Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae

Machida, Masayuki; Chang, Young‐Chae; Manabe, Masaru; Yasukawa, Mieko; Kunihiro, Sumiko; Jigami, Yoshifumi

doi:10.1007/s002940050152

Cited by 29 publications

(22 citation statements)

References 44 publications

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“…2. The probe for the enolase (enoA) gene (Machida et al 1996) was used as a loading control as its transcript levels are equal during cultivation on different carbon sources (Nakajima et al 2000). The glaB gene was transcribed to a very low level at 17 h in 2% WLM (lane 1) compared to that on 2% WSM (lanes 5-8).…”

Section: Resultsmentioning

confidence: 99%

Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation

Biesebeke

Biezen

Vos

et al. 2004

Appl Microbiol Biotechnol

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Solid-state fermentation (SSF) with Aspergillus oryzae results in high levels of secreted protein. However, control mechanisms of gene expression in SSF have been only poorly studied. In this study we show that both glucoamylase (glaB) and protease (alpA, nptB) genes are highly expressed during surface cultivation on wheatbased solid medium, and even higher during cultivation on wheat kernels. In wheat-based liquid medium, low levels of gene expression are observed. Typical SSF cultivation conditions, such as low water activity and the formation of aerial hyphae, did not contribute to the high-level gene expression on wheat-based solid medium. Analysis of wheat-based solid and liquid cultivations showed differences in carbon and nitrogen utilisation and external pH. The results presented show that the difference in regulation of transcription of the alpA and nptB genes in wheatbased liquid and solid medium could be pH dependent, involving a pH-dependent transcription regulator. The results obtained suggest that the difference in regulation of transcription of the glaB gene in wheat-based liquid and solid medium is caused by a difference in carbohydrate degradation and consumption under the different culture conditions.

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Section: Resultsmentioning

confidence: 99%

Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation

Biesebeke

Biezen

Vos

et al. 2004

Appl Microbiol Biotechnol

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“…Plasmids from three independent clones were isolated, and the sequence from each of these was compared with the VRG4 gene isolated by PCR from the isogenic parental strain and cloned in the same way to exclude PCR-derived mutations. DNA sequencing was performed as described (22) using the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech), using an automated LI-COR 4000L DNA sequencer.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Conserved Motif in the Yeast Golgi GDP-mannose Transporter Required for Binding to Nucleotide Sugar

Gao

Nishikawa

Dean

2001

Journal of Biological Chemistry

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Glycoproteins and lipids in the Golgi complex are modified by the addition of sugars. In the yeast Saccharomyces cerevisiae, these terminal Golgi carbohydrate modifications primarily involve mannose additions that utilize GDP-mannose as the substrate. The transport of GDP-mannose from its site of synthesis in the cytosol into the lumen of the Golgi is mediated by the VRG4 gene product, a nucleotide sugar transporter that is a member of a large family of related membrane proteins. Loss of VRG4 function leads to lethality, but several viable vrg4 mutants were isolated whose GDP-mannose transport activity was reduced but not obliterated. Mutations in these alleles mapped to a region of the Vrg4 protein that is highly conserved among other GDP-mannose transporters but not other types of nucleotide sugar transporters. Here, we present evidence that suggest an involvement of this region of the protein in binding GDP-mannose. Most of the mutations that were introduced within this conserved domain, spanning amino acids 280 -291 of Vrg4p, lead to lethality, and none interfere with Vrg4 protein stability, localization, or dimer formation. The null phenotype of these mutant vrg4 alleles can be complemented by their overexpression. Vesicles prepared from vrg4 mutant strains were reduced in luminal GDP-mannose transport activity, but this effect could be suppressed by increasing the concentration of GDP-mannose in vitro. Thus, either an increased substrate concentration, in vitro, or an increased Vrg4 protein concentration, in vivo, can suppress these vrg4 mutant phenotypes. Vrg4 proteins with alterations in this region were reduced in binding to guanosine 5-[␥-32 P]triphosphate ␥-azidoanilide, a photoaffinity substrate analogue whose binding to Vrg4-HAp was specifically inhibited by GDP-mannose. Taken together, these data are consistent with the model that amino acids in this region of the yeast GDP-mannose transporter mediate the recognition of or binding to nucleotide sugar prior to its transport into the Golgi.

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“…We have established that this gene encodes enolase and that the acuN356 mutation results from a translocation breakpoint in the 59 region separating glycolytic from gluconeogenic regulation, leading to loss of the ability to use gluconeogenic carbon sources while growth is unaffected on glycolytic carbon sources. Analysis of the 59 region of the enolase gene from A. oryzae showed transcription start points at À17, À38, and À45, and deletion analysis of the promoter indicated that a 100-bp sequence between À121 and À228 is necessary for expression on glucose (Machida et al 1996;Figure 6.-Effects of the acuK248 and acuM301 mutations on gene expression. (A) Effects on AcuF-LacZ expression.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Control of Gluconeogenesis in Aspergillus nidulans

et al. 2007

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Aspergillus nidulans can utilize carbon sources that result in the production of TCA cycle intermediates, thereby requiring gluconeogenesis. We have cloned the acuG gene encoding fructose-1,6 bisphosphatase and found that expression of this gene is regulated by carbon catabolite repression as well as by induction by a TCA cycle intermediate similar to the induction of the previously studied acuF gene encoding phosphoenolpyruvate carboxykinase. The acuN356 mutation results in loss of growth on gluconeogenic carbon sources. Cloning of acuN has shown that it encodes enolase, an enzyme involved in both glycolysis and gluconeogenesis. The acuN356 mutation is a translocation with a breakpoint in the 59 untranslated region resulting in loss of expression in response to gluconeogenic but not glycolytic carbon sources. Mutations in the acuK and acuM genes affect growth on carbon sources requiring gluconeogenesis and result in loss of induction of the acuF, acuN, and acuG genes by sources of TCA cycle intermediates. Isolation and sequencing of these genes has shown that they encode proteins with similar but distinct Zn(2) Cys(6) DNA-binding domains, suggesting a direct role in transcriptional control of gluconeogenic genes. These genes are conserved in other filamentous ascomycetes, indicating their significance for the regulation of carbon source utilization.

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Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae

Cited by 29 publications

References 44 publications

Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation

Different control mechanisms regulate glucoamylase and protease gene transcription in Aspergillus oryzae in solid-state and submerged fermentation

Identification of a Conserved Motif in the Yeast Golgi GDP-mannose Transporter Required for Binding to Nucleotide Sugar

Transcriptional Control of Gluconeogenesis in Aspergillus nidulans

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