Tandem-yeast expression system for engineering and producing unspecific peroxygenase
Tandem-yeast expression system for engineering and producing unspecific peroxygenase
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DOI：
10.1016/j.enzmictec.2015.03.004
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发表时间：
2015-06-01
影响因子：
3.4
通讯作者：
Alcalde, Miguel
中科院分区：
文献类型：
--
作者：
Molina-Espeja, Patricia;Ma, Su;Alcalde, Miguel
非特异性过氧酶（UPO）是一种高效的生物催化剂，具有过氧化物依赖的单加氧酶活性和许多生物技术应用，但缺乏合适的异种表达系统阻碍了其在不同工业环境中的应用。近年来，研究人员进化出了来自aegercybe的UPO，用于酿酒酵母（Saccharomyces cerevisiae）的分泌和活性。在目前的工作中，我们描述了一个用于UPO工程和大规模生产的串联酵母表达系统。利用酿酒酵母的定向进化过程，有益的分泌突变使毕赤酵母能够在甲醇诱导型醇氧化酶1启动子的控制下表达进化后的UPO。虽然两种酵母在瓶内发酵时的分泌水平相似（相似于8 mg/L），但重组酵母的UPO在分批补料发酵中（217 mg/L）的产量提高了27倍。在催化常数、pH活性谱和热稳定性方面，pastoris P. uPO变体保持了与酿酒酵母相似的生化特性。因此，这种串联酵母表达系统确保了upo的工程化，以便在未来的工业应用和大规模生产中使用它们。(c) 2015 Elsevier Inc.版权所有。
Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (similar to 8 mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217 mg/L). The P. pastoris uPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production. (c) 2015 Elsevier Inc. All rights reserved.