Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum-shifted luminescence-emitting mutants and their application in nuclear receptor-reporter assays.

Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum-shifted luminescence have not yet been reported.

Pinellic Acid Isolated from Quercetin-rich Onions has a Peroxisome Proliferator-Activated Receptor-Alpha/Gamma (PPAR-α/γ) Transactivation Activity.

Quercetin, a flavonol, is a functional compound that is abundant in onions and is known to have antioxidant and anti-inflammatory effects. Quercetin and its glucoside are known to function as peroxisome proliferator-activated receptor (PPAR) ligands and showed high PPAR- transactivation activity but little PPAR- transactivation activity in some reports. In this study, we demonstrated that an aqueous extract of a quercetin-rich onion cultivar increased transactivation activities not only of PPAR- but also of PPAR-.

Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in .

Background: Expression of d-xylose isomerase having high catalytic activity in () is a prerequisite for efficient and economical production of bioethanol from cellulosic biomass. Although previous studies demonstrated functional expression of several xylose isomerases (XI) in , identification of XIs having higher catalytic activity is needed. Here, we report a new strategy to improve xylose fermentation in the strain IR-2 that involves an evolutionary engineering to select top-performing XIs from eight previously reported XIs derived from various species.

Whole-Genome Sequence of an Isogenic Haploid Strain, Saccharomyces cerevisiae IR-2A30(a), Established from the Industrial Diploid Strain IR-2.

We present the draft genome sequence of an isogenic haploid strain, IR-2A30( ), established from IR-2. Assembly of long reads and previously obtained contigs from the genome of diploid IR-2 resulted in 50 contigs, and the variations and sequencing errors were corrected by short reads.

Systematic optimization of gene expression of pentose phosphate pathway enhances ethanol production from a glucose/xylose mixed medium in a recombinant Saccharomyces cerevisiae.

The pentose phosphate pathway (PPP) plays an important role in the synthesis of ribonucleotides and aromatic amino acids. During bioethanol production from cellulosic biomass composed mainly of D-glucose and D-xylose, the PPP is also involved in xylose metabolism by engineered Saccharomyces cerevisiae. Although the activities and thermostabilities of the four PPP enzymes (transaldolase: TAL1, transketolase: TKL1, ribose-5-phosphate ketol-isomerase: RKI1 and D-ribulose-5-phosphate 3-epimerase: RPE1) can affect the efficiency of cellulosic ethanol production at high temperatures, little is known about the suitable expression levels of these PPP genes.

Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in Saccharomyces cerevisiae IR-2 for high-temperature ethanol production.

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures.

Isolation and characterization of xylitol-assimilating mutants of recombinant Saccharomyces cerevisiae.

To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele.

Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae.

The secretion efficiency of foreign proteins in recombinant microbes is strongly dependent on the combination of the signal peptides (SPs) used and the target proteins; therefore, identifying the optimal SP sequence for each target protein is a crucial step in maximizing the efficiency of protein secretion in both prokaryotes and eukaryotes. In this study, we developed a novel method, named the SP optimization tool (SPOT), for the generation and rapid screening of a library of SP-target gene fusion constructs to identify the optimal SP for maximizing target protein secretion. In contrast to libraries generated in previous studies, SPOT fusion constructs are generated without adding the intervening sequences associated with restriction enzyme digestion sites.

Evidence of environmental and vertical transmission of Burkholderia symbionts in the oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae).

The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria.

Draft Genome Sequence of Saccharomyces cerevisiae NAM34-4C, a Lactic Acid-Assimilating Industrial Yeast Strain.

We determined the genome sequence of industrial Saccharomyces cerevisiae strain NAM34-4C, which would be useful for bioethanol production. The approximately 11.5-Mb draft genome sequence of NAM34-4C will provide remarkable insights into metabolic engineering for effective production of bioethanol from biomass.

Draft Genome Sequence of Saccharomyces cerevisiae IR-2, a Useful Industrial Strain for Highly Efficient Production of Bioethanol.

We sequenced the genome of Saccharomyces cerevisiae IR-2, which is a diploid industrial strain with flocculation activity and the ability to efficiently produce bioethanol. The approximately 11.4-Mb draft genome information provides useful insights into metabolic engineering for the production of bioethanol from biomass.

Total biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α: heterologous expression of four biosynthetic genes in Aspergillus oryzae.

Clustering of biosynthetic genes for producing fungal secondary metabolites, which frequently consist of less than ten genes, has been recognized with numerous genomes. The heterologous expression of whole genes in the clusters will therefore produce various types of natural products when using a suitable fungal host. We introduced the whole gene cluster for the biosynthesis of diterpene aphidicolin into the fungal quadruple auxotrophic host, Aspergillus oryzae, by using four different vectors (pTAex3, pPTRI, pUSA and pAdeA) which harbor a starch-inducible promoter/terminator to examine the expression conditions.

Chemical studies on the BRET system between the bioluminescence of Cypridina and quantum dots.

