Dataset Information

Transcriptome and Proteome Expression Analysis of the Metabolism of Amino Acids by the Fungus Aspergillus oryzae in Fermented Soy Sauce.

ABSTRACT: Amino acids comprise the majority of the flavor compounds in soy sauce. A portion of these amino acids are formed from the biosynthesis and metabolism of the fungus Aspergillus oryzae; however, the metabolic pathways leading to the formation of these amino acids in A. oryzae remain largely unknown. We sequenced the transcriptomes of A. oryzae 100-8 and A. oryzae 3.042 under similar soy sauce fermentation conditions. 2D gel electrophoresis was also used to find some differences in protein expression. We found that many amino acid hydrolases (endopeptidases, aminopeptidases, and X-pro-dipeptidyl aminopeptidase) were expressed at much higher levels (mostly greater than double) in A. oryzae 100-8 than in A. oryzae 3.042. Our results indicated that glutamate dehydrogenase may activate the metabolism of amino acids. We also found that the expression levels of some genes changed simultaneously in the metabolic pathways of tyrosine and leucine and that these conserved genes may modulate the function of the metabolic pathway. Such variation in the metabolic pathways of amino acids is important as it can significantly alter the flavor of fermented soy sauce.

SUBMITTER: Zhao G

PROVIDER: S-EPMC4405012 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Transcriptome and Proteome Expression Analysis of the Metabolism of Amino Acids by the Fungus Aspergillus oryzae in Fermented Soy Sauce.

Zhao Guozhong G Yao Yunping Y Wang Chunling C Tian Fengwei F Liu Xiaoming X Hou Lihua L Yang Zhen Z Zhao Jianxin J Zhang Hao H Cao Xiaohong X

BioMed research international 20150407

Amino acids comprise the majority of the flavor compounds in soy sauce. A portion of these amino acids are formed from the biosynthesis and metabolism of the fungus Aspergillus oryzae; however, the metabolic pathways leading to the formation of these amino acids in A. oryzae remain largely unknown. We sequenced the transcriptomes of A. oryzae 100-8 and A. oryzae 3.042 under similar soy sauce fermentation conditions. 2D gel electrophoresis was also used to find some differences in protein express ...[more]

PMID: 25945335

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Project description:Aspergillus oryzae, which generates numerous enzymes for the breakdown of raw materials, is an essential koji mold in soy sauce production. For better soy sauce productivity and flavor quality, China and Japan have developed their own industrial A. oryzae strains at distinct evolutionary branches for use in soy sauce production for decades. However, systematic comparison between the two national industrial strains has been poorly conducted, and thus we have not been able to generate adequate knowledge, especially regarding what are the key hydrolytic enzymes produced by A. oryzae during koji production. This study sequenced and assembled three high-quality genome sequences of industrial A. oryzae originating from China and Japan. Based on the genome sequences, a phylogenetic tree analysis was performed and revealed the evolutional distances between the two national industrial koji molds. Meanwhile, a comparative phenotypic analysis revealed that the two national industrial strains differed in growth and catalytic characteristics, particularly in proteolytic enzyme activities. To investigate the molecular mechanism underlying the phenotypic difference, we conducted systematic comparative genome and transcriptome investigations. We found minor differences in the quantity and diversity of proteolytic enzyme genes between Chinese and Japanese koji molds, while the protease secretion ratio and transcriptional level were dissimilar. We identified 58 potential important enzymes associated with high protein breakdown efficiency during industrial koji fermentation by combining comparative phenotypic and transcriptome data. More research is required to confirm the function of these putative key hydrolytic enzymes. IMPORTANCE Aspergillus oryzae is widely used as an industrial koji mold for soy sauce brewing due to its powerful raw material decomposition capability. Although various proteases in A. oryzae have been identified, it remains a challenge to find essential enzymes involved in soy sauce production. Generally, the industrial A. oryzae used in soy sauce brewing has excellent proteolytic activity. Based on this, we analyzed key proteolytic enzymes according to a comparison of the genome and transcriptome between three industrial strains. This study found little difference in gene numbers and mutations of proteolytic enzymes between three industrial A. oryzae strains. However, variations in protease secretion ratio and transcriptome were discovered between industrial strains. Based on that, we generated 58 candidate key proteolytic enzymes. This work comprehensively analyzed three industrial koji molds, revealing genome development under separate artificial domestication and helping in the study of key proteolytic enzymes during soy sauce production.

Project description:Cantonese soy sauce is an important type of traditional Chinese brewed soy sauce that was developed in southern China, mainly in Guangdong. Due to the long fermentation period and complex microbiota in Cantonese soy sauce, there are few reports on the microbial metaproteomics of Cantonese soy sauce. In this study, integrative metagenomic and metaproteomic analyzes were used to identify the changes in the dominant microbiota and amino acid synthesis-related enzymes and metabolism during Cantonese soy sauce fermentation. Metagenomic analysis revealed that Tetragenococcus halophilus, Weissella confusa, Weissella paramesenteroides, Enterobacter hormaechei, and Aspergillus oryzae were the dominant microbiota. Using the Top 15 dominant microbiota identified by metagenomics as the database, LTQ Orbitrap Velos Pro ETD mass spectrometry was used to obtain metaproteomic information about the microbes in the soy sauce, and the results indicated that the active enzymes involved in the metabolism of amino acids were secreted by microorganisms such as A. oryzae, T. halophilus, and Zygosaccharomyces rouxii. During the Cantonese soy sauce fermentation process. Among them, early fermentation (0-15d) was dominated by A. oryzae and T. halophilus, mid-term fermentation (60-90d) was dominated by Z. rouxii, A. oryzae, and T. halophilus, and late fermentation (90-120d) was dominated by A. oryzae, Z. rouxii, and T. halophilus. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the main enzymes involved in the metabolism of umami amino acids were aspartate aminotransferase, citrate synthase, aconitase, and isocitrate dehydrogenase, which were produced by Z. rouxii and A. oryzae during early fermentation (0-15 d) and the middle fermentation stage (60-90 d). This study constructed a regulatory network of enzymes potentially involved in the metabolism of flavor amino acids, which provided a theoretical basis for studying the amino acid metabolism of Cantonese soy sauce.

Project description:Background:Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNF? antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (Fc?R) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with Fc?RIIIa. Results:Adalimumab was expressed in A. oryzae by the fusion protein system with ?-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNF? neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the Fc?RIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions. Conclusion:These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient's welfare.

Dataset Information

Transcriptome and Proteome Expression Analysis of the Metabolism of Amino Acids by the Fungus Aspergillus oryzae in Fermented Soy Sauce.

Publications

Transcriptome and Proteome Expression Analysis of the Metabolism of Amino Acids by the Fungus Aspergillus oryzae in Fermented Soy Sauce.

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