Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris
Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris
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基因剂量和培养温度对重组毕赤酵母生产β-甘露聚糖酶的影响
DOI：
10.1007/s11771-019-3992-z
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发表时间：
2019
影响因子：
4.4
通讯作者：
Zhou Hongbo
中科院分区：
文献类型：
--
作者：
Tang Shizhe;Lin Fulai;Zheng Jia;Zhou Hongbo
β-甘露聚糖酶在毕赤酵母中得到了高效表达。在毕赤酵母蛋白表达系统中，影响β-甘露聚糖酶表达的两个重要因素是基因用量和培养温度。本研究的目的是通过适当增加基因用量和降低培养温度来提高β-甘露聚糖酶在毕赤酵母中的表达水平。为此，通过多重沸素浓度梯度筛选，获得了一组具有不同拷贝数的β-甘露聚糖酶基因的菌株，考察了基因拷贝数对酵母X33表达β-甘露聚糖酶的影响。以GAP启动子为启动子，研究了GAP启动子、GAP启动子和GAP启动子对GAP-甘露聚糖酶在毕赤酵母X33中的表达4个拷贝菌株的β-甘露聚糖酶产量是1个拷贝菌株的4.0 4倍，总分泌蛋白是1个拷贝菌株的1.83倍，但4个拷贝以上的拷贝数增加会导致表达下降。在摇瓶中研究了培养温度对β-甘露聚糖酶活性的影响，4个拷贝的菌株降低培养温度后，β-甘露聚糖酶活力分别是30℃时的1.80倍(26℃)和3.5倍(22℃)，并成功地用于高密度发酵，在5号L发酵罐中培养72h，β-甘露聚糖酶活力达到2124U/mL。
High-level expression of β-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level of β-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers of β-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression of β-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higher β-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold (26 °C) and 3.5-fold (22 °C) higher β-mannanase activity compared to that at 30 °C.A fed-batch strategy was successfully used for high cell-density fermentation and β-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.
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影响因子：
3.5
作者：
Waterham, HR;Digan, ME;Cregg, JM
通讯作者：
Cregg, JM
影响因子：
3.4
作者：
Rongrong Zhan;W. Mu;B. Jiang;L. Zhou;Tao Zhang
通讯作者：
Rongrong Zhan;W. Mu;B. Jiang;L. Zhou;Tao Zhang
影响因子：
5
作者：
Luo, Huiying;Wang, Yaru;Yao, Bin
通讯作者：
Yao, Bin
影响因子：
4.4
作者：
Ye Wang;Jia Zheng;Fu-lai Lin;Hong-bo Zhou
通讯作者：
Ye Wang;Jia Zheng;Fu-lai Lin;Hong-bo Zhou
影响因子：
2.7
作者：
Shi Yu;Zhezhe Li;Yaping Wang;Wanping Chen;Ling Fu;Wei Tang;Cheng Chen;Yunyun Liu;Xue Zhang;Lixin Ma
通讯作者：
Shi Yu;Zhezhe Li;Yaping Wang;Wanping Chen;Ling Fu;Wei Tang;Cheng Chen;Yunyun Liu;Xue Zhang;Lixin Ma