Johnson, A.O., Gonzalez-Villanueva, M., Tee, K.L. et al. (1 more author) (2018) An engineered constitutive promoter set with broad activity range for Cupriavidus necator H16. ACS Synthetic Biology, 7 (8). pp. 1918-1928. ISSN 2161-5063
Abstract
Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited. This limited number of characterized promoters poses a significant challenge during the engineering of C. necator H16 for biomanufacturing and biotechnological applications. In this article, we first examined the architecture and genetic elements of the four most widely used constitutive promoters of C. necator H16 (i.e., PphaC1, PrrsC, Pj5, and Pg25) and established a narrow 6-fold difference in their promoter activities. Next, using these four promoters as starting points and applying a range of genetic modifications (including point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration), we created a library of 42 constitutive promoters, all of which are functional in C. necator H16. Although these promoters are also functional in E. coli, they show different promoter strength and hierarchical rank of promoter activity. Subsequently, the activity of each promoter was individually characterized, using l-arabinose-inducible PBAD promoter as a benchmark. This study has extended the range of constitutive promoter activities to 137-fold, with some promoter variants exceeding the l-arabinose-inducible range of PBAD promoter. Not only has the work enhanced our flexibility in engineering C. necator H16, it presented novel strategies in adjusting promoter activity in C. necator H16 and highlighted similarities and differences in transcriptional activity between this organism and E. coli.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License (https://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 02 Jul 2018 10:51 |
Last Modified: | 20 Apr 2021 11:13 |
Status: | Published |
Publisher: | American Chemical Society |
Refereed: | Yes |
Identification Number: | 10.1021/acssynbio.8b00136 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:131750 |