Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter
Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter
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DOI：
10.1007/s10295-014-1516-2
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发表时间：
2014-11
影响因子：
3.4
通讯作者：
Rongrong Zhan;W. Mu;B. Jiang;L. Zhou;Tao Zhang
中科院分区：
文献类型：
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作者：
Rongrong Zhan;W. Mu;B. Jiang;L. Zhou;Tao Zhang
菊粉果糖转移酶(IFTase)是一种天然的低热量功能性甜味剂，它能催化菊粉水解制得二果糖酸酐(DFA III)，因而受到人们的广泛关注。在本研究中，我们首次描述了在甲醛脱氢酶1启动子(PFLD1)的控制下，IFTase在毕赤酵母中的表达。利用该系统，我们在3-L发酵罐上实现了四种补料分批策略的高效发酵。甲胺盐酸盐和甲醇共加诱导策略获得的最大胞外IFTase活力为62.72U/mL−1，是野生型菌株的3.2倍。除甲醇外，葡萄糖和甘油等碳源也可被PFLD1控制的磷利用。利用甲胺盐酸盐诱导生产IFTase的酵母菌。然而，我们发现甘油和葡萄糖应该分别严格控制在0.5-1.5%(v/v)和1-1.5%(w/v)的低浓度。甘油和葡萄糖的使用证明P。通过无甲醇培养策略，酵母菌对IFTase的生产也很有吸引力。本研究为该重组IFTase工业化生产DFA III奠定了基础。
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 inPichia pastorisunder 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 byPFLD1-controlledP. pastorisfor 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 thatP. pastoriswas 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.