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. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.

No abstract

No abstract

Here we report the cloning and characterization of cDNA for a different type of PLD (rat PLD2 (rPLD2)) from rat brain. We synthesized highly degenerate amplimers corresponding to the conserved regions of eukaryote PLDs and performed polymerase chain reaction on a rat brain cDNA library. Using the amplified sequence as the probe, we cloned a rat brain cDNA clone that contained an open reading frame of 933 amino acids with an M r of 105,992. The deduced amino acid sequence showed significant similarity to hPLD1 with a large deletion in the middle of the sequence. When the sequence was expressed in the fission yeast Schizosaccharomyces pombe, PLD activity was greatly increased. The activity was markedly stimulated by phosphatidylinositol 4,5-bisphosphate, but not by ADPribosylation factor 1 and RhoA. Rat brain cytosol known to stimulate small GTP-binding protein-dependent PLD did not stimulate rPLD2 expressed in S. pombe. The transcript was detected at significant levels in brain, lung, heart, kidney, stomach, small intestine, colon, and testis, but at low levels in thymus, liver, and muscle. Only a negligible level was found in spleen and pancreas. Thus rPLD2 is a novel type of PLD dependent on phosphatidylinositol 4,5-bisphosphate, but not on the small GTP-binding proteins ADP-ribosylation factor 1 and RhoA. Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline (1). A variety of signal molecules such as hormones, neurotransmitters, and growth factors are known to induce the activation of PLD in a wide range of cell types. Hence PLD is implicated in a broad spectrum of physiological processes and diseases, including metabolic regulation, inflammation, secretion, mitogenesis, oncogenesis, neural and cardiac stimulation, diabetes, and senescence (for reviews, see Ref.2). Despite its crucial importance in signal transduction, the molecular structure and characteristics of PLD enzyme are only poorly understood.Multiple PLD isoforms exist in mammalian tissues. Several factors were reported to stimulate PLD activity in vitro, including unsaturated fatty acid (3), phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (4), monomeric GTP-binding proteins (G proteins) such as ADP-ribosylation factor 1 (ARF1) (5, 6) and RhoA (7, 8), protein kinase C (9), and calmodulin (10). Massenburg et al. (11) showed that two major forms of PLD activity in rat brain membranes can be separated into ARF-dependent and oleate-dependent enzymes, clearly indicating that these are distinct isoforms. Both oleate-dependent and ARF-dependent types of PLD were recently highly purified from pig lung and brain, respectively (12, 13). In addition, there may be multiple forms of small G protein-dependent PLD including ARF-sensitive, RhoA-sensitive, and ARF-, RhoA-sensitive PLDs. Siddiqi et al. (14) reported that the cytosolic fraction of HL-60 cells contained a soluble PLD activated by ARF, but not RhoA. Malcolm et al. (8) showed rat liver plasma membrane PLD to be sensitive to RhoA, but not to ARF. PL...

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD + -dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52-and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k cat /K m with NADH)/(k cat /K m with NADPH)] compared with the wild-type (WT), which was due to decrease of k cat with NADPH in the R276H mutant and increase of K m with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP + and NADH/NAD + ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

Pichia stipitis NAD؉ -dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD ؉ to NADP ؉ and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp 207 ؉ -dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP ؉ .Xylose is one of the major components of hemicellulose, the second most abundant carbohydrate polymer in nature. Efficient utilization of xylose is required to develop economically viable processes for producing biofuels such as ethanol from biomass (for recent reviews, see Refs. 1-4). Yeasts have long been used for the production of alcoholic beverages such as wine, beer, and Japanese sake. In particular, Saccharomyces cerevisiae has been used widely because of the ability to produce high concentrations of ethanol and high inherent ethanol tolerance. The native strains, however, cannot ferment xylose as a carbon source. The major strategy for the generation of xylose-fermenting S. cerevisiae is to introduce genes involved in xylose metabolism from other organisms (Scheme 1). In xylosefermenting fungi such as Pichia stipitis, xylose is converted into xylulose by the sequential action of two oxidoreductases. First, xylose reductase (alditol:NADP ϩ 1-oxidoreductase, EC 1.1.1.21) catalyzes reduction of the C1 carbonyl group of xylose, yielding xylitol as the product. Xylitol is then oxidized by xylitol dehydrogenase (XDH 1 ; EC 1.1.1.9) to give xylulose. S. cerevisiae transformed with these two genes from P. stipitis could ferment xylose to ethanol. There is another problem; the excretion of xylitol occurs unless a co-metabolizable carbon source such as glucose is added. This is probably caused by several combined factors. In particular, intercellular redox imbalance due to a different coenzyme specificity of xylose reductase (with NADPH) and XDH (with NAD ϩ ) has been thought to be one of the main factors (2). The generation of an NADP ϩ -dependent XDH by protein engineering would avoid this problem.XDH is a medium-chain dehydrogenase/reductase (MDR), which constitutes a large enzyme superfamily consisting of about 1000 members (for reviews, see Refs. 5 and 6). The superfamily is classified into eight subfamilies based on amino acid sequence alignment and the structural similarity of substrates. XDH belongs to the polyol dehydrogenase ...

Many bacteria are able to grow with L-arabinose as the sole carbon and energy source, and the bacterial pathway of L-arabinose metabolism has been extensively investigated. Many bacteria including Escherichia coli depend on protein products of the araBAD operon, which contains araB (ribulokinase, EC 2.7.1.16), araA (L-arabinose isomerase, EC 5.3.1.4) and araD (L-ribulose-phosphate 4-epimerase, EC 5.1.3.4) to convert L-arabinose to D-xylulose 5-phosphate through L-ribulose and L-ribulose 5-phosphate (1). In a recently characterized fungal pathway (2-4), L-arabinose is also converted to D-xylulose 5-phosphate but through different intermediates by two reductases, two dehydrogenases, and a kinase. On the other hand, it is believed that a hypothetical pathway of L-arabinose metabolism is operative in some bacteria (5-13) (Fig. 1A). In this pathway, L-arabinose is oxidized to L-arabino-␥-lactone by NAD(P) ϩ -dependent dehydrogenase. The lactone is cleaved by a lactonase to L-arabonate, followed by two successive dehydration reactions forming L-2-keto-3-deoxyarabonate (L-KDA) 2 and ␣-ketoglutaricsemialdehyde(␣KGSA).ThelaststepistheNAD(P) ϩ -dependent dehydrogenation of ␣KGSA to ␣-ketoglutaric acid (referred to as the first pathway). Alternatively, L-KDA is cleaved through an aldolase reaction to glycolaldehyde and pyruvate (referred to as the second pathway).

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrPC) of PrP into an abnormal form (PrPSc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrPC is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrPSc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrPC, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.