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

Autor: Jackson HO; Department of Structural and Molecular Biology, University College London, London, UK., Taunt HN; Department of Structural and Molecular Biology, University College London, London, UK., Mordaka PM; Department of Plant Sciences, University of Cambridge, Cambridge, UK., Kumari S; DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai, India., Smith AG; Department of Plant Sciences, University of Cambridge, Cambridge, UK., Purton S; Department of Structural and Molecular Biology, University College London, London, UK.
Jazyk: angličtina
Zdroj: Biotechnology journal [Biotechnol J] 2022 Oct; Vol. 17 (10), pp. e2200088. Date of Electronic Publication: 2022 May 12.
DOI: 10.1002/biot.202200088
Abstrakt: 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.
(© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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