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Úloha izoenzymů v regulaci metabolismu sinic
BACHHAR, Anushree
Model cyanobacterium Synechocystis PCC 6803 is one of the most studied species of cyanobacteria, having a significant impact in biotechnology, which explains why it is also known as "green E. coli". Experimental studies could deliver in vitro characterization of isozymes, but nobody knows if and how accurate such parametrization is in comparison to in vivo conditions. Also, despite the single/multi-mutant studies, the extent of isoenzymes roles is still lacking. The reasons behind this issue are: i) single knock-out is likely to be compensated by remaining isozyme(s), ii) deactivation of all isozymes does not count for unknown multi functionalities or iii) metabolic plasticity of Synechocystis allowing the redirection of metabolic flux towards alternative pathways within the central carbon metabolism, e.g., via glycolytic pathways (Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, phosphoketolase pathway and oxidative pentose phosphate pathway). In this study, I aimed to decipher some of the regulatory mechanisms within the central carbon metabolism of Synechocystis with the aid of metabolic model, integrating fluxomic, metabolic and transcriptomic data from literature and various databases e.g., Uniprot, KEGG. In the first half of my PhD research, I have focused on the impact of the mostly ignored glycolytic phosphoketolase pathway. In particular, we have shown that carbon flux via phosphoketolase pathway could be above 250% of the flux via Embden-Meyerhof-Parnas glycolysis under autotrophic conditions ambient CO2, thus playing a crucial role by mitigating the decarboxylation, i.e., carbon loss, occurring in other glycolytic pathways. We predicted that role of phosphoketolase pathway under mixotrophic conditions is rather negligible in Synechocystis, despite phosphoketolase pathway is crucial for heterotrophic conditions in most bacteria. In addition, we supported the existence of putative phosphoketolase isoenzyme (PKET 2) and quantified the following results under autotrophic conditions ambient CO2: 1) 17% flux reduction via RuBisCO for delta pket1 and 2) 11.2-14.3% growth decrease for delta pket2 in turbulent environment and 3) the substrate preference for phosphoketolase pathway under a given growth conditions. In the second half of my research work, I have focused on the recently confirmed glycolytic route in Synechocystis, the Entner-Doudoroff pathway (ED-P). Entner-Doudoroff pathway was previously concluded to be a very common (~92%) pathway among cyanobacteria, but my bioinformatic analysis based on available data sources predicted, Entner-Doudoroff pathway occurrence is below 50%. Interestingly, we have also identified plausible isozymes within Entner-Doudoroff pathway for some cyanobacteria. In the case of Synechocystis, we provided the first estimation of flux via Entner-Doudoroff pathway based on the growth impairment data which cannot be currently detected by 13C labelling experiments due to current detection limit for lower concentrations metabolite levels in mass-spectrometry devices. Nevertheless, we have identified too many uncertainties (enzyme multifunctionalities and identity issues) that it become difficult to annotate or predict the extent of possible metabolic and regulatory functions of Entner-Doudoroff pathway in Synechocystis.
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