resumo
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, 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 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.
palavras-chave
CYANOBACTERIAL EXOPOLYSACCHARIDES; PSEUDOMONAS-AERUGINOSA; STRAIN; POLYSACCHARIDE; ENZYMES; MYXOXANTHOPHYLL; PURIFICATION; EXPRESSION; PLATFORM; GLYCANS
categoria
Biotechnology & Applied Microbiology; Microbiology
autores
Pissarra, J; Santos, M; Pereira, SB; Pacheco, CC; Pinto, F; Ferreira, SS; Monteiro, R; Nunes, C; Coimbra, MA; Cabanes, D; Mota, R; Tamagnini, P
nossos autores
Cláudia Nunes
Investigador Auxiliar
Sónia dos Santos Ferreira
Bolseiro de pós-DoutoramentoProjectos
Collaboratory for Emerging Technologies, CoLab (EMERGING TECHNOLOGIES)
agradecimentos
This work was conducted within the framework of the scholarship 2020.08663.BD (J.P.) funded by National Funds through FCT - Fundacao para a Ciencia e a Tecnologia, I.P., and by ESF - European Social Fund through the Programa POCH - Operacional Capital Humano within the framework of PORTUGAL2020, namely, through the NORTE 2020 Programa Operacional Regional do Norte. It was also conducted within the context of the Molecular and Cell Biology doctoral program from ICBAS and FCUP. FCT - Fundacao para a Ciencia e a Tecnologia, I.P., also funded this work under the project UIDB/04293/2020. FCT is also acknowledged for the assistant researcher contracts CEECIND/00259/2017 (C.C.P.) and 2020.01953.CEECIND (F.P.). This work was in part developed within the scope of the project CICECO-Aveiro Institute of Materials (UIDB/50011/2020, UIDP/50011/2020, and LA/PP/0006/2020) and LAQV-REQUIMTE (UIDB/50006/2020, UIDP/50006/2020) financed by national funds through the FCT/MEC (PIDDAC).