Dataset Information

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

ABSTRACT: Although the model agaricomycete Coprinopsis cinerea possess 17 different laccase genes, up to now only four C. cinerea laccases have been purified and characterized to some degree. By exchanging the nucleotide sequence of the deduced signal peptide of Lcc8 it was possible to homologously express lcc8 in C. cinerea under control of the Agaricus bisporus gdpII promoter and the C. cinerea lcc1 terminator. The purified Lcc8 showed two bands in the SDS-PAGE with a molecular weight of 64 kDa and 77 kDa, respectively. The IEF determined pI values of 3.3 and 3.4 for both bands. The optimal pH for oxidation of the substrates ABTS, 2,6-dimethoxyphenol, guaiacol and syringaldazine was pH 4.0, pH 5.0, pH 4.5 and pH 5.0, respectively. Best pH for enzyme storage was pH 8.0. The optimal temperature for oxidation of ABTS was 63 °C, while Lcc8 showed activity of at least 50% over 300 min at 50 °C. The comparable high stability of Lcc8 at alkaline pH and higher temperatures can be of interest for biotechnical applications.

SUBMITTER: Schulze M

PROVIDER: S-EPMC6420550 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Schulze Marcus M Geisler Lukas L Majcherczyk Andrzej A Rühl Martin M

AMB Express 20190315 1

Although the model agaricomycete Coprinopsis cinerea possess 17 different laccase genes, up to now only four C. cinerea laccases have been purified and characterized to some degree. By exchanging the nucleotide sequence of the deduced signal peptide of Lcc8 it was possible to homologously express lcc8 in C. cinerea under control of the Agaricus bisporus gdpII promoter and the C. cinerea lcc1 terminator. The purified Lcc8 showed two bands in the SDS-PAGE with a molecular weight of 64 kDa and 77 k ...[more]

PMID: 30874916

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Project description:Frequently, laccases are triggered during fungal cocultivation for overexpression. The function of these activated laccases during coculture has not been clarified. Previously, we reported that Gongronella sp. w5 (w5) (Mucoromycota, Mucoromycetes) specifically triggered the laccase Lcc9 overexpression in Coprinopsis cinerea (Basidiomycota, Agaricomycetes). To systematically analyze the function of the overexpressed laccase during fungal interaction, C. cinerea mycelia before and after the initial Lcc9 overexpression were chosen for transcriptome analysis. Results showed that accompanied by specific utilization of fructose as carbohydrate substrate, oxidative stress derived from antagonistic compounds secreted by w5 appears to be a signal critical for laccase production in C. cinerea. A decrease in reactive oxygen species (ROS) in the C. cinerea wild-type strain followed the increase in laccase production, and then lcc9 transcription and laccase activity stopped. By comparison, increased H2O2 content and mycelial ROS levels were observed during the entire cocultivation in lcc9 silenced C. cinerea strains. Moreover, lcc9 silencing slowed down the C. cinerea mycelial growth, affected hyphal morphology, and decreased the asexual sporulation in coculture. Our results showed that intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Lcc9 takes part in a defense strategy to eliminate oxidative stress during the interspecific interaction with w5. IMPORTANCE The overproduction of laccase during interspecific fungal interactions is well known. However, the exact role of the upregulated laccases remains underexplored. Based on comparative transcriptomic analysis of C. cinerea and gene silencing of laccase Lcc9, here we show that oxidative stress derived from antagonistic compounds secreted by Gongronella sp. w5 was a signal critical for laccase Lcc9 production in Coprinopsis cinerea. Intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Ultimately, Lcc9 takes part in a defense strategy to eliminate oxidative stress and help cell growth and development during the interspecific interaction with Gongronella sp. w5. These findings deepened our understanding of fungal interactions in their natural population and communities.

Project description:BackgroundThe transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development.ResultsWe evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom.ConclusionsWe implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.

Dataset Information

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

Publications

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

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