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1.
Gene ; 145(2): 179-87, 1994 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-8056329

RESUMEN

The regulation of transcription of the glucoamylase-encoding gene (glaA) of Aspergillus niger was studied. To facilitate this study a reporter strain containing a fusion of the glaA promoter (PglaA) of A. niger to the beta-glucuronidase-encoding gene (uidA) of Escherichia coli was constructed. To analyze whether regulatory proteins are involved in the regulation of glaA, multiple copies of PglaA were introduced into this reporter strain. Analysis of the resulting strains revealed that introduction of an increasing number of PglaA copies resulted in lower expression of the uidA reporter gene and the endogenous glaA gene in cultures cultivated on different inducing carbon sources. However, repression by xylose was not influenced by the copy number of PglaA. These results indicate that the expression of genes under control of PglaA are regulated by specific trans-acting regulatory protein(s). Deletion analysis of PglaA indicated that regulatory proteins interact with DNA sequences within 0.5-kb upstream from the ATG, whereas sequences between about 0.8- and 0.5-kb upstream from the ATG are required for high-level expression of glaA.


Asunto(s)
Aspergillus niger/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Glucano 1,4-alfa-Glucosidasa/genética , Aspergillus niger/enzimología , Secuencia de Bases , Metabolismo de los Hidratos de Carbono , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Inducción Enzimática , Represión Enzimática , Genes Reporteros , Vectores Genéticos , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Datos de Secuencia Molecular , Familia de Multigenes/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Xilosa/farmacología
2.
Gene ; 184(1): 89-97, 1997 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-9016957

RESUMEN

This report describes a transformation system leading to stable high copy number integration into the ribosomal DNA (rDNA) of the astaxanthin-producing yeast Phaffia rhodozyma. A plasmid was constructed that contains the transposon Tn5 encoded kanamycin resistance gene (KmR) fused in frame to the 5'-terminal portion of the Phaffia actin gene. This marker, driven by the Phaffia actin promoter, confers resistance to G418 (Geneticin). The plasmid also contains a rDNA portion that comprises the 18S rDNA and promotes high copy integration leading to stable Phaffia transformants that maintained the plasmid at high copy number after 15 generations of non-selective growth. Phaffia, strain CBS 6938, was found to contain the rDNA units in clusters distributed over three chromosomes with a total copy number of 61. Phaffia transformants were shown to have over 50 copies of pGB-Ph9 integrated in tandem in chromosomes that contain rDNA loci. The chromosomal shifts that occur as a result of these integrations as shown by pulsed field electrophoresis strongly suggest that Phaffia is haploid.


Asunto(s)
ADN Ribosómico/genética , Dosificación de Gen , Genes Fúngicos/genética , Actinas/genética , Southern Blotting , Clonación Molecular , Sondas de ADN , Resistencia a Medicamentos/genética , Electroforesis en Gel de Agar , Electroforesis en Gel de Campo Pulsado , Marcadores Genéticos/genética , Gentamicinas/farmacología , Kanamicina/farmacología , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Transformación Genética/genética
3.
Gene ; 146(2): 159-65, 1994 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-8076814

RESUMEN

The development of an improved gene cloning strategy by complementation of mutant alleles in Aspergillus niger is described. The strategy is based on the use of a fungal autonomously replicating vector, pAB4-ARp1. This vector was constructed by the introduction of a previously described sequence involved in autonomous replication (AMA1), into a pyrG integrative vector, pAB4-1. With vector pAB4-ARp1, a 10-100-fold increase in transformation frequency was obtained, as compared to pAB4-1. Furthermore, the transformation frequency of a co-transformed plasmid is also increased using pAB4-ARp1. A. niger transformants containing pAB4-ARp1 are mitotically unstable. Co-transformed plasmids strictly co-segregated with the autonomously replicating vector, as a result of recombination between both vectors. The use of pAB4-ARp1 in gene cloning was demonstrated by the complementation of two linkage group-VII-specific A. niger mutants. Complementation of a lysF mutant was achieved by co-transformation of pAB4-ARp1 with total genomic A. niger DNA ('instant bank'). A nicB-deficient A. niger was complemented by co-transformation with pAB4-ARp1 and an A. niger cosmid library. The complementing DNA was re-isolated from a Nic+ transformant by transforming Escherichia coli with total genomic DNA of this transformant. Gene disruption and genetic analysis was carried out to prove that the previously unknown A. niger nicB gene had been cloned.


