Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways

Abstract

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens . Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1- 13 C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31 P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. Conclusions Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism. Electronic supplementary material The online version of this article (10.1186/s12934-019-1178-x) contains supplementary material, which is available to authorized users.

Whole‐genome sequencing and genome‐scale metabolic modeling of Chromohalobacter canadensis 85B to explore its salt tolerance and biotechnological use

“…However, in high salinity, there is a high metabolic burden on this pathway due to the use of NADPH and ATP for the synthesis of compatible solutes. This allows the organism to use other pathways to meet other metabolic requirements (Pastor et al, 2019 ).…”

“…Salt tolerance is a hallmark phenotype of halophilic organisms with several mechanisms happening simultaneously for survival. The uptake and synthesis of compatible solutes constitute an important adaptation strategy for Chromohalobacter (Arahal & Ventosa, 2006;Piubeli et al, 2018). According to the genome annotation, C. canadensis 85B should be able to oxidize choline to betaine and synthesize ectoine de novo via the use of EctA, EctB, and EctC genes.…”

“…The Chromohalobacter models iFP764 (Piubeli et al, 2018) and iOA584 (Ates et al, 2011) is reported in Figure 3c, showing that iEB1159 has a larger number of reactions, genes, and metabolites.…”

“…In addition, these pathways also seem to be evolutionarily conserved in halophilic ectoine producers (Arahal & Ventosa, 2006;Piubeli et al, 2018). Demand reactions were also included to simulate the production of intracellular ectoine.…”

Characterization of Fructose-1,6-Bisphosphate Aldolase 1 of Echinococcus multilocularis

“…The energy metabolism of parasites mainly depends on the Embden-Meyerhof-Parnas pathway (EMP) [6,7]. Fructose-1, 6-bisphosphate aldolase (FBA) is a key enzyme that is involved in the first stage of glucose metabolism, where glucose is broken down into pyruvate.…”