Phosphoketolase (PKET) pathway is predominant in cyanobacteria (around 98%) but current opinion is that it is virtually inactive under autotrophic ambient CO2 condition (AC-auto). This creates an evolutionary paradox due to the existence of PKET pathway in obligatory photoautotrophs. We aim to answer the paradox with the aid of bioinformatic analysis along with metabolic, transcriptomic, fluxomic and mutant data integrated into a multi-level kinetic model. We discussed the problems linked to neglected isozyme, pket2 (sll0529) and inconsistencies towards the explanation of residual flux via PKET pathway in the case of silenced pket1 (slr0453) in Synechocystis sp. PCC 6803. Our in silico analysis showed: (1) 17% flux reduction via RuBisCO for Δpket1 under AC-auto, (2) 11.2–14.3% growth decrease for Δpket2 in turbulent AC-auto, and (3) flux via PKET pathway reaching up to 252% of the flux via phosphoglycerate mutase under AC-auto. All results imply that PKET pathway plays a crucial role under AC-auto by mitigating the decarboxylation occurring in OPP pathway and conversion of pyruvate to acetyl CoA linked to EMP glycolysis under the carbon scarce environment. Finally, our model predicted that PKETs have low affinity to S7P as a substrate.

The Entner-Doudoroff pathway (ED-P) was established in 2016 as the fourth glycolytic pathway in Synechocystis sp. PCC 6803. ED-P consists of two reactions, the first catalyzed by 6-phosphogluconate dehydratase (EDD), the second by keto3-deoxygluconate-6-phosphate aldolase/4-hydroxy-2-oxoglutarate aldolase (EDA). ED-P was previously concluded to be a widespread (∼92%) pathway among cyanobacteria, but current bioinformatic analysis estimated the occurrence of ED-P to be either scarce (∼1%) or uncommon (∼47%), depending if dihydroxy-acid dehydratase (ilvD) also functions as EDD (currently assumed). Thus, the biochemical characterization of ilvD is a task pending to resolve this uncertainty. Next, we have provided new insights into several single and double glycolytic mutants based on kinetic model of central carbon metabolism of Synechocystis. The model predicted that silencing 6-phosphogluconate dehydrogenase (gnd) could be coupled with ∼90% down-regulation of G6P-dehydrogenase, also limiting the metabolic flux via ED-P. Furthermore, our metabolic flux estimation implied that growth impairment linked to silenced EDA under mixotrophic conditions is not caused by diminished carbon flux via ED-P but rather by a missing mechanism related to the role of EDA in metabolism. We proposed two possible, mutually non-exclusive explanations: (i) Δeda leads to disrupted carbon catabolite repression, regulated by 2-keto3-deoxygluconate-6-phosphate (ED-P intermediate), and (ii) EDA catalyzes the interconversion between glyoxylate and 4-hydroxy-2-oxoglutarate + pyruvate in the proximity of TCA cycle, possibly effecting the levels of 2-oxoglutarate under Δeda. We have also proposed a new pathway from EDA toward proline, which could explain the proline accumulation under Δeda. In addition, the presented in silico method provides an alternative to 13C metabolic flux analysis for marginal metabolic pathways around/below the threshold of ultrasensitive LC-MS. Finally, our in silico analysis provided alternative explanations for the role of ED-P in Synechocystis while identifying some severe uncertainties.