Project description:The filamentous fungus Aspergillus oryzae is an important microbial cell factory for industrial production of useful enzymes, such as α-amylase. In order to optimize the industrial enzyme production process, there is a need to understand fundamental processes underlying protein production, here under how protein production links to metabolism through global regulatory structures. In this study, two α-amylase-producing strains of A. oryzae, a wild type strain and a transformant strain containing additional copies of the α-amylase gene, were characterized at a systematic level. Based on integrated analysis of ome-data together with genome-scale metabolic network and flux calculation, we identified key genes, key enzymes, key proteins, and key metabolites involved in the processes of protein synthesis and secretion, nucleotide metabolism, and amino acid metabolism that can be the potential targets for improving industrial protein production. Keywords: Two Aspergillus oryzae strains and two different carbon sources
Project description:The filamentous fungus Aspergillus oryzae is an important microbial cell factory for industrial production of useful enzymes, such as α-amylase. In order to optimize the industrial enzyme production process, there is a need to understand fundamental processes underlying protein production, here under how protein production links to metabolism through global regulatory structures. In this study, two α-amylase-producing strains of A. oryzae, a wild type strain and a transformant strain containing additional copies of the α-amylase gene, were characterized at a systematic level. Based on integrated analysis of ome-data together with genome-scale metabolic network and flux calculation, we identified key genes, key enzymes, key proteins, and key metabolites involved in the processes of protein synthesis and secretion, nucleotide metabolism, and amino acid metabolism that can be the potential targets for improving industrial protein production. Keywords: Two Aspergillus oryzae strains and two different carbon sources Two carbon sources (glucose, maltose) with three biological replicates for A. oryzae strain A1560 and strain CF1.1
Project description:The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.
Project description:The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. On a condition that induces mating-type genes descrived in growth protocol section, each cogenic MAT1-1 and MAT1-2 mating-type strains were processed into RNA extraction and hybridization on Affymetrix microarrays. In a speculation that genes involved in putative mating process were reguated in mating-type dependent manner, we designed the cogenic strains that differs only in mating-type gene locus to analyze differential gene expression of MAT1-1 vs MAT1-2 strains.
Project description:In this study, we achieved translocated chromosomal duplication and triplication of a 1.4-Mb targeted chromosomal region by directly introducing I-SceI meganuclease into A. oryzae protoplast cells. Strains with duplication and triplication of chromosome 2 showed substantial increases in the activity of protease and amylase. Gene dosage effects were enhanced by combining the structural gene and its regulatory gene, indicating that segmental duplications of chromosomes play important phenotypic roles in koji mold strains.
Project description:Aspergillus flavus and A. oryzae represent two unique species predicted to have spent centuries in vastly different environments. A. flavus is an important opportunistic plant pathogen known for contaminating crops with the carcinogenic mycotoxin, aflatoxin and A. oryzae is a domesticated fungus used in food fermentations. Remarkably, the genomes of these two species are still nearly identical. We have used the recently sequenced genomes of A. oryzae RIB40 and A. flavus NRRL3357 along with array based comparative genome hybridization (CGH) as a tool to compare genomes across several strains of these two species. A comparison of three strains from each species by CGH revealed only 42 and 129 genes unique to A. flavus and A. oryzae, respectively. Further, only 709 genes were identified as being polymorphic between the species. Despite the high degree of similarity between these two species, correlation analysis among all data from the CGH arrays for all strains used in this study reveals a species split. However, this view of species demarcation becomes muddled when focused on only those genes for secondary metabolism.
Project description:The Aspergillus oryzae, an important filamentous fungus used in food fermentation and enzyme industry, has been revealed to own prominent features in its genomic compositions by genome sequencing and various other tools. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions and confirmed most of the annotated genes. Moreover, with high resolution and sensitivity afforded by RNA-Seq, we were able to identify a substantial number of novel transcripts, new exons, untranslated regions, alternative upstream initiation codons (uATGs) and upstream open reading frames (uORFs), which serves a remarkable insight into the A. oryzae transcriptome. We also were able to assess the alternative mRNA isoforms in A. oryzae and found a large number of genes undergoing alternative splicing. Many genes or pathways that might involve in higher levels of protein production in solid-state culture than in liquid culture were identified by comparing gene expression levels between different cultures. Our analysis indicated that the transcriptome of A. oryzae was much more complex than previously anticipated and the results might provide a blueprint for further study of A. oryzae transcriptome.
Project description:Vongsangnak2008 - Genome-scale metabolic
network of Aspergillus oryzae (iWV1314)
This model is described in the article:
Improved annotation through
genome-scale metabolic modeling of Aspergillus oryzae.
Vongsangnak W, Olsen P, Hansen K,
Krogsgaard S, Nielsen J.
BMC Genomics 2008; 9: 245
Abstract:
BACKGROUND: Since ancient times the filamentous fungus
Aspergillus oryzae has been used in the fermentation industry
for the production of fermented sauces and the production of
industrial enzymes. Recently, the genome sequence of A. oryzae
with 12,074 annotated genes was released but the number of
hypothetical proteins accounted for more than 50% of the
annotated genes. Considering the industrial importance of this
fungus, it is therefore valuable to improve the annotation and
further integrate genomic information with biochemical and
physiological information available for this microorganism and
other related fungi. Here we proposed the gene prediction by
construction of an A. oryzae Expressed Sequence Tag (EST)
library, sequencing and assembly. We enhanced the function
assignment by our developed annotation strategy. The resulting
better annotation was used to reconstruct the metabolic network
leading to a genome scale metabolic model of A. oryzae.
RESULTS: Our assembled EST sequences we identified 1,046 newly
predicted genes in the A. oryzae genome. Furthermore, it was
possible to assign putative protein functions to 398 of the
newly predicted genes. Noteworthy, our annotation strategy
resulted in assignment of new putative functions to 1,469
hypothetical proteins already present in the A. oryzae genome
database. Using the substantially improved annotated genome we
reconstructed the metabolic network of A. oryzae. This network
contains 729 enzymes, 1,314 enzyme-encoding genes, 1,073
metabolites and 1,846 (1,053 unique) biochemical reactions. The
metabolic reactions are compartmentalized into the cytosol, the
mitochondria, the peroxisome and the extracellular space.
Transport steps between the compartments and the extracellular
space represent 281 reactions, of which 161 are unique. The
metabolic model was validated and shown to correctly describe
the phenotypic behavior of A. oryzae grown on different carbon
sources. CONCLUSION: A much enhanced annotation of the A.
oryzae genome was performed and a genome-scale metabolic model
of A. oryzae was reconstructed. The model accurately predicted
the growth and biomass yield on different carbon sources. The
model serves as an important resource for gaining further
insight into our understanding of A. oryzae physiology.
This model is hosted on
BioModels Database
and identified by:
MODEL1507180056.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.