alvaro R Lara  1 , Jose Utrilla  2 , Luz María Martínez  3 , Niels Krausch  4 , Lucas Kaspersetz  4 , David Hidalgo  2 , Nicolas Cruz-Bournazou  4 , Peter Neubauer  4 , Juan-Carlos Sigala  5 , Guillermo Gosset  3 , Jochen Büchs  6
Affiliations

Recombinant protein expression in proteome-reduced cells under aerobic and oxygen-limited regimes

Alvaro R Lara et al. Biotechnol Bioeng. 2024 Apr.

Abstract

Industrial cultures are hindered by the physiological complexity of the host and the limited mass transfer capacity of conventional bioreactors. In this study, a minimal cell approach was combined with genetic devices to overcome such issues. A flavin mononucleotide-based fluorescent protein (FbFP) was expressed in a proteome-reduced Escherichia coli (PR). When FbFP was expressed from a constitutive protein generator (CPG), the PR strain produced 47% and 35% more FbFP than its wild type (WT), in aerobic or oxygen-limited regimes, respectively. Metabolic and expression models predicted more efficient biomass formation at higher fluxes to FbFP, in agreement with these results. A microaerobic protein generator (MPG) and a microaerobic transcriptional cascade (MTC) were designed to induce FbFP expression upon oxygen depletion. The FbFP fluorescence using the MTC in the PR strain was 9% higher than that of the WT bearing the CPG under oxygen limitation. To further improve the PR strain, the pyruvate dehydrogenase complex regulator gene was deleted, and the Vitreoscilla hemoglobin was expressed. Compared to oxygen-limited cultures of the WT, the engineered strains increased the FbFP expression more than 50% using the MTC. Therefore, the designed expression systems can be a valuable alternative for industrial cultivations.

Keywords: gene circuits; genetic amplifier; microaerobic expression; minimal cells; proteome reduction; µRAMOS‐BioLector.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

References

REFERENCES

    1. Abdolmohammadi Khiav, L., & Zahmatkesh, A. (2021). Vaccination against pathogenic Clostridia in animals: A review. Tropical Animal Health and Production, 53(2), 284. https://doi.org/10.1007/s11250-021-02728-w
    1. Anand, A., Olson, C. A., Sastry, A. V., Patel, A., Szubin, R., Yang, L., Feist, A. M., & Palsson, B. O. (2021). Restoration of fitness lost due to dysregulation of the pyruvate dehydrogenase complex is triggered by ribosomal binding site modifications. Cell Reports, 35(1), 108961. https://doi.org/10.1016/j.celrep.2021.108961
    1. Basan, M., Hui, S., Okano, H., Zhang, Z., Shen, Y., Williamson, J. R., & Hwa, T. (2015). Overflow metabolism in Escherichia coli results from efficient proteome allocation. Nature, 528, 99–104. https://doi.org/10.1038/nature15765
    1. Baumann, K., Maurer, M., Dragosits, M., Cos, O., Ferrer, P., & Mattanovich, D. (2008). Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnology and Bioengineering, 100, 177–183. https://doi.org/10.1002/bit.21763
    1. Beg, Q. K., Vazquez, A., Ernst, J., de Menezes, M. A., Bar‐Joseph, Z., Barabási, A. L., & Oltvai, Z. N. (2007). Intracellular crowding defines the mode and sequence of substrate uptake by Escherichia coli and constrains its metabolic activity. Proceedings of the National Academy of Sciences, 104(31), 12663–12668. https://doi.org/10.1073/pnas.0609845104

Publication types

LinkOut - more resources