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Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

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

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Abstract

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.

摘要

使用GAP 启动子可以实现β-甘露聚糖酶基因在毕赤酵母中的组成型表达。为了提高β-甘露聚 糖酶在毕赤酵母中的产量,本研究通过梯度抗生素浓度筛选法构建了携带多拷贝β-甘露聚糖酶的毕赤 酵母菌株,并利用实时荧光定量PCR 技术确定了各菌株的拷贝数。考察了不同基因拷贝数对β-甘露 聚糖酶在毕赤酵母中表达的影响,同时研究了不同拷贝数下,培养温度对β-甘露聚糖酶分泌表达的影 响。研究获得了β-甘露聚糖酶基因拷贝数为1、3、4、5、7 的毕赤酵母重组菌株,结果显示β-甘露聚 糖酶的mRNA 水平随着拷贝数的增加而升高,β-甘露聚糖酶产量随着拷贝数的增加而提高,其中拷贝 数为4 时产酶水平最高,是单拷贝菌株的4.04 倍,当拷贝数大于4 时,酶的产量不再随着拷贝数的增 加而增加。培养温度实验结果显示,较低的温度对重组菌株生产β-甘露聚糖酶有促进作用,当温度降 低至22 °C 时,携带4 拷贝的重组菌株的酶活是30 °C 时的3.5 倍。为了探索上述实验结果在工业发 酵上的应用潜力,我们进行了5 L 反应器水平的发酵实验,4 拷贝菌株在发酵72 h 后,酶活达到2124 U/mL。

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Authors and Affiliations

  1. School of Minerals Processing and Bioengineering, Central South University, Changsha, 410083, China

    Shi-zhe Tang  (唐诗哲), Fu-lai Lin  (林福来), Jia Zheng  (郑甲) & Hong-bo Zhou  (周洪波)

  2. Key Laboratory of Biometallurgy of Ministry of Education, Changsha, 410083, China

    Hong-bo Zhou  (周洪波)

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  1. Shi-zhe Tang  (唐诗哲)
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  2. Fu-lai Lin  (林福来)
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Correspondence to Hong-bo Zhou  (周洪波).

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Foundation item: Project(31870115) supported by the National Natural Science Foundation of China; Project(2015JJ5006) supported by the Natural Science of Hunan Province & Changde City Joint Foundation, China; Projects(2015zzts268, ZY2015823) supported by the Fundamental Research Funds for the Central Universities, China

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Tang, Sz., Lin, Fl., Zheng, J. et al. Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris. J. Cent. South Univ. 26, 184–195 (2019). https://doi.org/10.1007/s11771-019-3992-z

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