Metabolic engineering of Gluconobacter oxydans for improved growth rate and growth yield on glucose by elimination of gluconate formation.

Gluconobacter oxydans N44-1, an obligatory aerobic acetic acid bacterium, oxidizes glucose primarily in the periplasm to the end products 2-ketogluconate and 2,5-diketogluconate, with intermediate formation of gluconate. Only a minor part of the glucose (less than 10%) is metabolized in the cytoplasm after conversion to gluconate or after phosphorylation to glucose-6-phosphate via the only functional catabolic routes, the pentose phosphate pathway and the Entner-Doudoroff pathway. This unusual method of glucose metabolism results in a low growth yield. In order to improve it, we constructed mutants of strain N44-1 in which the gene encoding the membrane-bound glucose dehydrogenase was inactivated either alone or together with the gene encoding the cytoplasmic glucose dehydrogenase. The growth and product formation from glucose of the resulting strains, N44-1 mgdH::kan and N44-1 DeltamgdH sgdH::kan, were analyzed. Both mutant strains completely consumed the glucose but produced neither gluconate nor the secondary products 2-ketogluconate and 2,5-diketogluconate. Instead, carbon dioxide formation of the mutants increased by a factor of 4 (N44-1 mgdH::kan) or 5.5 (N44-1 DeltamgdH sgdH::kan), and significant amounts of acetate were produced, presumably by the activities of pyruvate decarboxylase and acetaldehyde dehydrogenase. Most importantly, the growth yields of the two mutants increased by 110% (N44-1 mgdH::kan) and 271% (N44-1 DeltamgdH sgdH::kan). In addition, the growth rates improved by 39% (N44-1 mgdH::kan) and 78% (N44-1 DeltamgdH sgdH::kan), respectively, compared to the parental strain. These results show that the conversion of glucose to gluconate and ketogluconates has a strong negative impact on the growth of G. oxydans.