Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number
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BACKGROUND: Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. RESULTS: An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. CONCLUSIONS: This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.
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Polyploidy affects gene dosage: different chromosomal or native plasmid integration sites can produce different expression levels because replicon copy number and local genomic context influence the number and activity of inserted gene copies.
“Polyploidy also affects gene dosage: different chromosomal or native plasmid integration sites can produce different expression levels because replicon copy number and local genomic context influence the number and activity of inserted gene copies [136].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [136]
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Jaramillo, A., Satta, A., Pinto, F., Faraloni, C., Zittelli, G. C., Benavides, A. M. S., Torzillo, G., Schumann, C., Méndez, J. F., Berggren, G., et al. (2025) · Energy & Fuels · doi:10.1021/acs.energyfuels.4c04772 · Cited by 40 · see citing works
Cengic, I., Cañadas, I. C., Minton, N. P., & Hudson, E. P. (2022) · ACS Synthetic Biology · doi:10.1021/acssynbio.2c00375 · Cited by 33 · see citing works
Puzorjov, A., Ünal, S. M., Wear, M. A., & McCormick, A. J. (2021) · Bioresource Technology · doi:10.1016/j.biortech.2021.126459 · Cited by 34 · see citing works
Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria
Opel, F., Siebert, N. A., Klatt, S., Tüllinghoff, A., Hantke, J. G., Toepel, J., Bühler, B., Nürnberg, D. J., & Klähn, S. (2022) · ACS Synthetic Biology · doi:10.1021/acssynbio.1c00605 · Cited by 30 · see citing works
Wu, P., Hu, T., Sun, L., & Fan, J. (2023) · Chemical Engineering Journal · doi:10.1016/j.cej.2023.145974 · Cited by 29 · see citing works
Synthetic biology in marine cyanobacteria: Advances and challenges
Bourgade, B., & Stensjö, K. (2022) · Frontiers in Microbiology · doi:10.3389/fmicb.2022.994365 · Cited by 28 · see citing works
A toolbox to engineer the highly productive cyanobacterium <i>Synechococcus</i> sp. PCC 11901
Victoria, A. J., Selão, T. T., Moreno-Cabezuelo, J. Á., Mills, L. A., Gale, G. A. R., Lea-Smith, D. J., & McCormick, A. J. (2024) · Plant Physiology · doi:10.1093/plphys/kiae261 · Cited by 28 · see citing works
Customized molecular tools to strengthen metabolic engineering of cyanobacteria
Klähn, S., Opel, F., & Hess, W. R. (2024) · Green Carbon · doi:10.1016/j.greenca.2024.05.002 · Cited by 27 · see citing works
Zhou, C., Yang, G., Zhang, L., Zhang, H., Zhou, H., & Fuping, L. (2021) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2021.10.208 · Cited by 24 · see citing works
Blanc-Garin, V., Chenebault, C., Díaz‐Santos, E., Vincent, M., Sassi, J., Cassier‐Chauvat, C., & Chauvat, F. (2022) · Biotechnology for Biofuels and Bioproducts · doi:10.1186/s13068-022-02211-0 · Cited by 24 · see citing works
Yi, Y.-C., Xue, C., & Ng, I. (2021) · ACS Sustainable Chemistry & Engineering · doi:10.1021/acssuschemeng.1c05994 · Cited by 20 · see citing works
Hoffmann, U. A., Heyl, F., Rogh, S. N., Wallner, T., Backofen, R., Hess, W. R., Steglich, C., & Wilde, A. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab1161 · Cited by 19 · see citing works
Sakamaki, Y., Maeda, K., Nimura-Matsune, K., Chibazakura, T., & Watanabe, S. (2023) · Frontiers in Microbiology · doi:10.3389/fmicb.2023.1111979 · Cited by 11 · see citing works
Behle, A., Dietsch, M., Goldschmidt, L., Murugathas, W., Berwanger, L. L., Burmester, J., Yao, L., Brandt, D., Busche, T., Kalinowski, J., et al. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac1132 · Cited by 11 · see citing works
Ohdate, K., Sakata, M., Maeda, K., Sakamaki, Y., Nimura-Matsune, K., Ohbayashi, R., Hess, W. R., & Watanabe, S. (2024) · Frontiers in Microbiology · doi:10.3389/fmicb.2024.1311290 · Cited by 9 · see citing works
Simple transformation of the filamentous thermophilic cyanobacterium Leptolyngbya sp. KC45
Mahanil, K., Sattayawat, P., Pekkoh, J., Kameya, M., Ishii, M., & Pumas, C. (2022) · Algal Research · doi:10.1016/j.algal.2022.102758 · Cited by 9 · see citing works
The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories
Opel, F., Axmann, I. M., & Klähn, S. (2022) · Advances in Biochemical Engineering/Biotechnology · doi:10.1007/10_2022_210 · Cited by 7 · see citing works
The role of the 5’ sensing function of ribonuclease E in cyanobacteria
Hoffmann, U. A., Lichtenberg, E., Rogh, S. N., Bilger, R., Reimann, V., Heyl, F., Backofen, R., Steglich, C., Hess, W. R., & Wilde, A. (2024) · RNA Biology · doi:10.1080/15476286.2024.2328438 · Cited by 7 · see citing works
Nies, F., Wein, T., Hanke, D. M., Springstein, B. L., Alcorta, J., Taubenheim, C., & Dagan, T. (2023) · Environmental Microbiology Reports · doi:10.1111/1758-2229.13203 · Cited by 5 · see citing works
Cyanobacterial bioreporter of nitrate bioavailability in aquatic ecosystems
Wu, S., Zheng, H., Wang, Y., Wang, L., & Chen, W. (2023) · Water Research · doi:10.1016/j.watres.2023.120749 · Cited by 5 · see citing works
Özer, H., Wasser, D., Sandner, L., & Soppa, J. (2024) · Genes · doi:10.3390/genes15070861 · Cited by 4 · see citing works
Venero, O. M., Ndeh, R., Pan, A., Patrikainen, P., Eriksson, M., Akhgari, A., Kakko, L., Nedorezova, D., Mohamed, O., Thomson, L., et al. (2026) · BMC Biotechnology · doi:10.1186/s12896-025-01098-7 · Cited by 3 · see citing works
Self-citationWalshe, J., & Saha, S. K. (2026) · Current Issues in Molecular Biology · doi:10.3390/cimb48060638 · Cited by 1 · see citing works
“Polyploidy also affects gene dosage: different chromosomal or native plasmid integration sites can produce different expression levels because replicon copy number and local genomic context influence the number and activity of inserted gene copies [136].”
