Kenji Yamagishi

Production of the α-glycosidase inhibitor 1-deoxynojirimycin from Bacillus species

1-Deoxynojirimycin (DNJ), a potent α-glycosidase inhibitor, has therapeutic applications in treatments of HIV, Gauchers disease, and diabetes. DNJ has been extracted from natural sources (mulberry leaves) for therapeutic purposes; however, DNJ ingredients are in limited supply and are costly to obtain on a large scale. Since certain strains of Bacillus and Streptomyces species reportedly produce DNJ, they may serve as potential sources for high-yield DNJ production. In this study, we obtained evidence for a DNJ production in Bacillus subtilis DSM704 by hydrophilic interaction chromatography-tandem mass spectrometry. In addition, from a screen of 750 microorganisms, we identified additional Bacillus strains (Bacillus amyloliquefaciens AS385 and Bacillus subtilis B4) that produce DNJ in large quantities. Investigation of the effect of various culture conditions, using Bacillus subtilis DSM704 and the DNJ high-production Bacillus strains, provided evidence for the importance of sorbitol supplementation on the yield of the DNJ precursor, 2-amino-2-deoxy-D-mannitol, thereby increasing DNJ production. The role of sorbitol in increased DNJ production was supported by an observed increase in mRNA expression of the biosynthetic gene, gabT1. When Bacillus amyloliquefaciens AS385 was cultured in medium supplemented with sorbitol, extracellular DNJ concentration reached a maximum of 460 mg/l of medium (equivalent to 9.20mg/g of freeze-dried medium), indicating that this strain can serve as a source for food- and drug-grade products. These findings not only lead to a further understanding of the DNJ biosynthetic pathway, but also suggest a method for microbial mass production of DNJ for therapeutic applications.

Cloning and Homologous Expression of Novel Lignin Peroxidase Genes in the White-Rot Fungus Phanerochaete sordida YK-624

Two genes, encoding YK-LiP1 and YK-LiP2, were cloned from the white-rot fungus Phanerochaete sordida YK-624, and a homologous expression system for the gene was constructed. Two full-length cDNAs (ylpA and ylpB) were isolated by degenerate RT-PCR and RACE-PCR. The results of N-terminal amino acid sequence analysis of native YK-LiP1 and YK-LiP2 showed that ylpA and ylpB coded for YK-LiP2 and YK-LiP1 respectively. The promoter of glyceraldehyde-3-phosphate dehydrogenase cloned from P. sordida YK-624 (PsGPD) was used to drive the expression of ylpA. Expression vector pGPD-g-ylpA was transformed into a P. sordida YK-624 uracil auxotrophic mutant, UV-64. The YlpA protein was secreted in active form by the transformants after 4 d of growth in a medium containing an excessive nitrogen source, whereas endogenous YK-LiP1 and YK-LiP2 were not produced. The physical and catalytic properties of the purified YlpA protein were very similar to those of YK-LiP2. These results suggest that homologous expression of recombinant YK-LiP2 was successful.

Treatment of rice straw with selected Cyathus stercoreus strains to improve enzymatic saccharification

Seventeen Cyathus stercoreus isolates were tested for their ability to treat rice straw for improved enzymatic saccharification. These isolates showed a negative correlation between cellulase and xylanase activity and enzymatic saccharification yields. Incubation of rice straw pretreated at 60°C for 15 min with strain C. stercoreus TY-2 for 25 days resulted in an enzymatic saccharification yield of 57% as compared to a yield of 11% for the same straw in the absence of the fungus. These findings highlight the potential of this isolate for biological pretreatment of rice straw under conditions of low energy input.

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624.

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2u2009×u2009107 protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.

Elevation of intracellular cAMP levels by dominant active heterotrimeric G protein alpha subunits ScGP-A and ScGP-C in homobasidiomycete, Schizophyllum commune.

In many fungi, the heterotrimeric G protein alpha subunits, and/or small G protein (RAS) control intracellular cAMP levels. But it is not clear which types of G proteins modulate cAMP levels in homobasidiomycete (mushrooms). To explain the mechanism, we expressed dominant active RAS (a homolog of S. cerevisiae RAS1) in homobasidiomycete Schizophyllum commune and compared the cAMP levels in the transformed clones with those of clones expressing dominant active heterotrimeric G protein alpha subunits ScGP-A, B, and C. The results demonstrated that the dominant active ScGP-A and C elevated the intracellular cAMP levels. In contrast, the dominant active S. commune RAS gene did not affect the cAMP levels, even though colony growth and formation of fruiting bodies were apparently repressed. These data suggest that the heterotrimeric G protein alpha subunits are involved in the mechanism of cAMP regulation, and that RAS modulates another signal-transduction pathway regulating cell growth and differentiation.

