title: A water-forming NADH oxidase regulates metabolism in anaerobic fermentation.
abstract: BACKGROUND
  Water-forming NADH oxidase can oxidize cytosolic NADH to NAD(+), thus relieving cytosolic NADH accumulation in Saccharomyces cerevisiae. Previous studies of the enzyme were conducted under aerobic conditions, as O2 is the recognized electron acceptor of the enzyme. In order to extend its use in industrial production and to study its effect on anaerobes, the effects of overexpression of this oxidase in S. cerevisiae BY4741 and Clostridium acetobutylicum 428 (Cac-428) under anaerobic conditions were evaluated.
  
  
  RESULTS
  Glucose was exhausted in the NADH oxidase-overexpressing S. cerevisiae strain (Sce-NOX) culture after 26 h, while 43.51 ± 2.18 g/L residual glucose was left in the control strain (Sce-CON) culture at this time point. After 30 h of fermentation, the concentration of ethanol produced by Sce-NOX reached 36.28 ± 1.81 g/L, an increase of 56.38 % as compared to Sce-CON (23.20 ± 1.16 g/L), while the byproduct glycerol was remarkably decreased in the culture of Sce-NOX. In the case of the C. acetobutylicum strain (Cac-NOX) overexpressing NADH oxidase, glucose consumption, cell growth rate, and the production of acetone-butanol-ethanol (ABE) all decreased, while the concentrations of acetic acid and butyric acid increased as compared to the control strain (Cac-CON). During fermentation of Cac-CON and Cac-NOX in 100-mL screw-capped bottles, the concentrations of ABE increased with increasing headspace. Additionally, several alternative electron acceptors in C. acetobutylicum fermentation were tested. Nitroblue tetrazolium and 2,6-dichloroindophenol were lethiferous to both Cac-CON and Cac-NOX. Methylene blue could relieve the effect caused by the overexpression of the NADH oxidase on the metabolic network of C. acetobutylicum strains, while cytochrome c aggravated the effect.
  
  
  CONCLUSIONS
  The water-forming NADH oxidase could regulate the metabolism of both the S. cerevisiae and the C. acetobutylicum strains in anaerobic conditions. Thus, the recombinant S. cerevisiae strain might be useful in industrial production. Besides the recognized electron acceptor O2, methylene blue and/or the structural analogs may be the alternative elector acceptor of the NADH oxidase in anaerobic conditions.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Xin-Chi
authors.initials: XC
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Ya-Nan
authors.initials: YN
authors.lastname: Zou
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Bing-Bing
authors.initials: BB
authors.lastname: Li
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Jia-Hui
authors.initials: JH
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Xiao-Ning
authors.initials: XN
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing, 210009 People's Republic of China.
authors.forename: Han-Jie
authors.initials: HJ
authors.lastname: Ying
keywords: Alternative elector acceptor; Anaerobic fermentation; Clostridium acetobutylicum; NADH oxidase; Saccharomyces cerevisiae