A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901

Victoria, Angelo J., Selao, Tiago Toscano, Moreno-Cabezuelo, José Ángel, Mills, Lauren A., Gale, Grant A. R., Lea-Smith, David J. ORCID: https://orcid.org/0000-0003-2463-406X and McCormick, Alistair J. (2024) A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901. Plant Physiology. ISSN 0032-0889

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Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2, 4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

Item Type: Article
Additional Information: Accession Numbers: Sequence data from this article can be found in the GenBank data libraries under accession number GCA_005577135.1. Data Availability: Plasmid vectors in Supplementary Table S1 are available from Addgene (https://www.addgene.org/Alistair_McCormick), Addgene IDs: 203934-203955, 205441. Funding information: A.J.V. was funded by a postgraduate research scholarship from the Darwin Trust of Edinburgh. T.T.S. acknowledges funding from the Green Chemicals Beacon of Excellence, University of Nottingham. J.A.M.C. acknowledges funding support from a FEBS short term fellowship and University of Cordoba fellowship. L.A.M. acknowledges funding support from the UK Biotechnology and Biological Sciences Research Council (BBSRC) Norwich Research Park Doctoral Training Partnership program [BB/S507404/1]. D.J.L.-S. acknowledges funding from BBSRC grant [BB/S020365/1] and [BB/Y008332/1], and UK Natural Environmental Research Council grant [NE/X014428/1]. A.J.M acknowledges funding from the BBSRC grants [BB/S020128/1] and [BB/W003538/1].
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
Depositing User: LivePure Connector
Date Deposited: 26 Apr 2024 14:30
Last Modified: 08 May 2024 09:30
URI: https://ueaeprints.uea.ac.uk/id/eprint/95030
DOI: 10.1093/plphys/kiae261


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