Efficient expression of laccase gene from white‐rot fungus Schizophyllum commune in a transgenic tobacco plant

Ligninolytic enzymes produced by white-rot fungi are effective degraders of recalcitrant aromatic environmental pollutants. However, gene sequences of these enzymes are rich in CpG dinucleotides, which are particularly unfavorable to efficient expression in plants. In order to develop a phytoremediation technique with a ligninolytic enzyme-producing transgenic plant, laccase cDNA (scL) from white-rot fungus Schizophyllum commune was used as a model ligninolytic enzyme, and we attempted to obtain the efficient expression of scL in a transgenic tobacco plant by decreasing the CpG-dinucleotide motif content. We constructed a mutagenized scL sequence, scL12, decreasing the CpG-dinucleotide motif content by 12%, and scL12 was introduced into the tobacco plant. Much higher laccase activity was detected in transgenic scL12 plants than in transgenic scL plants and wild-type plants. Using reverse transcriptase-PCR analysis, scL12 was translated in transgenic scL12 plants whereas mRNA of scL was not detected in the transgenic scL plants, and scL, which is the product of the scL12 gene, was produced in the transgenic scL12 plants using native-polyacrylamide gel electrophoresis analysis. Moreover, transgenic scL12 plants were able to remove trichlorophenol more effectively than transgenic scL plants and wild-type plants. These results suggest that decreasing CpG-dinucleotide motif content in fungal target genes is a useful method for efficient expression of these genes in transgenic plants.

Molecular breeding of lignin-degrading brown-rot fungus Gloeophyllum trabeum by homologous expression of laccase gene

The basidiomycete Gloeophyllum trabeum KU-41 can degrade Japanese cedar wood efficiently. To construct a strain better suited for biofuel production from Japanese cedar wood, we developed a gene transformation system for G. trabeum KU-41 using the hygromycin phosphotransferase-encoding gene (hpt) as a marker. The endogenous laccase candidate gene (Gtlcc3) was fused with the promoter of the G. trabeum glyceraldehyde-3-phosphate dehydrogenase-encoding gene and co-transformed with the hpt-bearing pAH marker plasmid. We obtained 44 co-transformants, and identified co-transformant L#61, which showed the highest laccase activity among all the transformants. Moreover, strain L#61 was able to degrade lignin in Japanese cedar wood-containing medium, in contrast to wild-type G. trabeum KU-41 and to a typical white-rot fungus Phanerochaete chrysosporium. By using strain L#61, direct ethanol production from Japanese cedar wood was improved compared to wild type. To our knowledge, this study is the first report of the molecular breeding of lignin-degrading brown-rot fungus and direct ethanol production from softwoods by co-transformation with laccase overproduction constructs.

Expression of a manganese peroxidase isozyme 2 transgene in the ethanologenic white rot fungus Phlebia sp. strain MG-60

BackgroundThe white-rot fungus Phlebia sp. strain MG-60 was proposed as a candidate for integrated fungal fermentation process (IFFP), which unifies aerobic delignification and semi-aerobic consolidated biological processing by a single microorganism based on its ability to efficiently degrade lignin and ferment the sugars from cellulose. To improve IFFP, the development of a molecular breeding method for strain MG-60 is necessary. The purpose of this study is to establish the transformation method for the strain MG-60 and to obtain the over-expressing transformants of lignin-degrading enzyme, manganese peroxidase.FindingsIn the present study, the expression vector regulated by Phlebia brevispora glyceraldehyde-3-phosphate dehydrogenase promoter and terminator was constructed. A polyethylene glycol transformation method for the ethanol-fermenting white-rot fungus Phlebia sp. MG-60 was established with high transformation efficiency, and the manganese peroxidase isozyme 2 gene (MGmnp2) transformants were obtained, showing higher MnP activity than control transformants. MGmnp2 transformants showed higher selective lignin degradation on Quercus wood powder.ConclusionsThis first report of MG-60 transformation provides a useful methodology for widely accessible to interested researches. These results indicate the possibility of metabolic engineering of strain MG-60 for improving IFFP.

Construction of an endogenous selectable marker gene for the rice straw degrading white-rot basidiomycete Cyathus stercoreus.

Cyathus stercoreus has a superior ability to increase the cellulase saccharification yields of rice straw under biological pretreatment. As a first step to improve the biological pretreatment efficiency of C. stercoreus further, a new gene transformation system was constructed. Uracil auxotrophic mutant uv-180 was generated as host strain by ultraviolet radiation. It was transformed using plasmid pGE containing the orotate phosphoribosyltransferase (CsURA5) and enhanced green fluorescent protein (egfp). Many transformants exhibited strong fluorescence, indicating successful genetic transformation. An intron was needed for the expression of egfp. The EGFP fluorescence intensities of the three transformants did not decay even after subculturing on uracil-containing semisolid medium 5 times, suggesting that the transformed plasmids were stably retained in the absence of selective pressure.

Evaluation of the anti-hyperglycemic effect and safety of microorganism 1-deoxynojirimycin

1-Deoxynojirimycin (DNJ) is a potent α-glucosidase inhibitor and thus beneficial for prevention of diabetes. While we have succeeded in obtaining the culture supernatant extract (CSE) rich in DNJ from microorganism source, information regarding its anti-hyperglycemic effect and safety were still limited. Therefore, this study was aimed to evaluate the anti-hyperglycemic effect and safety of microorganism DNJ. Oral sucrose tolerance test was performed, and the result showed that CSE was able to significantly suppress the blood glucose elevation and suggested DNJ as the main active compound. To determine its safety, the absorption and excretion of microorganism DNJ were evaluated using 15N labeling method. Our findings investigated the recovery rate of 15N from DNJ reached 80% up to 48 hours after oral administration, suggesting its rapid excretion, suggesting the safety of DNJ. This study verified the functional properties and safety of DNJ from microorganisms, suggesting its potential use for functional purpose.