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CpPosNeg: A positive‐negative selection strategy allowing multiple cycles of marker‐free engineering of the Chlamydomonas plastome

Jackson, Harry O; Taunt, Henry N; Mordaka, Paweł M; Kumari, Sujata; Smith, Alison G; Purton, Saul; (2022) CpPosNeg: A positive‐negative selection strategy allowing multiple cycles of marker‐free engineering of the Chlamydomonas plastome. Biotechnology Journal , Article 2200088. 10.1002/biot.202200088. (In press). Green open access

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Abstract

The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( = plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations. This presents issues with respect to selectable markers, since there are a limited number available, they can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative selection system, known as CpPosNeg, for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (AadA: conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (CodA: conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the 3′UTR of the marker and the 3′UTR of the introduced transgene. We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome.

Type: Article
Title: CpPosNeg: A positive‐negative selection strategy allowing multiple cycles of marker‐free engineering of the Chlamydomonas plastome
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/biot.202200088
Publisher version: https://doi.org/10.1002/biot.202200088
Language: English
Additional information: © 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).
Keywords: 5-fluorocytosine, Chlamydomonas, chloroplast, marker recycling, plastome
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10148409
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