We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13°C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDCSS, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIUIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase.

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Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a beta-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n [TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeasts. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran's starch producing similar amounts of ethanol (5.3 +/- 0.14 g/L and 5.0 +/- 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisatio

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wildtype and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEMÒ-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k cat ) for D-xylose compared with the wild-type enzyme at 60°C, but they did not show any increase in catalytic eciency (k cat /K m ). For D-glucose, both the k cat and the k cat /K m values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K i ) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular¯exibility of the mutant enzymes at low temperatures.

The xynB gene encoding the Bacillus pumilus beta-xylosidase was expressed separately and jointly with the Trichoderma reesei beta-xylanase (xyn2) gene in the yeast Saccharomyces cerevisiae. Both genes were placed under the transcriptional control of the glucose-derepressible alcohol dehydrogenase 2 promoter (ADH2p) and terminator (ADH2T) sequences. The xynB gene was fused in frame to the yeast mating factor alpha1 secretion sequence (MFalpha1s) to effect secretion in S. cerevisiae. The fusion protein was designated Xlo1. Xlo1 produced in S. cerevisiae exhibited low affinity for xylobiose, but eventually hydrolyzed xylobiose and xylotriose to the monomeric constituent, D-xylose. Coproduction of Xyn2 and Xlo1 by S. cerevisiae led to a 25% increase in the amount of reducing sugars released from birchwood xylan compared to S. cerevisiae producing only the Xyn2 beta-xylanase. However, no D-xylose was produced from birchwood xylan, presumably due to very low Xlo1 beta-xylosidase activity and its low affinity for xylobiose.