Metabolic Engineering of Gluconobacter oxydans for Improved Growth Rate and Growth Yield on Glucose by Elimination of Gluconate Formation

Abstract

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 Δ mgdH 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 Δ mgdH 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 Δ mgdH sgdH :: kan ). In addition, the growth rates improved by 39% (N44-1 mgdH :: kan ) and 78% (N44-1 Δ mgdH 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 .