Asunto(s)
Aspergillus niger/genética , Clonación Molecular/métodos , Genes Fúngicos/genética , Vectores Genéticos/genética , Transformación Genética/genética , Escherichia coli/genética , Prueba de Complementación Genética , Vectores Genéticos/fisiología , Fenotipo , Transformación Bacteriana
4.
FEBS Lett ; 438(3): 293-6, 1998 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-9827564

RESUMEN

Recently, we reported the purification of the novel enzyme limonene-1,2-epoxide hydrolase involved in limonene degradation by Rhodococcus erythropolis DCL14. The N-terminal amino acid sequence of the purified enzyme was used to design two degenerate primers at the beginning and the end of the 50 amino acids long stretch. Subsequently, the complete limonene-1,2-epoxide hydrolase gene (limA) was isolated from a genomic library of R. erythropolis DCL14 using a combination of PCR and colony hybridization. The limA gene encoded a 149-residue polypeptide with a deduced molecular mass of 16.5 kDa. It was functionally expressed in Escherichia coli. The amino acid sequence of limA contains neither any of the conserved regions of the alpha,beta-hydrolase fold enzymes, to which most of the previously reported epoxide hydrolases belong, nor any of the conserved motifs present in leukotriene A4 hydrolase. The structural data presented in this paper confirm previous physical and biochemical findings [van der Werf et al. (1998) J. Bacteriol. 180, 5052-5057] that limonene-1,2-epoxide hydrolase is the first member of a new class of epoxide hydrolases.


Asunto(s)
Proteínas Bacterianas , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Rhodococcus/enzimología , Rhodococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Epóxido Hidrolasas/química , Escherichia coli , Genes Bacterianos , Biblioteca Genómica , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Rhodococcus/crecimiento & desarrollo
5.
J Biotechnol ; 36(2): 165-75, 1994 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-7765235

RESUMEN

To evaluate the possibility of improving glucoamylase (GLA) production in Aspergillus niger strains carrying multiple copies of the GLA encoding gene (glaA), additional glaA copies were introduced either by genetic recombination or retransformation. For strains to be used in such experiments a genetic analysis was first carried out. The results of this analysis clearly revealed that in each transformant integration had occurred at a chromosome corresponding to a single linkage group (LG). The GLA production per gene copy showed considerable variation in these strains, indicating a clear effect of the site of integration on gene expression. Introduction of additional gene copies by genetic recombination experiments was carried out for different combinations of strains, carrying glaA copies in different chromosomes. The introduction of additional glaA gene copies by genetic recombination did not result in a considerable increase in GLA production compared to the parental strains. In some strains recombination resulted in genetic instability, observed by the frequent loss of glaA copies. Also, retransformation of multi-copy glaA strains did not result in an increase in GLA production. In several strains even a decrease in GLA production was found after retransformation. Southern analysis of these transformants suggested that newly introduced gene copies were heavily rearranged, which partly explains why GLA production was not increased. Further analysis of one such transformant provided evidence that the overexpression of the glaA gene is limited by the amount of trans-acting regulatory protein(s) available.


Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/genética , Aspergillus niger/genética , Biotecnología , Estudios de Evaluación como Asunto , Amplificación de Genes , Genes Fúngicos , Técnicas Genéticas , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Transformación Genética
6.
Biotechnol Bioeng ; 63(6): 750-5, 1999 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-10397832

RESUMEN

The first carotenoid biosynthetic gene from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated by heterologous complementation in Escherichia coli. The isolated gene, denominated as crtI, was found to encode for phytoene desaturase. The coding region is interrupted by 11 introns. The deduced amino acid sequence showed significant homology with its bacterial and eukaryotic counterparts, especially those of fungal origin. A plasmid containing the geranylgeranyl diphosphate synthase and phytoene synthase encoding genes from Erwinia uredovora was introduced in E. coli together with the phytoene desaturase encoding cDNA from X. dendrorhous. As a result, lycopene accumulation was observed in these transformants. We conclude that in X. dendrorhous the four desaturase steps, by which phytoene is converted into lycopene, are carried out by a single gene product.


Asunto(s)
Basidiomycota/genética , Basidiomycota/metabolismo , Proteínas Fúngicas/genética , Oxidorreductasas/genética , beta Caroteno/análogos & derivados , Secuencia de Aminoácidos , Secuencia de Bases , Carotenoides/metabolismo , Clonación Molecular , Escherichia coli/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Licopeno , Datos de Secuencia Molecular , Oxidorreductasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Xantófilas , beta Caroteno/biosíntesis
7.
Appl Environ Microbiol ; 65(12): 5459-63, 1999 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-10584004

RESUMEN

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.


Asunto(s)
Basidiomycota/enzimología , Basidiomycota/genética , Epóxido Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Basidiomycota/crecimiento & desarrollo , Clonación Molecular , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Escherichia coli , Genes Fúngicos , Microsomas/enzimología , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Conejos , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido
8.
Appl Environ Microbiol ; 66(9): 3945-50, 2000 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10966413

RESUMEN

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i. e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100, 823-Da protein, including a 36-amino-acid signal sequence. The 96, 887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.


Asunto(s)
Bacillus/enzimología , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/metabolismo , Secuencia de Aminoácidos , Bacillus/genética , Liasas de Carbono-Oxígeno/química , Clonación Molecular , Sondas de ADN , ADN Bacteriano/genética , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polisacáridos Bacterianos/metabolismo , Análisis de Secuencia de ADN
9.
Appl Microbiol Biotechnol ; 53(4): 415-9, 2000 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10803897

RESUMEN

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the alpha/beta hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min(-1)(mg protein)(-1) towards 1,2-epoxyhexane.


Asunto(s)
Clonación Molecular , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Rhodotorula/enzimología , Rhodotorula/genética , Secuencia de Aminoácidos , Secuencia de Bases , Epóxido Hidrolasas/química , Escherichia coli/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN
10.
Yeast ; 13(13): 1231-42, 1997 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9364747

RESUMEN

The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC1.2.1.12)-encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938. Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P. rhodozyma. The complete nucleotide sequence of the coding, as well as the flanking non-coding regions was determined. The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids. The codon usage in the gpd gene of P. rhodozyma was highly biased and was significantly different from the codon usage in other yeasts. Phylogenetic analysis of different yeasts and filamentous asco- and basidiomycetes gpd sequences indicated that the gpd gene of P. rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes.


Asunto(s)
Genes Fúngicos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Hongos Mitospóricos/genética , Levaduras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Molecular , Dosificación de Gen , Código Genético , Biblioteca Genómica , Gliceraldehído-3-Fosfato Deshidrogenasas/clasificación , Hongos Mitospóricos/clasificación , Hongos Mitospóricos/enzimología , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Levaduras/clasificación , Levaduras/enzimología
11.
Mol Gen Genet ; 262(3): 453-61, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10589832

RESUMEN

The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crt YB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to beta-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein.


Asunto(s)
Transferasas Alquil y Aril/genética , Basidiomycota/genética , Carotenoides/biosíntesis , Genes Fúngicos , Liasas Intramoleculares/genética , Levaduras/genética , Secuencia de Aminoácidos , Basidiomycota/enzimología , Clonación Molecular , ADN Complementario/genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Datos de Secuencia Molecular , Fragmentos de Péptidos , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Xantófilas , Levaduras/enzimología , beta Caroteno/análogos & derivados , beta Caroteno/biosíntesis
12.
Appl Environ Microbiol ; 65(9): 4099-107, 1999 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10473422

RESUMEN

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.


Asunto(s)
Actinomycetales/enzimología , Actinomycetales/genética , Genes Bacterianos , Xilosidasas/genética , Xilosidasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Alineación de Secuencia , Análisis de Secuencia de ADN , Temperatura , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/química
13.
Appl Microbiol Biotechnol ; 52(2): 232-9, 1999 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10499263

RESUMEN

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.


Asunto(s)
Celulasa/genética , Genes Bacterianos , Bacilos Grampositivos Asporogénicos Irregulares/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Celulasa/metabolismo , Clonación Molecular , Sistema Digestivo/microbiología , Bacilos Grampositivos Asporogénicos Irregulares/enzimología , Concentración de Iones de Hidrógeno , Insectos/microbiología , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido
14.
Appl Microbiol Biotechnol ; 61(1): 55-60, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12658515

RESUMEN

Two alpha-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both alpha-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-alpha-glucoside. The K(m) for pnp-alpha-glucoside was 1.05 and 0.47 mM and the V(max) was 228 and 113 U mg(-1) for AglA and AglB, respectively. Using pnp-alpha-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37 degrees C. For AglB, values of pH 6.8 and 47 degrees C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant alpha-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.


Asunto(s)
Bifidobacterium/enzimología , Himecromona/análogos & derivados , alfa-Glucosidasas/análisis , alfa-Glucosidasas/genética , Secuencia de Aminoácidos , Bifidobacterium/clasificación , Clonación Molecular , Glucósidos/metabolismo , Glicosilación , Himecromona/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/análisis , Alineación de Secuencia , Especificidad por Sustrato , alfa-Amilasas/análisis , alfa-Amilasas/genética , alfa-Glucosidasas/química , alfa-Glucosidasas/aislamiento & purificación
15.
J Gen Microbiol ; 139(11): 2801-10, 1993 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8277260

RESUMEN

Continuous and recycling cultures were carried out with Aspergillus niger N402 wild-type and a glucoamylase overproducing transformant to investigate growth and product formation characteristics. In shake flask cultures, the amount of glucoamylase produced by the transformant was about five times more than by the wild-type strain. In contrast with these results, a twofold overproduction was found in glucose-limited continuous cultures, while no overproduction was found under maltodextrin-limitation. Two regions of specific growth rates could be distinguished, one at specific growth rates lower (domain I) and one at specific growth rates higher than 0.12 h-1 (domain II). In domain I changes in mycelium morphology and conidia formation were observed. It has been concluded that maintenance requirements are dependent on the specific growth rate over the whole range of measured growth rates. The deviation in linearity in the linear equation of substrate utilization, caused by this phenomenon, should be considered when continuous cultures with filamentous fungi are performed. In recycling cultures, xylose as limiting carbon source repressed glucoamylase production very strongly. Under maltodextrin-limitation a fivefold overproduction was found. After about 150 h , the total amount of glucoamylase produced was still increasing, while total amount of product, measured as carbon, remained constant. After this time no increase in the amount of biomass formed was observed. These results suggest autolysis and cryptic growth taking place in a recycling fermenter and cell death rate equalling growth rate.


Asunto(s)
Aspergillus niger/enzimología , Genes Fúngicos , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Aspergillus niger/genética , Aspergillus niger/crecimiento & desarrollo , Fermentación , Glucano 1,4-alfa-Glucosidasa/genética
16.
Transgenic Res ; 2(2): 84-92, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8513339

RESUMEN

A strategy, based on the usage of the amdS selection marker and a cosmid vector containing four copies of the glucoamylase gene (glaA), was developed to obtain glucoamylase (GLA)-overproducing A. niger strains. With this strategy, fungal strains carrying up to 200 copies of the glaA gene could be isolated at a relatively high frequency. In each transformant analysed, integration occurred in a single chromosome. A significant increase in the extracellular GLA production was observed in most of the transformants carrying multiple copies of the glaA gene. Further analysis showed that the amount of GLA that is produced was not proportional to the number of glaA copies in these transformants. However, the level of GLA production clearly correlated with the amount of glaA mRNA produced in these transformants. From these results it is concluded that GLA production is limited at the level of transcription.


Asunto(s)
Aspergillus niger/genética , Glucano 1,4-alfa-Glucosidasa/genética , Aspergillus niger/enzimología , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , Amplificación de Genes , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Ingeniería Genética , Metilación , Datos de Secuencia Molecular , Transformación Genética
17.
Mol Gen Genet ; 244(1): 75-80, 1994 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-8041364

RESUMEN

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, aglA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant strains for the purpose of chromosome assignment.


Asunto(s)
Aspergillus niger/genética , Cromosomas Fúngicos , Mapeo Cromosómico , Electroforesis en Gel de Agar , Ligamiento Genético , Cariotipificación
18.
Mol Gen Genet ; 230(1-2): 161-9, 1991 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-1660563

RESUMEN

A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.


Asunto(s)
Isomerasas Aldosa-Cetosa , Genes Bacterianos , Lactobacillus/genética , Fosfotransferasas (Aceptor de Grupo Alcohol) , Xilosa/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Secuencia de Bases , Carbohidrato Epimerasas/genética , Clonación Molecular , ADN Bacteriano/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Fosfotransferasas/genética , Plásmidos , Proteínas Represoras/genética , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Streptomyces/enzimología
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