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1.
Gene ; 95(1): 111-21, 1990 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-1979298

RESUMEN

The Schwanniomyces occidentalis glucoamylase (GAM)-encoding gene (GAM1) was isolated from a lambda Charon4A genomic library using synthetic oligodeoxynucleotides as probes. GAM1 contains an ORF of 2874 nucleotides (nt) coding for 958 amino acids. S1 mapping revealed that the transcript has only a very short 5'-untranslated leader of 8-12 nt. Disruption and displacement of the GAM1 gene in Sc. occidentalis resulted in loss of the ability to grow on starch efficiently. The gam1 strains still exhibit low GAM activity suggesting that at least a second weakly expressed GAM-encoding gene (GAM2) is present in Sc. occidentalis. Expression of the Sc. occidentalis GAM1 gene in Saccharomyces cerevisiae was achieved after promoter exchange. S. cerevisiae cells transformed with centromere plasmids carrying the GAM1 gene fused to promoters of different S. cerevisiae genes, namely GAL1, PDC1 and ADH1, efficiently secrete GAM and are able to grow with soluble starch as a sole carbon source. The essential enzymatic properties of the GAMs secreted from S. cerevisiae and Sc. occidentalis are identical, although the modifications of the proteins are different.


Asunto(s)
Genes Fúngicos , Glucano 1,4-alfa-Glucosidasa/genética , Levaduras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Expresión Génica , Glucano 1,4-alfa-Glucosidasa/metabolismo , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Transformación Genética
2.
FEBS Lett ; 294(1-2): 109-12, 1991 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-1743281

RESUMEN

Sequence comparison of the primary structure of the yeast Schwanniomyces occidentalis glucoamylase (GAM) with GAMs in different microorganisms did not reveal significant similarities. By contrast, striking similarities were, surprisingly, found with 3 mammalian secretory and integral membrane proteins: the 2 subunits of intestinal brush border sucrase-isomaltase and human lysosomal alpha-glucosidase. The similarities among these proteins are found as clusters of up to 8 amino acids and distributed all over the protein sequences. The major sequence differences are found in the N-terminal regions accounting, probably, for the different cellular locations of these proteins. The high level of similarities between sucrase, isomaltase, Sch. occidentalis GAM and human lysosomal alpha-glucosidase suggest that these proteins are derived from the same ancestral gene. To our knowledge, this is the first report that describes similarities between a yeast secretory protein and mammalian secretory and integral membrane proteins.


Asunto(s)
Evolución Biológica , Genes , Glucano 1,4-alfa-Glucosidasa/genética , Intestinos/enzimología , Lisosomas/enzimología , Saccharomycetales/genética , Complejo Sacarasa-Isomaltasa/genética , alfa-Glucosidasas/genética , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Conejos , Ratas , Saccharomycetales/enzimología , Homología de Secuencia de Ácido Nucleico
3.
Biotechnol Adv ; 10(2): 179-89, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-14544533

RESUMEN

The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.

4.
Biotechnology (N Y) ; 9(3): 291-5, 1991 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-1367303

RESUMEN

We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.


Asunto(s)
Clonación Molecular/métodos , Glucano 1,4-alfa-Glucosidasa/genética , Pichia/genética , Saccharomycetales/enzimología , Secuencia de Aminoácidos , Fermentación , Genes Fúngicos , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Glucano 1,4-alfa-Glucosidasa/metabolismo , Datos de Secuencia Molecular , Pichia/enzimología , Plásmidos
6.
Biochemistry ; 30(2): 472-8, 1991 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-1899027

RESUMEN

It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits. Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism. An ionizing group having an apparent pK value near 7.5 must be protonated for activity. The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site. These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A. 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine. The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits. These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate.


Asunto(s)
Escherichia coli/enzimología , Complejos Multienzimáticos/metabolismo , Triptófano Sintasa/metabolismo , Sitios de Unión , Glicerofosfatos/metabolismo , Concentración de Iones de Hidrógeno , Indoles/metabolismo , Cinética , Ligandos , Concentración Osmolar , Conformación Proteica , Serina/metabolismo , Análisis Espectral
7.
Eur J Biochem ; 244(1): 128-33, 1997 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-9063455

RESUMEN

Site-directed mutagenesis was performed to define the active site of the Schwanniomyces occidentalis glucoamylase. The mutated GAM1 genes were expressed in Saccharomyces cerevisiae, and enzymatic and growth properties of the transformants were determined. Mutants were transcribed and translated similar to the wild-type glucoamylase. Therefore, all effects on enzymatic activity could be referred to single amino acid substitutions. Asp470 was shown to be essential for the enzyme activity. Replacement of Asp470 by glycine led to a complete loss of activity. We suppose that Asp470 serves as a general acid-base and stabilizes the formation of the intermediate carbenium ion. Substitution of Trp468 by alanine affected predominantly the alpha-1,6 activity and not the alpha-1,4 activity of the enzyme. The exchange impaired substrate binding as well as enzymatic catalysis. An influence of amino acid 474 on the substrate specificity could not be demonstrated. Exchanges at position 474 exhibited K(m) and Vmax values similar to wild-type glucoamylase.


