Abstract
Background2-keto-d-gluconic acid (2KGA) is widely used as a chemical intermediate in the cosmetic, pharmaceutical and environmental industries. Several microbial fermentation processes have been developed for production of 2KGA but these suffer from substrate/product inhibition, byproduct formation and low productivity. In previous work, we showed that 2KGA can be specifically produced from glucose (Glu) or gluconic acid (GA) by resting wild-type Gluconobacter oxydans DSM2003 cells, although substrate concentration was relatively low. In this study, we attempted to improve 2KGA productivity by G. oxydans DSM2003 by overexpressing the ga2dh gene, which encodes the membrane-bound gluconate-2-dehydrogenase enzyme (GA2DH).ResultsThe ga2dh gene was overexpressed in G. oxydans DSM2003 under the control of three promoters, PtufB, Pga2dh or Pghp0169, respectively. Among the recombinant strains obtained, G. oxydans_tufB_ga2dh showed a similar growth rate to that of the control strain and displayed the highest specific productivity of 2KGA from GA, which was increased nearly twofold compared with that of the control strain during batch biotransformation. When biocatalysis conditions were optimized, with provision of sufficient oxygen during biotransformation, up to 480 g/L GA was completely utilized over 45 h by resting cells of G. oxydans_tufB_ga2dh and 453.3 g/L 2KGA was produced. A productivity of 10.07 g/L/h and a yield of 95.3 % were obtained. Overexpression of the ga2dh gene also significantly improved the conversion of Glu to 2KGA. Under optimized conditions, 270 g/L Glu was converted to 321 g/L 2KGA over 18 h, with a yield of 99.1 % and a productivity of 17.83 g/L/h. The glucose concentrations during the batch biotransformation and the 2KGA productivities achieved in this study were relatively high compared with the results of previous studies.ConclusionsThis study developed an efficient bacterial strain (G. oxydans_tufB_ga2dh) for the production of 2KGA by overexpressing the ga2dh gene in G. oxydans. Supply of sufficient oxygen enhanced the positive effect of gene overexpression on 2KGA production. Gluconobacter oxydans_tufB_ga2dh is thus a competitive species for use in 2KGA production.
Highlights
The results show that enhanced expression of the ga2dh gene in G. oxydans under the control of tufB promoter efficiently improved the production yield of 2-keto-d-gluconic acid (2KGA) from gluconic acid (GA)
The results clearly reveal that high oxygen levels suppressed the oxidative activity of resting cells toward GA, and an excess of oxygen during bioconversion may result in decreasing productivity
The final 2KGA titer reached 234.6 g/L at 21 h during the batch bioconversion of glucose by the engineered strain, corresponding to a productivity of 11.17 g/L/h, which amounted to a 407 % increase compared with that obtained using the control strain G. oxydans_pBBR1MCS5
Summary
Overexpression of ga2dh in G. oxydans DSM2003 Efficient expression of ga2dh (gox1230-1232) was essential for enhancing the 2KGA production in G. oxydans DSM2003. The results show that enhanced expression of the ga2dh gene in G. oxydans under the control of tufB promoter efficiently improved the production yield of 2KGA from GA. To increase DO levels, oxygen instead of air was supplied continuously to support the oxidation of 480 g/L GA by 30 g(wet wt)/L resting cells Under this condition, the agitation speed and oxygen flow rate were controlled at 600 rpm and 1 L/min, respectively. The final 2KGA titer reached 234.6 g/L at 21 h during the batch bioconversion of glucose by the engineered strain, corresponding to a productivity of 11.17 g/L/h, which amounted to a 407 % increase compared with that obtained using the control strain G. Because sufficient oxygen supply could enhance 2KGA productivity during GA conversion, oxygen instead of air was supplied continuously to support the oxidation of 270 g/L glucose at the same cell mass (Fig. 8b). All of the supplied glucose was converted to 321 g/L 2KGA over 18 h by the constructed strain G. oxydans_tufB_ga2dh, corresponding to GA 2KGA Glu
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