Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface‐active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino‐terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene‐disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low‐temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of HLA-A24-restricted epitopes with high affinities to Hsp70 using peptide arrays

Heat shock protein 70 (Hsp70) family members are known as facilitators of immune responses by interacting with receptors on antigen-presenting cells leading to Hsp70-peptide uptake and antigen cross priming. Here, identification of human leukocyte antigen (HLA)-A24-restricted epitopes was achieved using peptide arrays for evaluation of their affinities to Hsp70 and HLA-A24 binding prediction tools. Using Hsp70 as the model antigen, the GYPVTNAVI and VFQHGKVEI peptides were identified as antigens. These peptides actually bound to HLA-A24 in the stabilization assay using T2-A*2402 cells, and induced a strong peptide-reactive cytotoxic T lymphocyte response in HLA-A24 transgenic mice after vaccination.     

Phytoextraction by a high-Cd-accumulating rice: reduction of Cd content of soybean seeds

Soybeans (Glycine max (L) Merr.) are the major summer crop grown in Japanese upland fields (characterized by aerobic soil) that have been converted from paddies. To evaluate the effect of phytoextraction by rice on the seed cadmium (Cd) content of soybeans grown subsequently, we grew Milyang 23, a high-Cd-accumulating rice cultivar, and then grew soybeans in three paddy soils contaminated with moderate Cd concentrations (2.50-4.27 mg Cd kg(-1)). The rice accumulated 7-14% of the total soil Cd in its shoots. The soybean seed Cd contents were 24-46% less than those grown on control soils. Phytoextraction by Milyang 23 rice is thus a promising remediation method for reducing seed Cd contents of soybeans grown on paddy soils under aerobic soil conditions.     

Myosin is solubilized in a neutral and low ionic strength solution containing l-histidine

Myosin, one of the major myofibrillar proteins, is insoluble at low and physiological ionic strength and soluble at high ionic strength. In this study, the behavior and morphology of myosin solubilized in a low ionic strength solution containing l-histidine (l-His) was investigated. More than 80% of myosin was solubilized in a low ionic strength solution with dialysis against a solution containing 1 mM KCl and 5 mM l-His. Transmission electron microscopy with rotary shadowing demonstrated that the rod of myosin in a low ionic strength solution containing l-His is longer than that of myosin in a high ionic strength solution. The elongation of the myosin rod in a low ionic strength solution containing l-His would inhibit the formation of a filament, resulting in the solubilization of myosin.     

Distribution, diversity and regulation of alcohol oxidase isozymes, and phylogenetic relationships of methylotrophic yeasts

In this study, we attempted to classify the methylotrophic yeasts based on diversities of alcohol oxidase (AOD), i.e. zymogram patterns and partial amino acid sequences. According to zymogram patterns for AOD, members of the methylotrophic yeasts separate into two major lineages, one group involving strains having a single AOD and the other group, including Pichia methanolica, Candida pignaliae and C. sonorensis, showing nine AOD isozymes. Based on partial amino acid sequences of AOD, the methylotrophic yeasts could be divided into five groups, and this classification agrees mostly with grouping based on 26S domain D1/D2 rDNA nucleotide sequences, except for some strains. Moreover, the strains having AOD isozymes constitute one group with P. trehalophila, P. glucozyma and Pichia sp. strain BZ159, although these strains are divided into two types, based on amino acid sequences of second AODs. On the other hand, these AOD isozymes consist of two subunits; the first subunits are induced not only by methanol but also by glycerol and pectin, although the second subunits are mainly induced by methanol. These data indicate that AOD isozymes and second AOD genes distribute widely in several methylotrophic yeasts in the natural environment, and second AOD genes may have evolved as methylotrophic genes that can adapt to the environmental conditions of higher methanol concentrations.