Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation

A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.     

Enhancement of Coenzyme Q10 Accumulation by Mutation and Effects of Medium Components on the Formation of Coenzyme Q Homologs by Pseudomonas N842 and Mutants

Mutation of Pseudomonas N842 was carried out to increase CoQ₁₀ production. The productivity of CoQ₁₀ was improved considerably by repeated mutation, and the content of CoQ₁₀ per unit cell of the fifth generation mutant was approximately 6 times that of the wild strain, Pseudomonas N842. CoQ₁₁, which was hardly detectable in the wild strain, increased significantly by mutation, and the ratio of CoQ₁₁ to total CoQ exceeded 20% in the fourth generation mutant. Intermittent feeding of glucose to the culture medium during cultivation increased cell yield and CoQ production. When total glucose added was 5 times that of basal medium, cell yield and CoQ formation respectively increased about 3 and 4 times.     

Chloroplast DNA phylogeny of subtribe Dendrobiinae (Orchidaceae): Insights from a combined analysis based onrbcL sequences and restriction site variation

Phylogenetic analyses using two chloroplast DNA data sets, derived from variation of the ribulose-bisphosphate carboxylase gene (rbcL) and restriction sites, were performed to examine relationships among 13 taxa in subtribe Dendrobiinae, one of the most taxonomically complicated groups in Orchidaceae, and its putative sister groups. Owing to a limited number of informative substitutions, therbcL data set did not provide conclusive evidence in itself. The data set combiningrbcL and restriction site mutations, however, provided the following insights: (1)Pseuderia belongs with tribe Podochileae rather than tribe Dendrobieae. (2) Subtribe Dendrobiinae is monophyletic ifPseuderia is excluded. (3) ExcludingPseuderia, Dendrobiinae comprises three major clades: Clade 1 (Dendrobium sectionSpatulata, Cadetia, Diplocaulobium, andFlickingeria); Clade 2 (Dendrobium sectionsDendrobium andCallista); and Clade 3 (Epigeneium). (4)Epigeneium diverged early from the lineage including Clades 1 and 2. (5) Relative toCadetia, Diplocaulobium, andFlickingeria, Dendrobium is shown to be para-/polyphyletic. (6)Diplocaulobium andFlickingeria constitute a monophyletic clade, from which cladeDendrobium sectionSpatulata andCadetia form succesive sister groups. Among these results, (1) and (5) are especially stable in view of the congruence between the separate and combined analyses as well as robust internal support.     

A role of EphB4 receptor and its ligand,ephrin-B2, in erythropoiesis

Erythropoiesis is regulated not only by erythropoietin but also by microenvironments which are composed of transmembrane molecules. We have previously shown that a receptor tyrosine kinase EphB4 is predominantly expressed on human erythroid progenitors in bone marrow. EphB4 is expressed in approximately 45% of hematopoietic progenitor cells, which are CD34-positive and c-Kit-positive in human umbilical cord blood (hUCB). The transmembrane ligand for EphB4 or ephrin-B2 is expressed on bone marrow stromal cells and arterial endothelial cells. When such EphB4-positive hematopoietic progenitor cells were co-cultured with stromal cells which express ephrin-B2, they were immediately detached from stromal cells and differentiated to mature erythroid cells. At that time, expression of EphB4 immediately down-regulated. In contrast, on ephrin-B2 non-expressing stromal cells, they remained EphB4-positive cells and the generated number of mature erythroid cells was less than that on ephrin-B2 expressing stromal cells. Additionally, ephrin-B2 expression on endothelial cells up-regulated under hypoxic condition. Taken together, we propose that one of the molecular cues that regulate erythropoiesis is ephrin-B2 on stromal cells.     

Structure of the quinoline N-hydroxylating cytochrome P450 RauA,an essential enzyme that confers antibiotic activity on aurachin alkaloids

The cytochrome P450 RauA from Rhodococcus erythropolis JCM 6824 catalyzes the hydroxylation of a nitrogen atom in the quinolone ring of aurachin, thereby conferring strong antibiotic activity on the aurachin alkaloid. Here, we report the crystal structure of RauA in complex with its substrate, a biosynthetic intermediate of aurachin RE. Clear electron density showed that the quinolone ring is oriented parallel to the porphyrin plane of the heme cofactor, while the farnesyl chain curls into a U-shape topology and is buried inside the solvent-inaccessible hydrophobic interior of RauA. The nearest atom from the heme iron is the quinolone nitrogen (4.3 Å), which is consistent with RauA catalyzing the N-hydroxylation of the quinolone ring to produce mature aurachin RE.     

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d ‐xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro‐organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β‐d ‐glucosidase (Aspergillus aculeatus β‐glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5‐fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.     

Loss of Catalase-1 (Cat-1) results in decreased conidial viability enhanced by exposure to light in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Light is one of the most important factors inducing morphogenesis in Neurospora crassa. The reception of light triggers the generation of reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂). Catalase-1 (Cat-1) is one of three catalases known to detoxify H₂O₂ into water and oxygen. We reported that the photomorphogenetic characteristics of mutants in nucleoside diphosphate kinase-1 (NDK-1), a light signal transducer, are severely affected, and NDK-1 interacted with Cat-1 in a yeast two-hybrid assay. To disclose the function of Cat-1, we created a Cat-1 loss-of-function mutant (cat-1 ^RIP ) by the repeat induced point-mutation (RIPing) method. No Cat-1 activity was detected in the mutant strain. Forty guanines were replaced with adenines in the cat-1 gene of cat-1 ^RIP , which caused 30 amino acid substitutions. The mutant strain grew normally, but its conidia and mycelia were more sensitive to H₂O₂ than those of the wild type. The lack of Cat-1 activity also caused a significant reduction in the conidial germination rate. Furthermore, light enhanced this reduction in cat-1 ^RIP more than that in the wild type. Introduction of cat-1 into the mutant reversed all of these defective phenotypes. These results indicate that Cat-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.