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Title (Primary) Generation of synthetic shuttle vectors enabling modular genetic engineering of cyanobacteria
Author Opel, F.; Siebert, N.A.; Klatt, S.; Tüllinghoff, A.; Hantke, J.G.; Toepel, J.; Bühler, B.; Nürnberg, D.J.; Klähn, S.
Journal ACS Synthetic Biology
Year 2022
Department SOMA
Volume 11
Issue 5
Page From 1758
Page To 1771
Language englisch
Topic T7 Bioeconomy
Supplements https://pubs.acs.org/doi/suppl/10.1021/acssynbio.1c00605/suppl_file/sb1c00605_si_001.pdf
https://pubs.acs.org/doi/suppl/10.1021/acssynbio.1c00605/suppl_file/sb1c00605_si_002.zip
Keywords synthetic biology; shuttle vectors; cyanobacteria; (photo)biotechnology; genetic engineering; molecular tools
UFZ wide themes MIKAT;
Abstract Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer–Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria.
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=26012
Opel, F., Siebert, N.A., Klatt, S., Tüllinghoff, A., Hantke, J.G., Toepel, J., Bühler, B., Nürnberg, D.J., Klähn, S. (2022):
Generation of synthetic shuttle vectors enabling modular genetic engineering of cyanobacteria
ACS Synth. Biol. 11 (5), 1758 - 1771