RESUMO
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.
Assuntos
Bifidobacterium/enzimologia , Himecromona/análogos & derivados , alfa-Glucosidases/análise , alfa-Glucosidases/genética , Sequência de Aminoácidos , Bifidobacterium/classificação , Clonagem Molecular , Glucosídeos/metabolismo , Glicosilação , Himecromona/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Alinhamento de Sequência , Especificidade por Substrato , alfa-Amilases/análise , alfa-Amilases/genética , alfa-Glucosidases/química , alfa-Glucosidases/isolamento & purificaçãoRESUMO
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.
Assuntos
Bacillus/enzimologia , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Carbono-Oxigênio Liases/química , Clonagem Molecular , Sondas de DNA , DNA Bacteriano/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polissacarídeos Bacterianos/metabolismo , Análise de Sequência de DNARESUMO
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.
Assuntos
Clonagem Molecular , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Rhodotorula/enzimologia , Rhodotorula/genética , Sequência de Aminoácidos , Sequência de Bases , Epóxido Hidrolases/química , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
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.
Assuntos
Alquil e Aril Transferases/genética , Basidiomycota/genética , Carotenoides/biossíntese , Genes Fúngicos , Liases Intramoleculares/genética , Leveduras/genética , Sequência de Aminoácidos , Basidiomycota/enzimologia , Clonagem Molecular , DNA Complementar/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase , Dados de Sequência Molecular , Fragmentos de Peptídeos , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Xantofilas , Leveduras/enzimologia , beta Caroteno/análogos & derivados , beta Caroteno/biossínteseRESUMO
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.
Assuntos
Basidiomycota/enzimologia , Basidiomycota/genética , Epóxido Hidrolases/genética , Sequência de Aminoácidos , Animais , Basidiomycota/crescimento & desenvolvimento , Clonagem Molecular , Epóxido Hidrolases/química , Epóxido Hidrolases/metabolismo , Escherichia coli , Genes Fúngicos , Microssomos/enzimologia , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Dobramento de Proteína , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Celulase/genética , Genes Bacterianos , Bacilos Gram-Positivos Asporogênicos Irregulares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Celulase/metabolismo , Clonagem Molecular , Sistema Digestório/microbiologia , Bacilos Gram-Positivos Asporogênicos Irregulares/enzimologia , Concentração de Íons de Hidrogênio , Insetos/microbiologia , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Actinomycetales/enzimologia , Actinomycetales/genética , Genes Bacterianos , Xilosidases/genética , Xilosidases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Análise de Sequência de DNA , Temperatura , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/químicaRESUMO
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.
Assuntos
Basidiomycota/genética , Basidiomycota/metabolismo , Proteínas Fúngicas/genética , Oxirredutases/genética , beta Caroteno/análogos & derivados , Sequência de Aminoácidos , Sequência de Bases , Carotenoides/metabolismo , Clonagem Molecular , Escherichia coli/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Licopeno , Dados de Sequência Molecular , Oxirredutases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Xantofilas , beta Caroteno/biossínteseRESUMO
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.
Assuntos
Proteínas de Bactérias , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Epóxido Hidrolases/química , Escherichia coli , Genes Bacterianos , Biblioteca Genômica , Cinética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Rhodococcus/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Genes Fúngicos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Fungos Mitospóricos/genética , Leveduras/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Molecular , Dosagem de Genes , Código Genético , Biblioteca Genômica , Gliceraldeído-3-Fosfato Desidrogenases/classificação , Fungos Mitospóricos/classificação , Fungos Mitospóricos/enzimologia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Leveduras/classificação , Leveduras/enzimologiaRESUMO
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.
Assuntos
DNA Ribossômico/genética , Dosagem de Genes , Genes Fúngicos/genética , Actinas/genética , Southern Blotting , Clonagem Molecular , Sondas de DNA , Resistência a Medicamentos/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Campo Pulsado , Marcadores Genéticos/genética , Gentamicinas/farmacologia , Canamicina/farmacologia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Transformação Genética/genéticaRESUMO
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.
Assuntos
Aspergillus niger/genética , Clonagem Molecular/métodos , Genes Fúngicos/genética , Vetores Genéticos/genética , Transformação Genética/genética , Escherichia coli/genética , Teste de Complementação Genética , Vetores Genéticos/fisiologia , Fenótipo , Transformação BacterianaRESUMO
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.
Assuntos
Aspergillus niger/enzimologia , Glucana 1,4-alfa-Glucosidase/genética , Aspergillus niger/genética , Biotecnologia , Estudos de Avaliação como Assunto , Amplificação de Genes , Genes Fúngicos , Técnicas Genéticas , Glucana 1,4-alfa-Glucosidase/biossíntese , Transformação GenéticaRESUMO
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.
Assuntos
Aspergillus niger/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glucana 1,4-alfa-Glucosidase/genética , Aspergillus niger/enzimologia , Sequência de Bases , Metabolismo dos Carboidratos , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Repressão Enzimática , Genes Reporter , Vetores Genéticos , Glucana 1,4-alfa-Glucosidase/biossíntese , Dados de Sequência Molecular , Família Multigênica/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Xilose/farmacologiaRESUMO
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.
Assuntos
Aspergillus niger/genética , Cromossomos Fúngicos , Mapeamento Cromossômico , Eletroforese em Gel de Ágar , Ligação Genética , CariotipagemRESUMO
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.
Assuntos
Aspergillus niger/enzimologia , Genes Fúngicos , Glucana 1,4-alfa-Glucosidase/biossíntese , Aspergillus niger/genética , Aspergillus niger/crescimento & desenvolvimento , Fermentação , Glucana 1,4-alfa-Glucosidase/genéticaRESUMO
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.
Assuntos
Aspergillus niger/genética , Glucana 1,4-alfa-Glucosidase/genética , Aspergillus niger/enzimologia , Sequência de Bases , DNA Fúngico/química , DNA Fúngico/genética , Amplificação de Genes , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Engenharia Genética , Metilação , Dados de Sequência Molecular , Transformação GenéticaRESUMO
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.