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bioRxiv · 10.1101/2025.03.27.645739

Rhamnose biosynthesis is not impaired by the deletion of putative rfbC genes, slr0985 and slr1933, in Synechocystis sp. PCC 6803

Pissarra, J.·Santos, M.·B. Pereira, S.·C. Pacheco, C.·Pinto, F.·S. Ferreira, S.·Monteiro, R.·Nunes, C.·Coimbra, M. A.·Cabanes, D.·Mota, R.·Tamagnini, P.
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Abstract

Cyanobacterial extracellular polymeric substances (EPS), mainly composed by heteropolysaccharides, can be attached to the cell wall (CPS) or released to the environment (RPS). These polymers have an unusually highly diversified monosaccharidic composition, making them attractive for biotechnological/biomedical applications. However, their production is still poorly understood hindering their optimisation for industrial needs. This work aimed at better understanding the biosynthesis of the 6-deoxysugars fucose and rhamnose in the model cyanobacterium Synechocystis sp. PCC 6803. To that end, genes encoding proteins putatively involved in the biosynthesis of GDP-L-fucose [sll1213 (fucS)] and dTDP-L-rhamnose [slr0985 (rfbC1) and slr1933 (rfbC2)] were deleted. As previously observed, {Delta}fucS had significant growth impairment and its RPS did not contain any fucose or rhamnose. Here, we also showed that both deoxyhexoses pathways are completely impaired in {Delta}fucS. In contrast, both {Delta}rfbC1 and {Delta}rfbC1{Delta}rfbC2 although producing significantly less RPS and more CPS than the wild type, did not show major differences regarding the RPS monosaccharidic composition. These results strongly suggest that their gene products are not essential for rhamnose biosynthesis. Transcriptional analysis revealed that one of the gmd genes (slr1072), putatively encoding a GDP-mannose 4,6-dehydratase, was upregulated in all the knockout strains, and that the three EPS-related genes in the same operon as rfbC1 (slr0982, slr0983 and slr1610) were upregulated in both {Delta}rfbC strains. Altogether, our results reveal that rhamnose biosynthesis in Synechocystis depends on FucS but not on the putative RfbC enzymes, underlining the need to further elucidate the mechanisms involved in the biosynthesis of this deoxyhexose. IMPORTANCEThis study contributes to the overall knowledge of deoxyhexoses biosynthesis in Synechocystis sp. PCC 6803. Here we demonstrated that the {Delta}fucS strain not only produces EPS without fucose and rhamnose but that both pathways are completely impaired. Furthermore, we also showed that the deletion of both putative rfbC genes do not affect rhamnose biosynthesis, despite having an impact on carbohydrates production/export, shifting RPS to CPS production. Altogether, our results suggest that the rfbC genes are not correctly annotated and highlight the intricacies and/or potential crosstalk between the two deoxyhexoses pathways, yet to be completely unravelled in Synechocystis. The understanding of cyanobacterial EPS assembly and export is crucial for the optimisation of their production and tailoring for industrial/commercial applications.

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Pissarra, J., Santos, M., B. Pereira, S., C. Pacheco, C., Pinto, F., S. Ferreira, S., Monteiro, R., Nunes, C., Coimbra, M. A., Cabanes, D., Mota, R., Tamagnini, P.. 2025-03-30. Rhamnose biosynthesis is not impaired by the deletion of putative rfbC genes, slr0985 and slr1933, in Synechocystis sp. PCC 6803. https://doi.org/10.1101/2025.03.27.645739

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