Genetic Modifications in Cyanobacteria: Current Status and Future Prospects
Tripathi, K., & Mishra, Y. (2026) · IntechOpen eBooks · doi:10.5772/intechopen.1014041 · Cited by 1 · see citing works
CyanOperon: An Operon Building Expansion for the CyanoGate MoClo Toolkit
Astbury, M. J., Osorio, A. A. S., Victoria, A. J., & McCormick, A. J. (2026) · ACS Synthetic Biology · doi:10.1021/acssynbio.6c00162 · Cited by 1 · see citing works
Sahoo, A., Veeranki, V. D., & Patra, S. (2025) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2025.06.007 · Cited by 1 · see citing works
Pappert, I., Greulich, S., Joseph, F. M., Otto, B., & Kaldenhoff, R. (2023) · Journal of Advances in Biology & Biotechnology · doi:10.9734/jabb/2023/v26i4629
Influence of phosphorus on genome copy number of Synechocystis sp. PCC 6803
Zhao, J., Wang, M., Liang, C., & Lu, Y. (2025) · Letters in Applied Microbiology · doi:10.1093/lambio/ovaf087
Blanc-Garin, V., Chenebault, C., Díaz‐Santos, E., Vincent, M., Sassi, J., Cassier‐Chauvat, C., & Chauvat, F. (2022) · Figshare · doi:10.6084/m9.figshare.21341203
Kakko, L., Muth‐Pawlak, D., Patrikainen, P., Aro, E., & Kallio, P. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.01.09.698553
Self-citationOver 4 months of ethylene production using solid-state photosynthetic cell factories
Kosourov, S. N., Siitonen, V., Tóth, G. S., Levä, T., Tammelin, T., Kallio, P., & Allahverdiyeva, Y. (2026) · Trends in biotechnology · doi:10.1016/j.tibtech.2026.06.006
Self-citationGlycogen deficiency impairs diurnal energy metabolism and cell division in Synechocystis
Hofer, J. M., Schulze, T., Witting, L., Laker, B., Krueger, S., Westhoff, P., Kohlheyer, D., Weber, A. P. M., & Eisenhut, M. (2026) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiag560
- Authors
- Nagy, C., Thiel, K., Mulaku, E., Mustila, H., Tamagnini, P., Aro, E.-M., Pacheco, C. C., & Kallio, P.
- Affiliations
- University of Turku (FI)
- Universidade do Porto (PT)
- Corresponding author
- Kallio, Pauli
- Venue
- Microbial Cell Factories
- Published
- 2021-07-10
- Volume
- 20
- Issue
- 1
- Pages
- 130-130
- ISSN
- 1475-2859 (ISSN-L)
- PMC
- PMC8272380
- OpenAlex
- W3183011970
- Cited by
- 33
- References
- 96
- Citations by year
- 2026: 7 · 2025: 3 · 2024: 5 · 2023: 5 · 2022: 8 · 2021: 5
- FWCI
- 1.81
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Microbial Cell Factories (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UTUPub (University of Turku) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, cc-by)
- Funders
- Fundação para Ciência e Tecnologia (contract CEECIND/00259/2017)
- European Union's Horizon 2020 (Marie Skłodowska-Curie Action- Innovative Training Network-2017 (grant agreement No 764920))
- Center of Excellence of the Academy of Finland (CoE project #307335)
- NordForsk (NCoE project #82845)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- chromosomal integration, megaplasmid, plasmid copy number, gene dosage, gene expression efficiency, site-specific integration, plasmid maintenance
- MeSH terms
- Chromosomes, Bacterial, Genetic Engineering, Plasmids, Recombination, Genetic, Transformation, Bacterial, Gene Expression, Synechocystis
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