RESUMO
Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-beta gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5-17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium 'Kozak'-like sequence increased expression levels approximately 1.5-fold. Thus, using both codon optimization and 'Kozak' adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/genética , Dictyostelium/genética , Hormônio Foliculoestimulante/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gonadotropina Coriônica Humana Subunidade beta/biossíntese , Códon/genética , Ensaio de Imunoadsorção Enzimática , Hormônio Foliculoestimulante/biossíntese , Técnicas Genéticas , Humanos , Dados de Sequência Molecular , Elongação Traducional da Cadeia Peptídica , Proteínas Recombinantes/biossíntese , Ribossomos/metabolismoRESUMO
The expression of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungus Aspergillus niger. A strain with a loss-of-function mutation in the xlnR gene encoding the transcriptional activator XlnR and a strain with multiple copies of this gene were investigated in order to define which genes are controlled by XlnR. The data presented in this paper show that the transcriptional activator XlnR regulates the transcription of the xlnB, xlnC, and xlnD genes encoding the main xylanolytic enzymes (endoxylanases B and C and beta-xylosidase, respectively). Also, the transcription of the genes encoding the accessory enzymes involved in xylan degradation, including alpha-glucuronidase A, acetylxylan esterase A, arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A, was found to be controlled by XlnR. In addition, XlnR also activates transcription of two endoglucanase-encoding genes, eglA and eglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.
Assuntos
Aspergillus niger/genética , Celulase/genética , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Glucosidases/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Aspergillus niger/metabolismo , Celulase/biossíntese , Celulose/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Fúngicos , Glucosidases/biossíntese , Mutação , Xilanos/metabolismoRESUMO
The xlnD gene encoding the 85-kDa beta-xylosidase was cloned from Aspergillus nidulans. The deduced primary structure of the protein exhibits considerable similarity to the primary structures of the Aspergillus niger and Trichoderma reesei beta-xylosidases and some similarity to the primary structures of the class 3 beta-glucosidases. xlnD is regulated at the transcriptional level; it is induced by xylan and D-xylose and is repressed by D-glucose. Glucose repression is mediated by the product of the creA gene. Although several binding sites for the pH regulatory protein PacC were found in the upstream regulatory region, it was not clear from a Northern analysis whether PacC is involved in transcriptional regulation of xlnD.
Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Genes Fúngicos , Xilosidases/genética , Sequência de Aminoácidos , Aspergillus nidulans/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Complementation by transformation of an Aspergillus niger mutant lacking xylanolytic activity led to the isolation of the xlnR gene. The xlnR gene encodes a polypeptide of 875 amino acids capable of forming a zinc binuclear cluster domain with similarity to the zinc clusters of the GAL4 superfamily of transcription factors. The XlnR-binding site 5'-GGCTAAA-3' was deduced after electrophoretic mobility shift assays, DNase I footprinting and comparison of various xylanolytic promoters. The importance of the second G within the presumed XlnR binding site 5'-GGCTAAA-3' was confirmed in vitro and in vivo. The 5'-GGCTAAA-3' consensus sequence is found within several xylanolytic promoters of various Aspergillus species and Penicillium chrysogenum. Therefore, this sequence may be an important and conserved cis-acting element in induction of xylanolytic genes in filamentous fungi. Our results indicate that XlnR is a transcriptional activator of the xylanolytic system in A. niger.
Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica/fisiologia , Xilanos/metabolismo , Xilosidases/genética , Sequência de Aminoácidos , Aspergillus niger/enzimologia , Sequência de Bases , Ligação Competitiva , Northern Blotting , Southern Blotting , Western Blotting , Pegada de DNA , DNA Fúngico/química , Desoxirribonuclease I , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Transativadores/genética , Transcrição Gênica , Transformação Genética , Xilano Endo-1,3-beta-Xilosidase , Xilosidases/química , Xilosidases/metabolismo , Dedos de Zinco/genética , Dedos de Zinco/fisiologiaRESUMO
Two proteins exhibiting beta-D-xylosidase activity were identified upon fractionation and purification of a culture filtrate of an arabinoxylan-grown Aspergillus niger. A single band of 110 kDa by SDS/PAGE was obtained in both cases and these were active on xylo-oligosaccharides and on xylan. Partial xlnD cDNA clones were immunochemically identified and isolated from a lambda cDNA expression library. Sequence analysis showed that all cDNA clones correspond to a single gene. A genomic clone was isolated and overexpressed in A. niger and A. nidulans. The xlnD gene has an ORF of 2412 nucleotides, encodes a protein of 804 amino acids and contains a potential signal peptide of 26 amino acids. This results in a mature protein of 778 amino acids with a predicted molecular mass of 85 kDa and an isoelectric point of 4.5. The protein is N-glycosylated and contains 15 potential N-glycosylation sites. Sequence similarity is found with beta-D-glucosidases both of bacterial and fungal origin. Both beta-xylosidase proteins purified have high activity on the artificial substrate p-nitrophenyl beta-D-xylopyranoside (XylNp) and a side activity on p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl beta-D-glucopyranoside. A niger strains in which the xlnD gene was disrupted accumulate mainly xylobiose and xylotriose when grown on xylan and have no significant beta-xylosidase activity in the culture medium, indicating that this gene encodes the major extracellular beta-xylosidase.
