Expression of alternative nitrogenases in Rhodopseudomonas palustris is enhanced using an optimized genetic toolset for rapid, markerless modifications

du Toit, Jan-Pierre, Lea-Smith, David J. ORCID:, Git, Anna, Hervey, John R. D., Howe, Christopher J. and Pott, Robert W. M. (2021) Expression of alternative nitrogenases in Rhodopseudomonas palustris is enhanced using an optimized genetic toolset for rapid, markerless modifications. ACS Synthetic Biology, 10 (9). 2167–2178. ISSN 2161-5063

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The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.

Item Type: Article
Uncontrolled Keywords: rhodopseudomonas palustris,alternative nitrogenases,biohydrogen,gene overexpression,genetic toolset,insertional mutagenesis,biomedical engineering,biochemistry, genetics and molecular biology (miscellaneous),sdg 7 - affordable and clean energy ,/dk/atira/pure/subjectarea/asjc/2200/2204
Faculty \ School: Faculty of Science > School of Biological Sciences
University of East Anglia Research Groups/Centres > Theme - ClimateUEA
UEA Research Groups: Faculty of Science > Research Groups > Molecular Microbiology
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Depositing User: LivePure Connector
Date Deposited: 16 Sep 2021 11:15
Last Modified: 13 Apr 2023 14:32
DOI: 10.1021/acssynbio.0c00496

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