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

Cyanobacterial extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides can be attached to the cell wall as capsular polysaccharides (CPS) or released to the environment as released polysaccharides (RPS). These polymers have an unusually high diversified monosaccharidic composition, making them attractive for biotechnological/biomedical applications. However, their production is still poorly understood, hindering their optimization for industrial needs. This work aimed to better understand the biosynthesis of the 6-deoxy sugars, fucose and rhamnose, in the model cyanobacterium <i>Synechocystis</i> sp. PCC 6803. To that end, genes encoding proteins putatively involved in the biosynthesis of GDP-L-fucose [<i>sll1213</i> (<i>fucS</i>)] and dTDP-L-rhamnose [<i>slr0985</i> (<i>rfbC1</i>) and <i>slr1933</i> (<i>rfbC2</i>)] were deleted. As previously observed, Δ<i>fucS</i> 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 Δ<i>fucS</i>. In contrast, both Δ<i>rfbC1</i> and Δ<i>rfbC1</i>Δ<i>rfbC2</i>, 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 <i>gmd</i> genes (<i>slr1072</i>) 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 <i>rfbC1</i> (<i>slr0982</i>, <i>slr0983</i>, and <i>slr1610</i>) were upregulated in both Δ<i>rfbC</i> strains. Altogether, our results reveal that rhamnose biosynthesis in <i>Synechocystis</i> 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 <i>Synechocystis</i> sp. PCC 6803. Here, we demonstrated that the Δ<i>fucS</i> 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 <i>rfbC</i> genes does not affect rhamnose biosynthesis despite having an impact on carbohydrate production/export, shifting RPS to CPS production. Altogether, our results suggest that the <i>rfbC</i> genes are not correctly annotated and highlight the intricacies and/or potential crosstalk between the two deoxyhexose pathways, yet to be completely unraveled in <i>Synechocystis</i>. The understanding of the cyanobacterial EPS assembly and export is crucial for the optimization of their production and tailoring for industrial/commercial applications.