title: High-level extracellular expression of inulin fructotransferase in Pichia pastoris for DFA III production.
abstract: BACKGROUND
  Inulin fructotransferase (IFTase) catalyzes inulin conversion to difructose anhydride (DFA III), which is a natural low-calorie sweetener. Although heterologous expression of IFTase was achieved in Escherichia coli, the extracellular enzyme activity was very low, which limited the commercialization of IFTase.
  
  
  RESULTS
  Active IFTase of about 43 kDa molecular mass of subunit was extracellularly expressed by Pichia pastoris and was greatly regulated by the IFTase gene copy number integrated into the P. pastoris genome and by the methanol concentration in the induction phase. Under optimized culture conditions, multicopy P. pastoris exhibited a maximum extracellular IFTase activity of 105.4 U mL(-1) in a 5 L fermenter, which was 8.9-fold the activity in shake flasks and 5.3-fold that obtained from wild-type strain.
  
  
  CONCLUSIONS
  IFTase was expressed in a eukaryotic P. pastoris system for the first time and achieved high-level extracellular expression using a high-cell-density fed-batch cultivation strategy. This demonstrated that P. pastoris was a good candidate for potential DFA III production as a novel IFTase expression system.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
authors.forename: Rongrong
authors.initials: R
authors.lastname: Zhan
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.forename: Yungao
authors.initials: Y
authors.lastname: Li
authors.forename: Liuming
authors.initials: L
authors.lastname: Zhou
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
mesh_terms: D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D019149:Bioreactors; D004187:Disaccharides / Q000096:biosynthesis*; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007444:Inulin / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D013549:Sweetening Agents / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D004187:Disaccharides; D013549:Sweetening Agents; C087758:difructose anhydride III; D007444:Inulin; D006602:Hexosyltransferases; D000432:Methanol
keywords: Pichia pastoris; difructose anhydride; extracellular expression; fed-batch fermentation; inulin fructotransferase