Asunto(s)
Glucano 1,4-alfa-Glucosidasa/química , Saccharomycetales/enzimología , Secuencia de Aminoácidos , Ácido Aspártico/metabolismo , Sitios de Unión , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Saccharomycetales/genética , Triptófano/metabolismo
8.
Appl Microbiol Biotechnol ; 50(3): 331-8, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9802218

RESUMEN

Two non-Saccharomyces yeasts have been developed as hosts for heterologous gene expression. The celD gene from Clostridium thermocellum, encoding a heat-stable cellulase, served as the test sequence. The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis. The systems comprise auxotrophic host strains (trp5 in the case of S. occidentalis; trp5-10, his3 in the case of P. stipitis) and suitable transformation vectors. Vector components consist of an S. occidentalis-derived autonomously replicating sequence (SwARS) and the Saccharomyces cerevisiae-derived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation and selection in a bacterial host. A range of vectors has been engineered employing different promoter elements for heterologous gene expression control in both species. Homologous elements derived from highly expressed genes of the respective hosts appeared to be of superior quality: in the case of S. occidentalis that of the GAM1 gene, in the case of P. stipitis that of the XYL1 gene. Further elements tested are the S. cerevisiae-derived ADH1 and PDC1 promoter sequences.


Asunto(s)
Celulasa/genética , Clostridium/genética , Vectores Genéticos , Pichia/genética , Saccharomycetales/genética , Transformación Genética , Celulasa/metabolismo , Clostridium/enzimología , Expresión Génica , Genes Bacterianos , Genes Reporteros , Mapeo Físico de Cromosoma , Pichia/enzimología , Pichia/crecimiento & desarrollo , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomycetales/enzimología , Saccharomycetales/crecimiento & desarrollo , Regiones Terminadoras Genéticas
9.
Curr Genet ; 15(5): 319-25, 1989 May.
Artículo en Inglés | MEDLINE | ID: mdl-2676205

RESUMEN

High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called "SwARS1". The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S. occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene. Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.


Asunto(s)
Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Transformación Genética , alfa-Amilasas/biosíntesis , Southern Blotting , Clonación Molecular , Replicación del ADN , ADN de Hongos/genética , Regulación de la Expresión Génica , Genes Fúngicos , Prueba de Complementación Genética , Cinética , Mutación , Plásmidos , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomycetales/enzimología , Homología de Secuencia de Ácido Nucleico , Triptófano Sintasa/genética
10.
Appl Microbiol Biotechnol ; 44(3-4): 377-85, 1995 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8597538

RESUMEN

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.


Asunto(s)
Hirudinas/metabolismo , Pichia/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Braquiuros/genética , Fermentación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Glucano 1,4-alfa-Glucosidasa/genética , Hirudinas/biosíntesis , Hirudinas/genética , Hormonas de Invertebrados/biosíntesis , Hormonas de Invertebrados/genética , Factor de Apareamiento , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Biosíntesis de Péptidos , Péptidos/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética
11.
Antonie Van Leeuwenhoek ; 62(1-2): 79-93, 1992 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-1444338

RESUMEN

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.


Asunto(s)
Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Levaduras/genética , Levaduras/metabolismo , Expresión Génica , Vectores Genéticos , Kluyveromyces/genética , Kluyveromyces/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Eur J Biochem ; 184(3): 699-706, 1989 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-2806251

RESUMEN

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.


Asunto(s)
Ascomicetos/enzimología , Transformación Genética , Levaduras/enzimología , alfa-Amilasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , alfa-Amilasas/análisis , alfa-Amilasas/metabolismo
13.
Biotechnol Bioeng ; 63(3): 373-81, 1999 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-10099617

RESUMEN

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.


Asunto(s)
6-Fitasa/biosíntesis , Pichia/metabolismo , 6-Fitasa/genética , Aspergillus/enzimología , Aspergillus/genética , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/genética , Biomasa , Biotecnología/economía , Biotecnología/métodos , Clonación Molecular/métodos , Análisis Costo-Beneficio , Medios de Cultivo , Fermentación , Glucosa/metabolismo , Glicerol/metabolismo , Cinética , Pichia/genética , Pichia/crecimiento & desarrollo , Plásmidos , Proteínas Recombinantes/biosíntesis
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