title: Overexpression of membrane-bound gluconate-2-dehydrogenase to enhance the production of 2-keto-D-gluconic acid by Gluconobacter oxydans.
abstract: BACKGROUND
  2-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).
  
  
  RESULTS
  The ga2dh gene was overexpressed in G. oxydans DSM2003 under the control of three promoters, P tufB , P ga2dh or P ghp0169 , 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Kefei
authors.initials: K
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Xinlei
authors.initials: X
authors.lastname: Mao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China. jplin@ecust.edu.cn.
authors.forename: Jinping
authors.initials: J
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Biotechnology Institute, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Shengli
authors.initials: S
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D020600:Gluconobacter oxydans / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013400:Sugar Acids; D002237:Carbohydrate Dehydrogenases; C020577:gluconate 2-dehydrogenase; C011491:provitamin C; D005947:Glucose
keywords: 2-keto-D-gluconic acid; Gluconobacter oxydans; Overexpression; Resting cell