High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors.,Applied Microbiology and Biotechnology

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High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors.
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2020-09-19 , DOI: 10.1007/s00253-020-10878-4
Nina Jankowski ₁ , Katja Koschorreck ₁ , Vlada B Urlacher ₁

Affiliation

Abstract

The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 °C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM⁻¹ s⁻¹, respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications.

Key points

• Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l.

• Purified enzyme exhibited stability over a broad pH and temperature range.

• Oxidation products cuminaldehyde and piperonal are of biotechnological interest.

Graphical abstract

中文翻译：

杏鲍菇侧耳的芳醇氧化酶2在巴斯德毕赤酵母中的高表达，用于生产香料和生物活性前体。

摘要

真菌分泌组包含各种氧化酶，参与木质纤维素生物质的降解，这是碳循环的重要步骤。在分泌的酶中，芳基醇氧化酶（AAOs）对于生物技术应用是令人感兴趣的，包括生产塑料，生物活性化合物以及调味剂和香料的生物基前体。使用甲基营养酵母巴斯德毕赤酵母（最近重新归类为Komagataella phaffii），从杏鲍菇侧耳真菌真菌中的芳醇氧化酶2（Pe AAO2）异源表达和分泌，是迄今为止报道的最高产量之一，为315 mg / l 。糖基化PeAAO2在pH 3.0至9.0的宽pH范围内均显示出高稳定性，并在高达55°C的温度下具有高热稳定性。用41种化合物进行的底物筛选显示，Pe AAO2氧化了典型的AAO底物，如对茴香醇，藜芦醇和反式，反式-2,4-己二醇，其活性比苄醇高出8倍。尚未报道作为AAOs底物的几种化合物也被Pe AAO2氧化。其中，枯草醇和胡椒基醇被氧化为异丙苯甲醛和胡椒醛，催化效率高达84.1和600.2 mM ^-1 s ^-1，分别。尽管芳香和风味化合物胡椒醛也可作为农用化学品和药物构建基块的原料，但由于异丙苯醛具有多种积极的健康作用，包括抗癌，抗糖尿病和神经保护作用。因此，Pe AAO2是用于生物技术应用的有前途的生物催化剂。