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

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 Electronic supplementary material The online version of this article (10.1007/s00253-020-10878-4) contains supplementary material, which is available to authorized users.


Compounds and solvents used in substrate screening
In total, 41 different benzylic, cyclic, heterocyclic and aliphatic alcohols were used at 1 mM final concentration in the coupled ABTS-HRP assay. The solutions of tested compounds were prepared using the following solvents (Table S1).  For measurements of absorbance at the corresponding maxima of the aldehydes, further dilutions were prepared: Cuminaldehyde dilutions ranged from 0.2 mM to 0.02 mM, and piperonal dilutions ranged from 0.1 to 0.01 mM in 100 mM sodium phosphate buffer pH 6.0. The respective absorption maxima were determined and plotted against the concentration of cuminaldehyde or piperonal. A linear fit of the data was conducted using the program OriginPro 9.0 (OriginLab Corporation, Northampton, USA) and the molar extinction coefficient was deduced from the fitted data as slope of the regression curve. According to Lambert-Beer-Law with A = ε * c * d, where A is absorbance, ε is the molar extinction coefficient, c is the concentration and d is the path length (1 cm). In a plot with A vs. c, the extinction coefficient is described as the = * .

Native PAGE of purified PeAAO2
Blue native PAGE of 5 µg of purified PeAAO2 was carried out using the SERVAGel N Native starter kit (SERVA Electrophoresis GmbH, Heidelberg, Germany) with 4-16 % gel according to the manufacturer's protocol. The gel was stained with Coomassie Brilliant Blue R250.

Influence of pH on enzyme activity
The effect of pH on activity towards p-anisyl alcohol, benzyl alcohol, cinnamyl alcohol, cumic alcohol,

Molar extinction coefficient of cuminaldehyde
The spectra of cumic alcohol and cuminaldehyde were recorded from 200 to 400 nm (Fig. S1). The substrate cumic alcohol showed one major absorbance maximum at 218 nm and a minor maximum at 262 nm, whereas the product cuminaldehyde showed a more pronounced absorbance maximum at 262 nm and a second maximum at 212 nm. Therefore, the maximum at 262 nm was used for determination of the molar extinction coefficient of cuminaldehyde. The absorbance at 262 nm for different cuminaldehyde concentrations was measured (Fig. S2). Cuminaldehyde concentration [mM]

Molar extinction coefficient of piperonal
The spectra of piperonyl alcohol and piperonal were recorded from 250 to 400 nm (Fig. S3). The absorbance at 317 nm for different piperonal concentrations was measured (Fig. S4). Native PAGE of purified PeAAO2 The purified PeAAO2 was investigated under non-denaturing conditions in a blue native PAGE (Fig. S5).

Fig. S5
Gel after Blue Native PAGE of purified PeAAO2. 5 µg of sample were loaded and separated in a 4-16 % gel Recombinant PeAAO2 moved as a single band between 67 and 146 kDa, and therefore indicates that PeAAO2 is present in a monomeric form.

Influence of pH on activity of PeAAO2
The routinely used buffer in the AAO activity assay mixture was exchanged for 100 mM Britton-Robinson buffer at different pH values. The activity of PeAAO2 towards several substrates at different pH was measured (Fig. S6).

Fig. S6
Influence of pH on activity of PeAAO2. 100 mM Britton-Robinson buffer at the corresponding pH was used instead of the assay buffer -100 mM sodium phosphate pH 6.0. Values were put in relation to the control experiment with assay buffer. All measurements were done in triplicate PeAAO2 converted the substrates benzyl alcohol, cinnamyl alcohol, cumic alcohol, trans,trans-2,4-hexadienol and veratryl alcohol best at pH 6.0, while for p-anisyl alcohol and piperonyl alcohol a slightly more acidic pH 5.0 was best. Relative activities towards p-anisyl alcohol and piperonyl alcohol at lower pH were higher compared with other substrates. Overall, the pH activity profile for p-anisyl alcohol and piperonyl alcohol appeared shifted towards more acidic pH. Sequence alignment and homology model The two P. eryngii derived AAOs PeAAO und PeAAO2 differ only in seven amino acid positions (Fig. S7). One of these positions is a potential N-glycosylation site (motif Asn-X-Thr/Ser, where X is any amino acid except for proline) in PeAAO2 (residue Asn361), whereas PeAAO lacks this site as an aspartic acid is present instead of asparagine. The catalytically active histidine residues His529 and His573 as well as the main residues involved in regulating substrate accessibility to the active site (Tyr119, Phe424 and Phe528, as described for PeAAO in Fernández et al. 2009) are conserved in both aryl-alcohol oxidases. The high similarity of both aryl-alcohol oxidases from P. eryngii, especially in terms of residues involved in substrate accessibility and catalytically active residues, indicate that both AAOs share similar catalytic properties.
Using the crystal structure of PeAAO expressed in E. coli as template (PDB entry 3FIM), a homology model of PeAAO2 was created and the differing residues were marked in purple (Fig. S8). The seven differing residues are located on or near the surface of the protein and the active site is conserved among both AAOs.