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pCAT vectors overcome inefficient electroporation of Cupriavidus necator H16

Lookup NU author(s): Christopher Azubuike, Professor Angharad MR GatehouseORCiD, Dr Thomas HowardORCiD



This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND).


Cupriavidus necator H16 is a chemolithoautotroph with a range of industrial biotechnological applications. Advanced metabolic engineering in the bacterium, however, is impeded by low transformation efficiency, making it difficult to introduce and screen new genetic functions rapidly. This study systematically characterized the broad host range plasmids pBHR1, pBBR1MCS-2 and pKT230 used frequently for C. necator engineering. Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. necator low electroporation transformation efficiency. Consequently, a series of modular minimal plasmids, named pCAT, were constructed. pCAT vectors transform C. necator H16 with a > 3000-fold higher efficiency (up to 107 CFU/μg DNA) compared to control plasmids. Further, pCAT vectors are highly stable, expressing reporter proteins over several days of serial cultivation in the absence of selection pressure. Finally, they can be assembled rapidly from PCR or synthesized DNA fragments, and restriction-ligation reactions can be efficiently electroporated directly into C. necator, circumventing the requirement to use Escherichia coli for plasmid maintenance or propagation. This study demonstrates that an understanding of the behaviour of the constituent parts of plasmids in a host is key to efficient propagation of genetic information, while offering new methods for engineering a bacterium with desirable industrial biotechnological features.

Publication metadata

Author(s): Azubuike CC, Gatehouse AMR, Howard TP

Publication type: Article

Publication status: Published

Journal: New Biotechnology

Year: 2021

Volume: 65

Pages: 20-30

Print publication date: 25/11/2021

Online publication date: 29/07/2021

Acceptance date: 24/07/2021

Date deposited: 30/07/2021

ISSN (print): 1871-6784

ISSN (electronic): 1876-4347

Publisher: Elsevier BV


DOI: 10.1016/j.nbt.2021.07.003


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Funder referenceFunder name
Department for International Development (DFID)