Takaiku Sakamoto

Changes in the Gene Expression of the White Rot Fungus Phanerochaete chrysosporium Due to the Addition of Atropine

We constructed a LongSAGE (Long Serial Analysis of Gene Expression) library from a 3-d culture of Phanerochaete chrysosporium supplemented with atropine, which inhibits the production of lignin-degrading enzymes. The library (the atropine library) contains 13,108 LongSAGE tags and 6,783 unique tags. The gene expression profile represented by the tags was compared with those of two previously constructed libraries, one of which was constructed using 2-d cultures in which the fungus had not yet produced ligninolytic enzymes (the 2-d library) and the other was constructed using 3-d cultures in which the fungus had just started to produce the enzymes (the 3-d library). We found a total of 595 genes that were at least twice more highly or at least twice less highly expressed in the 3-d library than in the 2-d library or the atropine library, and the fluctuations were statistically significant. The relationships among these 595 genes were considered using cluster analysis. Of the 595 genes, 164 showed expression patterns similar to those of four ligninolytic enzyme genes, which were more expressed on day 3 than under any other conditions. Many of these 164 genes comprised genes possibly involved in lignin degradation, lipid metabolism, xenobiotic degradation, stress response, or signal transduction pathways.

Gene targeting in the oil-producing fungus Mortierella alpina 1S-4 and construction of a strain producing a valuable polyunsaturated fatty acid

To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.

Effects of calmodulin on expression of lignin-modifying enzymes in Pleurotus ostreatus

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn2+-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.

The White-Rot Fungus Pleurotus ostreatus Transformant Overproduced Intracellular cAMP and Laccase

Transformation of Pleurotus ostreatus PC9 with the mutated heterotrimeric G protein alpha subunit (Gα) gene resulted in higher laccase (Lac) activity and intracellular cyclic adenosine monophosphate (cAMP) concentrations as compared to those in wild-type PC9. The transformant also exhibited higher Lac activity than the wild type when cultured in a medium containing known Lac inducers CuSO4 and ferulic acid.

Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids

The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain.