Cerevisiae Invertase Research Articles

Uptake of dimannoside clusters and oligomannosides by human dendritic cells.

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the alpha-amino group of epsilon-fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six alpha-D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys5-Ala-Cys-NH2). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.

Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.

Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.

Immobilization of invertase within calcium alginate gel capsules

Saccharomyces cerevisiae invertase was immobilized in alginate capsules. The immobilization resulted in 87% relative activity and activity for 36 days without decrease in activity. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4.3 and 60°C, respectively. Immobilized invertase was however, more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. Since the process is simple and invertase does not leak out of capsules, this method can be used for the industrial production of invert sugar.

High-level production of recombinant fungal endo-beta-1,4-xylanase in the methylotrophic yeast Pichia pastoris.

Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.

Metabolic engineering of invertase activities in different subcellular compartments affects sucrose accumulation in sugarcane cells

Transgenic sugarcane (Saccharum officinarum L.) lines were created to express altered invertase isoform activity to elucidate the role(s) of invertase in the sucrose accumulation process. A sugarcane soluble acid invertase cDNA (SCINVm, AF062734) in the antisense orientation was used to decrease invertase activity. The Saccharomyces cerevisiae invertase gene (SUC2), fused with appropriate targeting elements, was used to increase invertase activity in the apoplast, cytoplasm and vacuole. A callus/liquid culture system was established to evaluate change in invertase activity and sugar concentration in the transgenic lines. Increased invertase activity in the apoplast led to rapid hydrolysis of sucrose and rapid increase of hexose in the medium. The cellular hexose content increased dramatically and the sucrose level decreased. Cells with higher cytoplasmic invertase activity did not show a significant change in the sugar composition in the medium, but did significantly reduce the sucrose content in the cells. Transformation with the sugarcane antisense acid invertase gene produced a cell line with moderate inhibition of soluble acid invertase activity and a 2-fold increase in sucrose accumulation. Overall, intracellular and extracellular sugar composition was very sensitive to the change in invertase activities. Lowering acid invertase activity increased sucrose accumulation.

Raman spectroscopic studies of native and pressure‐ or temperature‐denatured invertase

A classical Raman vibrational spectroscopic study of conformational changes in Saccharomyces cerevisiae inver-tase after denaturation by temperature or pressure is described. Spectra were obtained from enzyme solutions and from lyophilized forms for native invertase and after pressure- or temperature-induced deactivation. For the native invertase spectra, tentative assignments of the main characteristic protein vibrations, as polypeptidic backbone, sugars, amides and amino acids vibrations are proposed. The irreversible conformational changes monitored by Raman spectroscopy for temperature- or pressure-induced deactivation were also followed. High-pressure denaturation of invertase leads to an increase in low-wavenumber scattering in solution and to a shift of amide I bands to lower wavenumbers, observed by recording spectra after lyophilization of pressure-treated samples. However, temperature denaturation does not show such modifications in the spectra, either for low-wavenumber scattering experiments in solution or for spectra recorded after lyophilization. Thus the microscopic processes involved in inactivation are different for temperature and pressure treatments and different conformational modifications are induced. © 1998 John Wiley & Sons, Ltd.

Raman spectroscopic studies of native and pressure- or temperature‐denatured invertase

A classical Raman vibrational spectroscopic study of conformational changes in Saccharomyces cerevisiae inver-tase after denaturation by temperature or pressure is described. Spectra were obtained from enzyme solutions and from lyophilized forms for native invertase and after pressure- or temperature-induced deactivation. For the native invertase spectra, tentative assignments of the main characteristic protein vibrations, as polypeptidic backbone, sugars, amides and amino acids vibrations are proposed. The irreversible conformational changes monitored by Raman spectroscopy for temperature- or pressure-induced deactivation were also followed. High-pressure denaturation of invertase leads to an increase in low-wavenumber scattering in solution and to a shift of amide I bands to lower wavenumbers, observed by recording spectra after lyophilization of pressure-treated samples. However, temperature denaturation does not show such modifications in the spectra, either for low-wavenumber scattering experiments in solution or for spectra recorded after lyophilization. Thus the microscopic processes involved in inactivation are different for temperature and pressure treatments and different conformational modifications are induced. © 1998 John Wiley & Sons, Ltd.

