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Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

ABSTRACT: The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families.

SUBMITTER: Matityahu A

PROVIDER: S-EPMC2546648 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

Matityahu Avi A Hadar Yitzhak Y Dosoretz Carlos G CG Belinky Paula A PA

Applied and environmental microbiology 20080707 17

The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies ...[more]

PMID: 18606804

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Project description:The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCEPhanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

Dataset Information

Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

Publications

Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

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