Entner-Doudoroff pathway in PCC 6803: Proposed regulatory roles and enzyme multifunctionalities.

The Entner-Doudoroff pathway (ED-P) was established in 2016 as the fourth glycolytic pathway in . 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 . The model predicted that silencing 6-phosphogluconate dehydrogenase () could be coupled with ∼90% down-regulation of G6P-dehydrogenase, also limiting the metabolic flux 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 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) Δ 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 Δ. We have also proposed a new pathway from EDA toward proline, which could explain the proline accumulation under Δ. In addition, the presented method provides an alternative to C metabolic flux analysis for marginal metabolic pathways around/below the threshold of ultrasensitive LC-MS. Finally, our analysis provided alternative explanations for the role of ED-P in while identifying some severe uncertainties.