An aryl-alcohol oxidase of Pleurotus sapidus: heterologous expression, characterization, and application in a 2-enzyme system

Abstract

Aryl-alcohol oxidases (AAOs) are enzymes supporting the degradation of lignin by fungal derived class II peroxidases produced by white-rot fungi. AAOs are able to generate H2O2 as a by-product via oxidation of an aryl-alcohol into its correspondent aldehyde. In this study, an AAO was heterologously expressed in a basidiomycete host for the first time. The gene for an AAO of the white-rot fungus Pleurotus sapidus, a close relative to the oyster mushroom Pleurotus ostreatus, was cloned into an expression vector and put under control of the promotor of the glyceraldehyde-3-phosphate dehydrogenase gene 2 (gpdII) of the button mushroom Agaricus bisporus. The expression vector was transformed into the model basidiomycete Coprinopsis cinerea, and several positive transformants were obtained. The best producing transformants were grown in shake-flasks and in a stirred tank reactor reaching enzymatic activities of up to 125 U L(-1) using veratryl alcohol as a substrate. The purified AAO was biochemically characterized and compared to the previously described native and recombinant AAOs from other Pleurotus species. In addition, a two-enzyme system comprising a dye-decolorizing peroxidase (DyP) from Mycetinis scorodonius and the P. sapidus AAO was successfully employed to bleach the anthraquinone dye Reactive Blue 5.

Signal peptide replacement resulted in recombinant homologous expression of laccase Lcc8 in Coprinopsis cinerea

“…By exchanging the N-terminal sequence of the Lcc8 with the signal peptide of Lcc1 (Additional file 1: Figures S1 and S2), known to be constantly expressed, a successful expression of Lcc8 was possible. Although in contrast to previous studies on recombinant enzyme production in C. cinerea (Kilaru et al 2006b; Galperin et al 2016), co-transformation experiments in this work resulted only in one distinct positive clone out of 80 tested clones accounting for a poor co-transformation efficiency of only 1.25%. Generally, efficiencies of more than 40% and 50% are possible as we could show in earlier co-transformations with C. cinerea using pCc1001 as marker plasmid (Kilaru et al 2006a, b; Galperin et al 2016).…”

“…It was cultivated in Lennox-LB media (Carl Roth, Karlsruhe, Germany) supplemented with 50 µg L −1 ampicillin (Carl Roth) at 37 °C and 180 rpm. Saccharomyces cerevisiae strain RH1385 ( MATa∆ura3 , Mösch et al 1990) was used for homologous recombination of expression plasmids (Gietz and Schiestl 2007) as described previously (Galperin et al 2016). Coprinopsis cinerea strain FA2222 (DSM 28333) was used as the expression host.…”

“…The PCR products and the linear fragments derived from pYSK7 were detected via agarose gel electrophoresis and purified from gel pieces using the NucleoSpin PCR cleanup Kit (Macherey-Nagel, Düren, Germany). Yeast strain RH1385 was transformed with the PCR product of pYMS33_for/rev and the linearized pYSK7 for homologous recombination as described elsewhere (Galperin et al 2016) to generate the final plasmid pYMS33. Clones were analyzed by colony PCR using pYSK7 expression cassette specific primers ( gpdII _for 5′-CCATCTCCGTTTTCTCCCATC-3′; lcc1 -term_rev 5′-CTGGCCCTCTGGTCAACTATAAT-3′).…”

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

“…This variant was transferred to Pichia pastoris for high-level production, leading to 25.5 mg/l of enzyme (Viña-Gonzalez et al 2018 ). Using a basidiomycete as expression host, an AAO from Pleurotus sapidus was heterologously produced in Coprinopsis cinerea with a yield of 1.4 mg/l (Galperin et al 2016 ). In order to fully elucidate fungal AAOs promising properties as biocatalysts in biotechnological processes, a high-yield expression system needs to be established.…”

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

“…AAO activity was first detected in Trametes versicolor cultures [10] and since then, it has been found in several other species, including Fusarium species [11] , [12] , Pleurotus species [13] , [14] , [15] , [16] , [17] , Bjerkandera adusta [18] , Botrytis cinerea [19] , Geotrichum candidum dec 1 [20] , Phanerochaete chrysosporium [21] and Ustilago maydis [22] . Initially located on the hyphal surface, the monomeric extracellular AAO acts on lignin-derived molecules, as well as on aromatic fungal metabolites.…”