Release of Pleurotus ostreatus Versatile-Peroxidase from Mn2+ Repression Enhances Anthropogenic and Natural Substrate Degradation

Abstract

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn2+ supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn2+ on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn2+-deficient media, whereas strongly repressed (to approximately 1%) in Mn2+-supplemented media. Accordingly, in-vitro Mn2+-independent activity was found to be negligible. We tested whether release of mnp4 from Mn2+ repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the β-tubulin promoter was produced. Now, despite the presence of Mn2+ in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to β-tubulin) and the activity was comparable to the typical activity of PC9 in Mn2+-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [14C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn2+ suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

Redundancy among Manganese Peroxidases in Pleurotus ostreatus

“…In parallel, gene expression analysis showed that Mn 2ϩ amendment results in a drastic increase in the transcript levels of the predominantly expressed genes mnp3 and mnp9 (while transcription of mnp4 was reduced), suggesting that the two corresponding short MnPs play significant roles in these processes (10,12,13,(18)(19)(20).…”

“…MnP oxidizes Mn 2ϩ to Mn 3ϩ , the former being an obligatory cosubstrate for this enzyme, as it is required to complete the catalytic cycle, whereas VP, in addition to preferentially having MnP activity and being structurally related to MnP at the gene and protein levels, can directly oxidize aromatic substrates (2)(3)(4)(6)(7)(8)(9)(10)(11)(12)(13). Hence, to date, many VP-encoding genes are classified as part of the MnP gene family (mnp genes) (3,(9)(10)(11)(12)(13). Accordingly, we have used this nomenclature in the current report.…”

“…This diversity, along with the broad range of substrate specificity and their remarkable degradative potential, makes these peroxidases attractive versatile biocatalysts for biotechnological and environmental applications, e.g., in pulping and bleaching, in removal of hazardous wastes, and in synthesis of certain organic compounds. Effective application of these enzymes is dependent on establishing a comprehensive understanding of their properties and mode of action (2,5,13,14).…”

“…Previous studies have shown that Mn 2ϩ amendment to P. ostreatus growth media enables Mn 2ϩ -mediated decolorization of the azo dye orange II (OII) and increases fungal lignin degradation capacity (10,13,(18)(19)(20)(21). In parallel, gene expression analysis showed that Mn 2ϩ amendment results in a drastic increase in the transcript levels of the predominantly expressed genes mnp3 and mnp9 (while transcription of mnp4 was reduced), suggesting that the two corresponding short MnPs play significant roles in these processes (10,12,13,(18)(19)(20).…”

“…However, direct evidence linking a specific mnp to the various enhanced lignocellulose and xenobiotic degradative activities observed under Mn 2ϩ -amended culture conditions (10,13,(18)(19)(20)(21)(22)) has yet to be provided. Here, we have studied the functionality of the predominantly expressed MnPs, focusing on characterization of ⌬mnp3 strains.…”