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

## TL;DR

This study investigates sugar biosynthesis in cyanobacteria and finds that certain genes are not essential for rhamnose production.

## Contribution

The study reveals that rhamnose biosynthesis in Synechocystis is not impaired by deleting putative rfbC genes.

## Key findings

- Deletion of fucS completely impairs biosynthesis of fucose and rhamnose.
- Deletion of rfbC genes does not affect rhamnose biosynthesis but alters EPS production/export.
- Transcriptional analysis shows upregulation of certain genes in knockout strains.

## Abstract

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 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, ΔfucS 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 ΔfucS. In contrast, both ΔrfbC1 and ΔrfbC1ΔrfbC2, 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 ΔrfbC 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.

This study contributes to the overall knowledge of deoxyhexoses’ biosynthesis in Synechocystis sp. PCC 6803. Here, we demonstrated that the ΔfucS 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 does not affect rhamnose biosynthesis despite having an impact on carbohydrate 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 deoxyhexose pathways, yet to be completely unraveled in Synechocystis. The understanding of the cyanobacterial EPS assembly and export is crucial for the optimization of their production and tailoring for industrial/commercial applications.