Influence of Additives on High Pressure Stability of β-Galactosidase from Kluyveromyces Lactis and Invertase from Saccharomyces Cerevisiae

High pressure denaturation of two yeast enzymes, Kluyveromyces lactis β-galactosidase and Saccharomyces cerevisiae invertase in aqueous solutions of salts and polyols was investigated. Polyols (more than salts) were shown to act as very effective agents against pressure denaturation for both enzymes, leading to an increase in half-lives by factors between 1–10,000. NaBr and KBr were essentially destabilizing compounds, leading to protective effects near zero in the case of β-galactosidase. The maximum stability was obtained when KCl was used as additive. The β-galactosidase half-life is increased by a factor of 60 in a 3 m KCl solution and the invertase half-life by a factor of 40. Whereas NaCl had no stabilizing effect on β-galactosidase up to 2 m, this salt was of greater efficiency for invertase whatever the concentration, leading to a protective effect of almost 30 at 3 m. The use of polyols (glycerol, erythritol, xylitol, and sorbitol) allows β-galactosidase and invertase to be stabilized against pressure deactivation. This stabilizing effect increases with the polyol concentration. For invertase in the presence of xylitol, the half-life is increased by a factor of 7 at 1 m and by a factor near 50 at 2 m. The increase in pressure resistance due to the presence of polyols was always greater with β-galactosidase than with invertase. The protective effect of a 2 m xylitol solution is above 10,000 for β-galactosidase whereas it is about 50 for invertase. After the systematic study of the influence of salts and polyols, certain hypotheses are advanced on the possible mechanisms by which additives stabilize proteins to pressure denaturation.

Schizosaccharomyces pombe fragile mutants as a host for heterologous protein production

In order to gain information about the potential interest of the Schizosaccharomyces pombe srb1 fragile mutants as a host for heterologous protein production, the extracellular secretion of homologous and heterologous invertases was investigated. Under catabolic derepression the fragile srb1 mutants released into the extracellular medium 5–6-fold more invertase than the parental strain. When transformed with the SUC2 gene, which codes for Saccharomyces cerevisiae invertase, the srb1-3 fragile mutant, grown under catabolic repression, released into the medium 3-fold more invertase than the wild-type transformant, even though the majority of the enzyme remained associated with the cell wall. Electrophoretic analysis revealed the presence in the fragile strains of some invertase forms with molecular weights smaller than their parallel wild-type strains, suggesting that the srb1 mutants may underglycosylate not only their homologous but also the heterologous proteins.

Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae

A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L. Li and L. G. Ljungdahl, Appl. Environ. Microbiol. 60:3160-3166, 1994). Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium. S. cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction. Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4. These proteins were recognized by anti-APX-II antibody. The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S. cerevisiae. Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media. No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region. A comparison of the A. pullulans and S. cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S. cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S. cerevisiae cells.

Secretion of Active Fc Fragments of Immunoglobulin E Directed by the Yeast Invertase Signal Sequence in Mammalian Cells.

The human Fc fragments of immunoglobulin E (IgE-Fc) expressed in the mammalian COS cells, those mainly consisting of C epsilon 3-C epsilon 4 chains with or without Cys-328 known to be responsible for interchain disulfide bonding, were secreted into the culture medium directed by the yeast Saccharomyces cerevisiae invertase (SUC2) signal sequence (SUC2/Ig2 or SUC2/Ig, respectively) as well as by the human interleukin 2 receptor alpha chain signal sequence (IL2R/Ig2 or IL2R/Ig, respectively). For the binding activity to the human soluble alpha chain of high affinity receptor for IgE, IgE-Fc with Cys328, SUC2/Ig2 and IL2R/Ig2, were active but had smaller activity than native IgE. By comparison IgE-Fc without Cys328, SUC2/Ig and IL2R/Ig, showed lower activity than SUC2/Ig2 and IL2R/Ig2. Immunoprecipitation analysis revealed both SUC2/Ig and SUC2/Ig2 had molecular weights (M(r)s) and degrees of glycosylation similar to IL2R/Ig and IL2R/Ig2, respectively. These results suggested that in mammalian cells the SUC2 signal sequence was functional in directing the heterologous multimeric proteins to the endoplasmic reticulum, resulting in secretion of the active proteins. This finding might show merits on heterologous protein secretion systems.

The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2×10 10 cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae α-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens, and the P. pastoris system offers an efficient method of production.

Expression of an ?-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).

Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study.

