Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis.

Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities. Mass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification. LC/MS/MS was found to be more sensitive and to provide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation.

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A.

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery.

Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins. Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus alpha-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted alpha-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the alpha-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiently obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast.

Cello-oligosaccharide hydrolysis by cellobiohydrolase II from Trichoderma reesei. Association and rate constants derived from an analysis of progress curves.

The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1-12 s-1 at 27 degrees C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for collotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169.

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.

Characterization of a double cellulose-binding domain. Synergistic high affinity binding to crystalline cellulose.

Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide-stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli, the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD components was investigated on different types of cellulose substrates as well as chitin. Under saturating conditions, nearly 20 micromol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhibited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase.

Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins.

The promoter of alcohol dehydrogenase I of the yeast Saccharomyces cerevisiae was studied using Bacillus amyloliquefaciens alpha-amylase as a marker protein. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase. When 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (Ruohonen et al. (1991) Yeast 7, 337-346). We have now restored 300 bp (from -414 bp to -700 bp) upstream of the deletion site and obtained expression from the ADH1 promoter throughout the yeast growth cycle. The restored sequence allowed alpha-amylase expression to start during early exponential growth phase indicating that it is required for activation of the ADH1 promoter during ethanol production growth phase, possibly through glucose induction. On ethanol, all the promoters were active, but the short promoter was temporally activated first, suggesting that the restored sequence is not required for promoter activity during early oxidative growth.

The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei.

Cellulose is the major polysaccharide of plants where it plays a predominantly structural role. A variety of highly specialized microorganisms have evolved to produce enzymes that either synergistically or in complexes can carry out the complete hydrolysis of cellulose. The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has been determined and refined to 1.8 angstrom resolution. The molecule contains a 40 angstrom long active site tunnel that may account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose. The active site residues were identified by solving the structure of the enzyme complexed with an oligosaccharide, o-iodobenzyl-1-thio-beta-cellobioside. The three-dimensional structure is very similar to a family of bacterial beta-glucanases with the main-chain topology of the plant legume lectins.

Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.

Cloning and sequencing of the yeast Saccharomyces cerevisiae SEC1 gene localized on chromosome IV.

The SEC1 gene of yeast Saccharomyces cerevisiae was cloned by complementing the temperature-sensitive mutation of sec1-1 at 37 degrees C, and its nucleotide sequence was determined. SEC1 is a single copy gene and encodes a protein of 724 amino acids and 83,490 daltons with a predicted pI value of 6.11. Hydrophobicity plotting showed no clearly hydrophobic regions suggesting a soluble nature for the protein. Amino acid sequence comparisons revealed no obvious homologies with the proteins in the SWISSPROT databank. Two consensus sequence for the cdc2 encoded protein kinase recognition site were revealed within Sec1p. The codon usage suggests a low expression level for SEC1. The 5' non-translated region contains two TATA-like sequences at -52 and -215 nucleotides from the translation start site. Two potential regulatory sequences for DNA binding proteins were found in the non-coding 5' region: a HAP2/HAP3 consensus recognition sequence at nucleotide-154 and a BAF1 consensus recognition sequence at nucleotide-136. The SEC1 specific probe detected a 2400 nucleotides long transcript, which was in reasonable agreement with the 2172 nucleotides long open reading frame.

Optimization of Bacillus alpha-amylase production by Saccharomyces cerevisiae.

Production of Bacillus amyloliquefaciens alpha-amylase by Saccharomyces cerevisiae using the multicopy plasmid pAAH5 and ways of improving the yields of secreted enzyme were studied. In standard non-buffered medium, alpha-amylase was rapidly inactivated but stabilization of the pH at 6 led to stable accumulation of alpha-amylase in the culture medium. Removal of 1100 bp of the upstream sequence of the ADH1 promoter present on pAAH5 resulted in delayed but increased alpha-amylase production: 29-fold in selective medium, two-fold in non-selective medium. With the original ADH1 promoter, accumulation of alpha-amylase in the medium started to level off before the cultures reached stationary phase and was very low when exponentially growing cells were transferred from glucose to ethanol. This coincided with the appearance of a mRNA larger than the alpha-amylase messenger. With the shortened promoter, the normal-size alpha-amylase mRNA was detected under all growth conditions and alpha-amylase was efficiently secreted into the medium also late in stationary phase and after transfer to ethanol. Highest total yields of alpha-amylase were obtained with the short promoter in non-selective glucose-containing medium; this may be explained by the greater final cell density obtained. However, the production of alpha-amylase per cell mass was higher in ethanol-containing selective medium. Seventy to eighty per cent of the alpha-amylase activity was secreted into the medium independent of the total amount produced.

Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from Saccharomyces cerevisiae host cells [corrected].

The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.

Nonstructural proteins of Semliki Forest virus: synthesis, processing, and stability in infected cells.

The synthesis of the nonstructural (ns) proteins of Semliki Forest virus was studied in vivo. The fourth ns protein, ns60, was identified and isolated. The order of translation (NH2-ns70-ns86-ns60-ns72-COOH) was determined by using various labeling procedures after or in the presence of a hypertonic block of translation initiation. A sequential labeling procedure was devised to specifically label defined segments of the polyprotein. The specific labeling procedures allowed isolation of the four ns proteins in radiochemically pure form by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The four ns proteins were shown to have different primary structures by digestion with V8 protease of Staphylococcus aureus. The processing of the ns polyprotein and the stability of the mature ns proteins were studied by pulse-chase experiments. The cleavage of each of the proteins from the polyprotein took place within 2 to 3 min after the translation of the polypeptide chain. The N-terminal protein, ns70, appeared in its mature form later than ns86, which follows it in the polyprotein, suggesting that ns70 undergoes a post-translational modification. The migration of the C-terminal protein, ns72, immediately after a pulse was slightly faster than after a chase, suggesting that ns72 also undergoes a post-translational modification other than a cleavage. The half-life of ns72 was shorter than that of the other ns proteins.