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Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench.

ABSTRACT: Aryl-alcohol oxidase (AAO) is a fungal GMC flavoprotein secreted by white-rot fungi that supplies H2O2 to the ligninolytic consortium. This enzyme can oxidize a wide array of aromatic alcohols in a highly enantioselective manner, an important trait in organic synthesis. The best strategy to adapt AAO to industrial needs is to engineer its properties by directed evolution, aided by computational analysis. The aim of this review is to describe the strategies and challenges we faced when undertaking laboratory evolution of AAO. After a comprehensive introduction into the structure of AAO, its function and potential applications, the different directed evolution enterprises designed to express the enzyme in an active and soluble form in yeast are described, as well as those to unlock new activities involving the oxidation of secondary aromatic alcohols and the synthesis of furandicarboxylic acids.

SUBMITTER: Vina-Gonzalez J

PROVIDER: S-EPMC7358221 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench.

Viña-Gonzalez Javier J Alcalde Miguel M

Computational and structural biotechnology journal 20200629

Aryl-alcohol oxidase (AAO) is a fungal GMC flavoprotein secreted by white-rot fungi that supplies H<sub>2</sub>O<sub>2</sub> to the ligninolytic consortium. This enzyme can oxidize a wide array of aromatic alcohols in a highly enantioselective manner, an important trait in organic synthesis. The best strategy to adapt AAO to industrial needs is to engineer its properties by directed evolution, aided by computational analysis. The aim of this review is to describe the strategies and challenges we ...[more]

PMID: 32695272

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Project description:The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2-9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds. KEY POINTS: • MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l. • MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). • Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.

Dataset Information

Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench.

Publications

Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench.

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