Two adjacent C-terminal mutations enable expression of aryl-alcohol oxidase from Pleurotus eryngii in Pichia pastoris

Nina Jankowski,Vlada B Urlacher,Katja Koschorreck

doi:10.1007/s00253-021-11585-4

Nina Jankowski, Vlada B Urlacher + Show 1 more

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https://doi.org/10.1007/s00253-021-11585-4

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Abstract

Fungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding.Key points• PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions.• Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence.• Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris.

Highlights

Flavin-dependent oxidases build a diverse group of enzymes that have been successfully used in biocatalysis and biosensors (Dijkman et al 2013)
Despite the low volumetric activity of PeAAO1 variant K583E compared to PeAAO2 wild-type with 61 U/l, the successful expression of this variant in P. pastoris was a good starting point for further mutagenesis
Six PeAAO1 double mutants were created by introducing mutations R152G, T265I, D361N, V367A, D512N, and Q584R, respectively, into the PeAAO1 variant K583E and screened for improved volumetric activity towards veratryl alcohol

Summary

Introduction

Flavin-dependent oxidases build a diverse group of enzymes that have been successfully used in biocatalysis and biosensors (Dijkman et al 2013). An important prerequisite for the application of these enzymes is their availability at high quantities In this regard, heterologous expression in microbial hosts has been recognized as the most efficient approach which on the one hand, opens the way to high-scale processes and, on the other hand, when combined with protein engineering enables production and. Applied Microbiology and Biotechnology (2021) 105:7743–7755 was applied for the production of the flavor and fragrance compound trans-2-hexenal (de Almeida et al 2019; van Schie et al 2018). This enzyme was used for the conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid as precursor for bioplastics in multi-enzyme cascades (Carro et al 2014; Serrano et al 2019a). A recent review summarized different approaches of directed evolution to unlock PeAAO1’s full potential for biotechnological purposes aiming at enhanced expression or acceptance of new substrates (Viña-Gonzalez and Alcalde 2020)

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