A Novel Production Method for High-Fructose Glucose Syrup from Sucrose-Containing Biomass by a Newly Isolated Strain of Osmotolerant Meyerozyma guilliermondii.

One osmotolerant strain from among 44 yeast isolates was selected based on its growth abilities in media containing high concentrations of sucrose. This selected strain, named SKENNY, was identified as Meyerozyma guilliermondii by sequencing the internal transcribed spacer regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA. SK-ENNY was utilized to produce high-fructose glucose syrup (HFGS) from sucrose-containing biomass.

Quantitative analysis of location- and sequence-dependent deamination by APOBEC3G using real-time NMR spectroscopy.

The human antiretroviral factor APOBEC3G (A3G) deaminates the newly synthesized minus strand of the human immunodeficiency virus 1 (HIV-1), which results in the abolition of the infectivity of virus-infectivity-factor (Vif)-deficient HIV-1 strains.1-6 A unique property of A3G is that it deaminates a CCC hot spot that is located close to the 5' end more effectively than one that is less close to the 5' end. However, the mechanism of this process is elusive as it includes nonspecific binding of A3G to DNA and sliding of A3G along the DNA strand.

A protein adaptor to locate a functional protein dimer on molecular switchboard.

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard.

Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP(+)-dependent xylitol dehydrogenase.

The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+).

Anti-prion activity of an RNA aptamer and its structural basis.

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir.

Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR.

'Intelligent' ribozyme whose activity is altered in response to K+ as a result of quadruplex formation.

The structure of r(GGAGGAGGAGGA) (R12) changes from a single-stranded form to a compact quadruplex one in response to K(+). In a hammerhead ribozyme, two portions of the catalytic core are linked with the stem and are located in close proximity in order to exert activity. In this study, the stem was replaced by R12 (or R11, which lacks the terminal A residue) with or without linker residues.

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S.

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis.

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation.

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes.

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO(2)-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5'-end of single stranded DNAs compared to when other nucleobases were in this position.

Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives.

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S.

Stabilization of the immobilized linkers and DNA probes for DNA microarray fabrication by end-capping of the remaining unreacted silanol on the glass.

High stability of the oligonucleotides immobilized on the glass is essential for the reliable DNA microarray analysis. In the present study, effect of end-capping of the unreacted silanol, remaining after the surface amine-functionalization, was explored: (1) Cy3-NHS (N-hydorxysuccinincimide) dye was spotted on the surface and change in the fluorescent intensity was measured. (2) DNA probes were immobilized by the reactivity of oxanine linked at the 5'-end, the complementary oligonucleotides with Cy5-fluorescence at the 5'-end was hybridized, and the time-dependence of the fluorescence intensity was observed.

Reinvestigation of the molecular influence of hypoxanthine on the DNA cleavage efficiency of restriction endonucleases BglII, EcoRI and BamHI.

Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base.

Efficient bioethanol production by a recombinant flocculent Saccharomyces cerevisiae strain with a genome-integrated NADP+-dependent xylitol dehydrogenase gene.

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

Reactive oxygen species generation through NADH oxidation by pterin derivatives.

Pterin is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities. In previous study, we have elucidated that 6FP derivatives in which the 3-position is modified possess reactive oxygen species (ROS), which are involved in the modulation of a variety of cell functions, generation activities through the oxidation of NADH to NAD(+) in the dark at neutral pH.

Reactivity of oxanine: efficient fabrication of DNA microarray by using oxanine-containing DNA oligomer as probe molecule.

Oxanine (Oxa), a mutagenic lesion generated from guanine by nitrosative oxidation, can make a covalent bonding with -NH(2) or -SH group since Oxa possesses O-acylisourea conformation in the base-ring structure. We employed such unique reactive functionality of Oxa for fabrication of DNA-immobilized system. When DNA oligomer with Oxa at 5'-end (Oxa-end DNA) is spotted on amine-functionalized surface, the DNA probe can be immobilized without any involvement of activation step or any treatment of chemical linker.

Efficient preparation of xanthine-containing oligodeoxynucleotide from oxanine-containing oligodeoxynucleotide, catalyzed by N alpha-acetyl-L-histidine.

Xanthine (Xan) and oxanine (Oxa) are major damage products from guanine (Gua) by NO- or HNO(2)-induced nitrosative deamination. Xan- and Oxa-containing oligodeoxynucleotides are essential substrates for the biochemical studies to reveal genotoxicity or mutagenesis raised by nitrosative oxidation. In previous study, we have developed chemical synthesis method for obtaining Oxa-ODN.

Expression of protein engineered NADP+-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae.

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S.

Bioethanol production from xylose by recombinant Saccharomyces cerevisiae expressing xylose reductase, NADP(+)-dependent xylitol dehydrogenase, and xylulokinase.

We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.

Construction of X-ray-inducible promoters through cis-acting element elongation and error-prone polymerase chain reaction.

Background: A promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA.

Biophysical stability and enzymatic recognition of oxanine in DNA.

Oxanine (Oxa), which is one of the major products generated from guanine by nitrosative oxidation and is as long-lived as Gua in DNA, has been thought to be one of the major causes for NO-induced DNA damage. In the present study, using several synthetic Oxa-containing oligodeoxynucleotides, biophysical stability and enzymatic recognition of Oxa was investigated in DNA strands. It was found that Oxa did not mediate marked distortion in the whole DNA structure although Oxa pairing with 4 normal bases decreased thermal stability of the DNA duplexes compared to Gua:Cyt base pair.

Comparison of enzymatic recognition of DNA-duplexes containing NO-induced lesions by DNA-relevant enzymes.

Nitric oxide (NO) produced in inflamed tissues is known to cause DNA damage by nitrosation or oxidation of base moieties. For investigating the biophysical and biochemical properties of DNA lesions induced by nitric oxide, we prepared synthetic DNA oligomers and analyzed melting temperature (T(m)) or enzymatic recognition of DNA strands containing oxanine (Oxa) and hypoxanthine (Hyp), using guanine (Gua) or adenine (Ade) as control bases, respectively. For enzymatic reaction by DNA-relevant enzymes, we employed T4 polynucleotide kinase, T4 DNA ligase and DNA polymerase (Klenow fragment (exo-)).

Development of stable DNA microarray platforms suitable for quantitative analysis.

DNA microarray has become one of the most indispensable tools for genome analysis, medical diagnosis and molecular biological studies. In this study, to make DNA microarray more reliable and efficient, we employed additional silanization modification on aminoalkoxysilane-treated surface. In addition, oxanine was used as a linker of DNA probe, which does not require any chemical activation step in probe immobilization.

Chemical natures of 6-formylpterin nucleoside analogs.

Pterin is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological activities. In the present study, we have developed the synthetic procedure for nucleoside analogs of 6FP and prepared 1-(beta-D-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF) and 1-(beta-D-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF) in which the 1-position is glycosylated.

Functional reactivity of oxanine: its biological meanings and biotechnological applications.

Oxanine (Oxa), generated as one of the major products from guanine by nitrosative oxidation, has been focused as a mutagenic lesion. Here, Oxa was explored in terms of its unique property to react with - NH2 or -SH group since Oxa possesses O-acylisourea conformation in the base-ring structure. In particular, Oxa has been characterized in terms of its formation and mechanism of DNA-protein cross-link (DPC).