Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae

The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae. When the mature portion of ROL (mROL) fused to the pre-alpha-factor leader sequence was expressed, secretion of active mROL was not observed. However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed. The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite. From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone. The results using the fragment of the prosequence confirmed that these residues (20-57) were significant for in vivo folding and secretion of active mROL.     

Cloning and screening of the putative hexokinase genes from Rhizopus oryzae and their heterologous expression in Saccharomyces cerevisiae

Molecular Biology Reports - A filamentous fungus, Rhizopus oryzae (R. oryzae) is one of the ideal candidates for ethanol and lactic acid production due to its ability to grow on renewable carbon...     

High level expression of proinsulin in the yeast, Saccharomyces cerevisiae

Human proinsulin (PI) has been expressed to a high level (100 mg/liter) as a human superoxide dismutase-PI fusion protein in the yeast, Saccharomyces cerevisiae. At the junction of the two proteins is a methionine residue, allowing PI to be released from the fusion by reaction with cyanogen bromide. The fusion is expressed using a regulated, hybrid promoter containing the regulatory region of the alcohol dehydrogenase II promoter and the 3' end of a glyceraldehyde-3-phosphate dehydrogenase promoter, allowing the recombinant yeast cells to be stably maintained. Production of the fusion protein is induced by growth in medium lacking a fermentable carbon source. The heterologous fusion protein is probably insoluble within the cell, since electron microscopy reveals the presence of 'inclusion bodies'. In a cell-free extract the fusion protein is also insoluble, but can be solubilized with sodium dodecyl sulfate, and cleaved with cyanogen bromide. The PI that is produced contains incorrect disulfide bonds. After sulfitolysis, the product can be easily purified, renatured, and processed to yield insulin.     

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.     

High-level expression of Rhizopus niveus lipase in the yeast Saccharomyces cerevisiae and structural properties of the expressed enzyme

Rhizopus niveus lipase (RNL) has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain). To improve this enzyme's properties by protein engineering, we have developed a new expression system for the production of recombinant lipase in the yeast Saccharomyces cerevisiae. For the present study, we developed a more efficient expression system using the strain ND-12B and the multicopy-type plasmid pJDB219. We purified two types of recombinant lipases, each to a single peak by gel-filtration HPLC, although they were found to be heterogeneous by SDS-PAGE. Analysis of reversed-phase HPLC, N-terminal amino acid sequence, and sugar content showed that the difference between the two types of lipases was due mainly to their sugar content (high or low mannose type). Moreover, there were two species within each type of lipase. One kind was processed to the A-chain and B-chain as in the native lipase, while the other remained unprocessed. Although these yeast-purified lipases contained several posttranslational modifications and different glycosylations, their secondary structures were the same as those of the native lipase as measured by circular dichroism spectra and determination of disulfide bonding. This suggests that protein folding of the recombinant lipase occurred correctly in yeast.     

Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae

A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%–56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADH1). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.     

Synthesis and secretion of bacterial alpha-amylase by the yeast Saccharomyces cerevisiae

Alpha-amylase from Bacillus amyloliquefaciens, synthesized in yeast Saccharomyces cerevisiae without substitution of the signal sequence, is efficiently secreted from yeast cells: 60-70% of the overall amount of the enzyme is found in the culture fluid. In contrast to many yeast secretory proteins, which accumulate in the periplasmic space and in the cell wall, intracellular alpha-amylase is localized mainly in the cytoplasm. Obviously, transfer across the cell wall is not a rate-limiting step in alpha-amylase export from the cell. The glycosylated forms of proteins are predominantly found both inside the cell and in the culture medium.     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

Heterologous expression of Aspergillus oryzae xylose reductase and xylitol dehydrogenase genes facilitated xylose utilization in the yeast Saccharomyces cerevisiae

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase

Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1(+): Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.     

Expression and secretion of barley α-amylase and A.niger glucoamylase in Saccharomyces cerevisiae

cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol. Project supported by the Guangdong Natural Science Foundation.     

Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells. The partially purified enzyme was active against glycogen, starch, maltotriose and maltose. It exhibited maximum catalytic activity against glycogen at pH 5.5. The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose. SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques. SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.     

Direct involvement of phosphatidylinositol 4-phosphate in secretion in the yeast Saccharomyces cerevisiae

The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37 degrees C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34 degrees C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1(ts) mutants were examined. Upon shift to restrictive temperature (37 degrees C), the PtdIns(4)P levels dropped by about 60% in both pik1(ts) strains within 1 h. During the same period, cells displayed a reduction (40-50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-to-plasma membrane stage of secretion.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path

GA (glucoamylase) hydrolyses starch and polysaccharides to beta-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with beta-cyclodextrin (SBD-betaCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp(47)) and II (Tyr(32)), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr(32) was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.     

Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.     

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.