Previously, Man8-14GlcNAc oligosaccharides were isolated from highly purified Saccharomyces cerevisiae invertase and shown by one-dimensional 1H NMR spectroscopy and alpha 1,2-linkage-specific mannosidase digestion to constitute a homologous series of nearly homogeneous compounds, which appeared to define the intermediates in oligosaccharide core synthesis in yeast (Trimble, R.B. and Atkinson, P.H. (1986) J. Biol. Chem., 261, 9815-9824). To evaluate whether invertase oligosaccharides reflected global core processing of yeast glycans, the soluble glycoprotein pool of disrupted log-phase cells was digested with endo-beta-N-acetyl-glucosaminidase H and Man8-13GlcNAc were isolated by Bio-Gel P-4 chromatography. Although analysis of each size class by one-dimensional 400 MHz and two-dimensional 500 MHz phase-sensitive COSY 1H NMR spectroscopy revealed considerable structural heterogeneity in all but Man8GlcNAc, the major positional isomer in Man9-13GlcNAc (approximately 50%) was identical to that previously elucidated on invertase. The heterogeneity resided in four families of oligosaccharides: (i) Glc3Man9GlcNAc----Man8 GlcNAc trimming intermediates; (ii) alpha-mannosidase degradation products of the principal isomers; (iii) mannan elongation intermediates; (iv) core structures with the alpha 1,2-linked mannose usually removed by the processing alpha-mannosidase. The potential for the vacuolar alpha-mannosidase (AMS1 gene product) to generate heterogeneity in vitro was confirmed by isolating oligosaccharides from AMS1 and ams1 yeast strains in the presence of a Man13GlcNAc[3H]-ol marker (where GlcNAc[3H]-ol is N-acetylglucosamin [1-3H]itol). Degradation of the Man13GlcNAc[3H]-ol to Man9-12GlcNAc[3H]-ol occurred in the former, but not in the latter. A role for the vacuolar alpha-mannosidase in generating at least some heterogeneity in vivo was inferred from the 1H NMR spectrum of the AMS1 Man11GlcNAc pool, which showed more structural isomerism than seen in the spectrum of a comparable ams1 Man11GlcNAc preparation. Thus, the principal biosynthetic pathway of inner core mannan in Saccharomyces is defined by the Man8-13GlcNAc oligosaccharides found on external invertase, while structural heterogeneity in these size classes results from precursor processing in the endoplasmic reticulum, core extension in the Golgi and metabolic degradation in the vacuole.

Optimisation of a host/vector system for heterologous gene expression by Hansenula polymorpha

For the methylotrophic yeast, Hansenula polymorpha, expression vectors with different origins of replication have been constructed in order to analyse their influence on transformation and integration efficiency. The constructed plasmids are identical except for their origin of replication, which involve, respectively, that of the Saccharomyces cerevisiae 2-μm plasmid and a H. polymorpha ARS sequence (HARS2). A plasmid with no origin of replication served as a control. The plasmids also contained the α-galactosidase expression cassette, consisting of the Cyamopsis tetragonoloba α-galactosidase gene, the H. polymorpha methanol oxidase promoter and terminator, and the S. cerevisiae invertase signal sequence. The transformation frequencies of the expression vectors containing the 2-μm and the HARS2 origins of replication, and no origin of replication, were 2,50 and 15 per μg of DNA respectively, which demonstrates the negative effect of the 2-μm sequence on the transformation frequency. Autonomously replicating plasmids could be isolated from the transformants obtained with the plasmid containing either the 2-μm or the HARS2 sequence. Integration of the 2-μm based plasmid into the H. polymorpha genome could not be established using a standard procedure. This is in contrast with transformants containing a plasmid bearing the HARS2 sequence or else with no origin of replication, which shows that the 2-μm sequence negatively influences the integration of the expression vector into the H. polymorpha genome. Integration of expression plasmids occurred in 50% of the analysed integrants on the homologous methanol oxidase locus, and tandem integration was favoured. The level of specific mRNA, and the expression of the α-galactosidase protein by these integrants, was proportional to the number of integrated copies of the expression plasmid in the H. polymorpha genome.

Efficiency and Diversity of Protein Localization by Random Signal Sequences

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

Efficiency and diversity of protein localization by random signal sequences.

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

Synthesis of Saccharomyces cerevisiae invertase by Schizosaccharomyces pombe

In order to gain information on the ability of the glycosylation system of Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of Saccharomyces cerevisiae invertase in the former yeast was studied. Sc. pombe cells are able to produce enzymatically active invertase from the S. cerevisiae SUC2 gene introduced by transformation and the enzyme is glycosylated and secreted into the cell wall. However, Sc. pombe transformants do not glycosylate the heterologous enzyme as their own invertase since it is not bound by the lectin from Bandeiraea simplicifolia seeds, which indicates the absence of terminal galactose residues. Moreover, the electrophoretic mobility of the heterologous invertase is similar to that of the large enzyme from S. cerevisiae, both in its native form and after being deglycosylated with Endo H. These results suggest that the polypeptide chain of S. cerevisiae invertase is the primary factor for the glycosylation in Sc. pombe cells.

Many random sequences functionally replace the secretion signal sequence of yeast invertase.

In the process of protein secretion, amino-terminal signal sequences are key recognition elements; however, the relation between the primary sequence of an amino-terminal peptide and its ability to function as an export signal remains obscure. The limits of variation permitted for functional signal sequences were determined by replacement of the normal signal sequence of Saccharomyces cerevisiae invertase with essentially random peptide sequences. Since about one-fifth of these sequences can function as an export signal the specificity with which signal sequences are recognized must be very low.

Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences.

Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0). Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment. This compartment contained most of the beta-lactamase activity present in the cell. Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S. cerevisiae, can provide the same function in E. coli. The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences.