CyanoConstruct: simple platform for cyanobacterial expression construct assembly and translational tuning
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BACKGROUND: Despite the advances in synthetic biology, the construction of efficient and balanced artificial pathways remains challenging and a general bottleneck in bacterial strain development. Although translational tuning can be applied for balancing consecutive catalytic steps at protein expression level, the potential remains underexplored in cyanobacterial engineering. To complement existing modular cloning systems for this purpose the objective here was to simplify the construct assembly procedure to make translational tuning more accessible for expression optimization in cyanobacteria. RESULTS: This study describes the design and use of a one-pot DNA construct assembly system (CyanoConstruct) for the generation transformation-ready multi-gene expression plasmids in a single Golden Gate reaction. This approach allows the user to select the ribosome binding site element (RBS) for each target gene, thus serving as a tool for independently modulating the translation efficiency of the individual overexpressed enzymes. For easy adaptation, a custom online tool ( www.cyanoconstruct.com ) guides the sequence design of new compatible parts and the assembly of constructs from user-specified parts in silico. We demonstrate the use of the system by assembling different two-gene and three-gene expression constructs from parts selected specifically for optimal performance in Synechocystis sp PCC 6803; the constructs functioned as intended in vivo and showed different pathway fluxes construed by alternative RBS combinations. The efficiency and specificity of the assembly were shown to be high, enabling the generation of the final expression plasmids from the library parts in one assembly cycle. CONCLUSIONS: CyanoConstruct offers a simple strategy for building bacterial operon-based expression constructs, specifically facilitating the use of modular cloning systems for RBS optimization in routine cyanobacterial engineering. With the help of the web-based design tool that also serves as a sequence repository, the part library described in this work (https://www.addgene.org/browse/article/28263931/) can be easily expanded with user-specified sequences. By increasing the throughput for generating pathway variants with different translational patterns, the system is expected to advance the design of more efficient strains with higher flux to the desired end-product, thereby contributing to the development of next-generation biotechnologies.
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In operon-based pathway design, multiple genes are regulated under a single promoter and co-transcribed as a polycistronic mRNA.
“In such systems, multiple genes are regulated under a single promoter and co-transcribed as a polycistronic mRNA [149].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [149]
Variations in translation efficiency among genes within an operon can affect the relative levels of individual enzymes, potentially impacting pathway efficiency.
“However, variations in translation efficiency among genes within an operon can affect the relative levels of individual enzymes, potentially impacting pathway efficiency [149].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [149]
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“However, variations in translation efficiency among genes within an operon can affect the relative levels of individual enzymes, potentially impacting pathway efficiency [149].”
- Authors
- Venero, O. M., Ndeh, R., Pan, A., Patrikainen, P., Eriksson, M., Akhgari, A., Kakko, L., Nedorezova, D., Mohamed, O., Thomson, L., Bonde, A., Kelanne, N., Aro, E.-M., Adamala, K. P., & Kallio, P.
- Affiliations
- University of Minnesota (US)
- University of Turku (FI)
- Corresponding author
- Adamala, Katarzyna P.
- Kallio, Pauli
- ORCID
- Venue
- BMC Biotechnology
- Published
- 2026-02-16
- Volume
- 26
- Issue
- 1
- ISSN
- 1472-6750 (ISSN-L)
- PMC
- PMC13041217
- OpenAlex
- W7129084097
- Cited by
- 3
- References
- 89
- Citations by year
- 2026: 3
- FWCI
- 6.20
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- BMC Biotechnology (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- UTUPub (University of Turku) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Alfred P. Sloan Foundation (G-2024-22710)
- Division of Molecular and Cellular Biosciences (2419641)
- NordForsk (82845)
- Jane ja Aatos Erkon Säätiö
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- ribosome binding site optimization, Golden Gate cloning, Synechocystis sp. PCC 6803, web-based design tool
- MeSH terms
- Cyanobacteria, Cloning, Molecular, Genetic Engineering, Plasmids, Protein Biosynthesis, Synechocystis, Synthetic Biology
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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