title: Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter.
abstract: Inulin fructotransferase (IFTase) has received considerable attention due to its ability to catalyse inulin hydrolysis to difructose anhydride (DFA III), a natural low-calorie functional sweetener. In the present study, for the first time, we describe the expression of IFTase in Pichia pastoris under the control of the formaldehyde dehydrogenase 1 promoter (PFLD1). Using this system, we achieved efficient secretion with four substrate fed-batch strategies in a 3-L fermenter. The co-feeding induction strategy with methylamine hydrochloride and methanol achieved the maximum extracellular IFTase activity of 62.72 U mL(-1), which was 3.2-fold higher than that obtained with the wild-type strain. In addition to methanol, carbon sources such as glucose and glycerol could also be utilised by PFLD1-controlled P. pastoris for IFTase production using methylamine hydrochloride induction. However, we found that glycerol and glucose should be strictly controlled at low concentrations of 0.5-1.5 % (v/v) and 1-1.5 % (w/v), respectively. The use of glycerol and glucose demonstrated that P. pastoris was also attractive for IFTase production via methanol-free cultivation strategies. This study may provide the basis for the industrial use of this recombinant IFTase for the production of DFA III.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, Jiangsu, 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: Liuming
authors.initials: L
authors.lastname: Zhou
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D019149:Bioreactors; D005990:Glycerol / Q000378:metabolism; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics*; D007444:Inulin / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008744:Methylamines / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D008744:Methylamines; D011994:Recombinant Proteins; D007444:Inulin; C027451:methylamine; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D006602:Hexosyltransferases; D005990:Glycerol; D000432:Methanol