We report a bioluminescence resonance energy transfer (BRET) system between Cypridina luciferase (Cluc) and QDs. After coupling of the biotinylated Cluc with streptavidin-QDs, a BRET emission of QDs through the resonance energy transfer from the oxidation of Vargula (Cypridina) luciferin was observed. A luciferin analogue having a naphtyl group at C6-position showed a large blue shift of the emission spectrum, which improved the ratio of this BRET system.

Introduction of amino acid residues at the N-terminus of the zeocin-resistance protein increases its expression in Saccharomyces cerevisiae.

Nucleotide sequences proximal to the initiation codon of a gene are known to affect the expression efficiency of that gene. We screened 10-bp random sequences upstream of the initiation codon of the zeocin-resistance gene to identify sequences that could enhance its expression in Saccharomyces cerevisiae. Of the isolated sequences, 20 sequences exhibited a common feature, i.

Sensitive and convenient yeast reporter assay for high-throughput analysis by using a secretory luciferase from Cypridina noctiluca.

The yeast reporter assay has been widely used in various applications such as detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system. The molecular characteristics of the reporter enzyme are critical determinants for this assay. We herein report the establishment of a novel yeast reporter assay using a secretory luciferase, Cypridina noctiluca luciferase (CLuc), as an alternative to the conventional beta-galactosidase.

Molecular cloning, expression, and characterization of a major 38-kd cochineal allergen.

Background: Carmine is a natural red pigment obtained from dried gravid female cochineal insects (Dactylopius coccus or Coccus cacti). There have been several reports of allergies to carmine, but the major allergens responsible have not been identified.

Objective: To identify the major allergenic proteins in cochineal.

Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins.

Yeast cells producing mammalian-type N-linked oligosaccharide show severe growth defects and the decreased protein productivity because of the disruption of yeast-specific glycosyltransferases. This decreased protein productivity in engineered yeast strains is an obstacle to the development of efficient glycoprotein production in yeast. For economic and effective synthesis of such therapeutic glycoproteins in yeast, the development of appropriate strains is highly desirable.

Yeast mutant with efficient secretion identified by a novel secretory reporter, Cluc.

Yeast is an important host for the production of pharmaceutical or industrial proteins by virtue of its genetic information and easy handling. A number of heterologous proteins have been produced and purified from yeast cell cultures as secreted forms. Here, we describe a novel screening system of Saccharomyces cerevisiae and its application to improve the secretion efficiency of yeast.

Preparation of biotinylated cypridina luciferase and its use in bioluminescent enzyme immunoassay.

Cypridina luciferase, a well-known secretory enzyme involved in the bioluminescence of marine ostracod, is used to monitor gene expression in mammalian cells. Here we report the preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay (BLEIA). Recombinant Cypridina luciferase was expressed in yeast and successfully purified to near homogeneity.

Relationship between the size of the bottleneck 15 A from iron in the main channel and the reactivity of catalase corresponding to the molecular size of substrates.

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase.

Cultivation of microorganisms in the cultural medium made from squid internal organs and accumulation of polyunsaturated fatty acids in the cells.

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs.

Knowledge-assisted recognition of cluster boundaries in gene expression data.

Background And Motivation: DNA microarray technology has made it possible to determine the expression levels of thousands of genes in parallel under multiple experimental conditions. Genome-wide analyses using DNA microarrays make a great contribution to the exploration of the dynamic state of genetic networks, and further lead to the development of new disease diagnosis technologies. An important step in the analysis of gene expression data is to classify genes with similar expression patterns into the same groups.

Isoform-specific expression and induction of udp-glucuronosyltransferase in immunoactivated peritoneal macrophages of the rat.

Phase I drug-metabolizing enzymes such as cytochrome P450 in immunocytes are known to play a role in metabolic activation of toxic and immunosuppressive compounds such as polycyclic aromatic hydrocarbon (PAH). UDP-glucuronosyltransferase (UGT), a drug-metabolizing phase II enzyme, accelerates elimination of these compounds; however, there is little information on the expression and function of UGT in immunocytes. In this study, we investigated the expressions of UGT isoforms in rat peritoneal macrophages and the role of UGT in macrophage functions.

Bitter gourd seed fatty acid rich in 9c,11t,13t-conjugated linolenic acid induces apoptosis and up-regulates the GADD45, p53 and PPARgamma in human colon cancer Caco-2 cells.

Bitter gourd (Momordica charantia) seed oil (BGO) is a unique oil which contains 9cis, 11trans, 13trans-conjugated linolenic acid (9c,11t,13t-CLN) at a high level of more than 60%. In this study, we investigated the anti-proliferative and apoptosis-inducing effects of free fatty acids prepared from BGO (BGO-FFA) using colon cancer Caco-2 cells. BGO-FFA and purified 9c,11t,13t-CLN remarkably reduced the cell viability of Caco-2.

A part of ice nucleation protein exhibits the ice-binding ability.

We generated a recombinant 96-residue polypeptide corresponding to a sequence Tyr176-Gly273 of ice nucleation protein from Pseudomonas syringae (denoted INP96). INP96 exhibited an ability to shape an ice crystal, whose morphology is highly similar to the hexagonal-bipyramid generally identified for antifreeze protein. INP96 also showed a non-linear, concentration-dependent retardation of ice growth.