Polymorphic extracellular glucoamylase genes and their evolutionary origin in the yeast Saccharomyces diastaticus.

DNA coding for extracellular glucoamylase genes STA1 and STA3 was isolated from DNA libraries of two Saccharomyces diastaticus strains, each carrying STA1 or STA3. Cells transformed with a plasmid carrying either the STA1 or STA3 gene secreted glucoamylases having the same enzymatic and immunological properties and the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases. Southern blot analysis of genomic DNA from S. diastaticus and a glucoamylase-non-secreting yeast, Saccharomyces cerevisiae, revealed that the STA1 and STA3 loci of S. diastaticus showed a high degree of homology, and that both yeast species (S. diastaticus and S. cerevisiae) contained DNA segments highly homologous to those of the extracellular glucoamylase genes. Restriction maps of the homologous DNA segments suggested that the extracellular glucoamylase genes of S. diastaticus may have arisen from recombination among the resident DNA segments in S. cerevisiae.     

The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview

Saccharomyces cerevisiae has been used widely both as a model system for unraveling the biochemical, genetic, and molecular details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest. The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and the broadening of the substrate range of S. cerevisiae. The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by alpha- and beta-amylases, glucoamylases, and debranching enzymes, e.g., pullulanases. Starch utilization in the yeast S. cerevisiae var. diastaticus depends on the expression of the three unlinked genes, STA1 (chr. IV), STA2 (chr. II), and STA3 (chr. XIV), each encoding one of the extracellular glycosylated glucoamylases isozymes GAI, GAII, or GAIII, respectively. The restriction endonuclease maps of STA1, STA2, and STA3 are identical. These genes are absent in S. cerevisiae, but a related gene, SGA1, encoding an intracellular, sporulation-specific glucoamylase (SGA), is present. SGA1 is homologous to the middle and 3' regions of the STA genes, but lacks a 5' sequence that encodes the domain for secretion of the extracellular glucoamylases. The STA genes are positively regulated by the presence of three GAM genes. In addition to positive regulation, the STA genes are regulated negatively at three levels. Whereas strains of S. diastaticus are capable of expressing the STA genes, most strains of S. cerevisiae contain STA10, whose presence represses the expression of the STA genes in an undefined manner. The STA genes are also repressed in diploid cells, presumably by the MATa/MAT alpha-encoded repressor. STA gene expression is reduced in liquid synthetic media, it is carbon catabolite repressed by glucose, and is inhibited in petite mutants.     

Improving the amylolytic activity of Saccharomyces cerevisiae glucoamylase by the addition of a starch binding domain

Glucoamylase produced by amylolytic strains of Saccharomyces cerevisiae (var. diastaticus) lacks a starch binding domain that is present in homologous glucoamylases from Aspergillus niger and other filamentous fungi. The absence of the binding domain makes the enzyme inefficient against raw starch and hence unsuitable for most biotechnological applications. We have constructed a hybrid glucoamylase-encoding gene by in-frame fusion of the S. cerevisiae STA1 gene and DNA fragment that encodes the starch binding domain of A. niger glucoamylase. The hybrid enzyme resulting from expression of the chimeric gene in S. cerevisiae has substrate binding capability and hydrolyses insoluble starch, properties not present in the original yeast enzyme.     

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.     

酵母糖化酶基因高效表达的剂量效应研究

通过原生质体融合和秋水仙素加倍技术,将糖化酵母的３个等效异位基因分别构建成交配型位点等痊基因纯合和糖化酶基因各自纯各加倍的纯合二倍体（ＳＴＡ１／ＳＴＡ１、ＳＴＡ２／ＳＴＡ２和ＳＴＡ３ＳＴＡ３）以及糖化酶基因相互组织加倍的组合二倍体（ＳＴＡ１／ＳＴＡ２、ＳＴＡ２／ＳＴＡ３和ＳＴＡ１／ＳＴＡ３）。研究了这３个糖化酶基因表达的剂量效及其相互关系。糖化酶酶活测定的结果表明,在交配型位点等位基因纯合状态下,     

Localization of glucoamylase genes of Saccharomyces cerevisiae by pulsed field gel electrophoresis

The chromosomal locations of four glucoamylase-specifying genes in the yeastSaccharomyces cerevisiae have been determined. Chromosomes were separated by pulsed field gel electrophoresis and blots were probed with radiolabelledSTA2 and marker DNA from specific yeast chromosomes. The three genes encoding extracellular glucoamylases,STA1 (DEX2), STA2 (DEX1) andSTA3 (DEX3) are located on chromosomes IV, II and XIV, respectively.SGA, specifying the sporulation-specific glucoamylase, was positioned on chromosome IX.     

Expression of cloned Saccharomyces diastaticus glucoamylase under natural and inducible promoters

Any one of three homologous genes - STA1, STA2 and STA3 - encoding glucoamylase isozymes I, II and III respectively, allows the Saccharomyces species to utilize starch as a sole carbon source. We show in this paper that glucoamylase II production can be increased 4-fold over the level produced by STA2 strains, by using a two-step fermentation and a yeast strain transformed with a high-copy-number plasmid carrying the STA2 gene. The accumulation of anomalous STA2 mRNA species, mainly differing at their 5' ends, and saturation of step(s) in the secretory pathway appear to be among the major factors limiting glucoamylase expression in synthetic media.     

Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.     

Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species.

Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species. S. cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W. J. Colonna and P. T. Magee, J. Bacteriol. 134:844-853, 1978; I. Yamashita and S. Fukui, Mol. Cell. Biol. 5:3069-3073, 1985). In this study we examined some of the physiological and genetic factors that affect glucoamylase expression. It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch. Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin. When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels. In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria. Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase. In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA. Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10. Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid. The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids. A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe. The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.     

Secretion of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae using the signal sequence from the glucoamylase-encoding STA2 gene

The budding yeast Saccharomyces cerevisiae is a safe and widely used host for the production of recombinant DNA-derived proteins. We have used the signal sequence from the S. diastaticus STA2 gene, encoding glucoamylase II, to secrete Escherichia coli beta-galactosidase, encoded by the lacZ gene. In frame STA2/lacZ gene fusions have been constructed and expressed in S. cerevisiae under the control of either the STA2 or the galactose inducible GAL1-10 upstream promoters. Fairly high amounts of the enzyme (up to 76% of total activity, depending on the growth conditions) are secreted in the periplasmic space. Adding yeast extract and peptone to the growth medium results in a dramatic increase in both synthesis and secretion of beta-galactosidase.     

Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus.

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.     

Allelism within the DEX and STA gene families in Saccharomyces diastaticus

Summary Saccharomyces diastaticus produces an extracellular glucoamylase and is therefore capable of hydrolyzing and fermenting starch. Tamaki (1978) studied starch utilization in S. diastaticus and found three polymeric genes controlling this function: STA1, STA2 and STA3. Independently, Erratt and Stewart (1978) studied dextrin utilization by the yeast S. diastaticus and designated the gene, which they identified, DEX1. Erratt and Stewart (1981a, b) later described two other genes which controlled glucoamylase production in S. diastaticus: DEX2 and a third which was allelic to STA3. At that time STA1 and STA2 were not available to test for allelism in the DEX gene family. In this study strains containing the remaining 4 genes have been examined to determine if further allelism exists between the two gene families. It was ascertained that DEX1 is allelic to STA2 and DEX2 is allelic to STA1. Therefore, no new gene controlling starch utilization has been identified and these two nomenclatures can now be consolidated into one. Based on the fact that the glucoamylase from S. diastaticus can hydrolyze both dextrin and starch, dextrin being the term used to described partially hydrolyzed starch, and the more wide use of the nomenclature STA, we propose to retain STA as the designation for genes coding for glucoamylase production in S. diastaticus.     

A DEX gene conferring production of extracellular amyloglucosidase on yeast

A DEX gene from Saccharomyces diastaticus (strain BRG536 alpha DEX1) has been cloned in the hybrid vector pJDB207. The gene is included within a 3.6-kb fragment and confers production of extracellular amylo-alpha-1,4-glucosidase (AMG) and, thereby the ability to hydrolyse starch or dextrins on Dex- strains of Saccharomyces cerevisiae. The cloned gene hybridizes to three fragments produced by ClaI digestion of DNA from BRG536; one of these (11 kb) cosegregates in crosses with DEX1, while another (10 kb) is present in all Dex+ and Dex- strains examined. Accumulation of extracellular AMG by Dex+ transformants is up to five-fold that of BRG536, and escapes regulation by the CDX1 gene under conditions of excess glucose. The enzyme produced by Dex+ transformants resembles that of BRG536 with respect to Mr (approx. 150 X 10(3)) and effects of temperature and pH. The cloned DEX gene can be used as a selectable marker for introducing recombinant plasmids into wild-type strains of S. cerevisiae.     

Expansion and contraction of the DUP240 multigene family in Saccharomyces cerevisiae populations

The influence of duplicated sequences on chromosomal stability is poorly understood. To characterize chromosomal rearrangements involving duplicated sequences, we compared the organization of tandem repeats of the DUP240 gene family in 15 Saccharomyces cerevisiae strains of various origins. The DUP240 gene family consists of 10 members of unknown function in the reference strain S288C. Five DUP240 paralogs on chromosome I and two on chromosome VII are arranged as tandem repeats that are highly polymorphic in copy number and sequence. We characterized DNA sequences that are likely involved in homologous or nonhomologous recombination events and are responsible for intra- and interchromosomal rearrangements that cause the creation and disappearance of DUP240 paralogs. The tandemly repeated DUP240 genes seem to be privileged sites of gene birth and death.