Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a Δ edd Δ eda Mutant on Mannitol

Abstract

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H oxidizes sugars and sugar alcohols primarily in the periplasm, and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is nonfunctional, and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a Δgnd mutant lacking the oxidative PPP and a Δedd Δeda mutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as the carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The Δedd Δeda mutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP also. These Δgnd zwf* mutants (with a mutation in the zwf gene causing inactivation of the glucose-6-phosphate dehydrogenase) were almost totally disabled in fructose catabolism but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression of gnd in the reference strain improved biomass formation in a similar manner as deletion of edd and eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism.