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.

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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.