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Genomic and transcriptomic comparison of Aspergillus oryzae strains: a case study in soy sauce koji fermentation.

ABSTRACT: The filamentous fungus Aspergillus oryzae is used in soy sauce koji making due to its high productivity of hydrolytic enzymes. In this study, we compared the genomes and transcriptomes of an industrial strain RD2 and a strain with decreased fermentation performance TS2, aiming to explain their phenotypic differences at the molecular level. Under the regulation of conidiation and fermentation conditions, the enhanced hydrolytic enzyme production and flavor precursor formation in RD2 described a complete expression profile necessary to maintain desirable fermentation performance. By contrast, central carbon metabolism was up-regulated in TS2 for fast growth, suggesting a conflicting relationship between mycelium growth and fermentation performance. Accumulation of mutations also lowered the fermentation performance of TS2. Our study has deepened the understanding of the metabolism and related regulatory mechanisms in desirable koji fermentation. A list of potential molecular markers identified here could facilitate targeted strain maintenance and improvement for better koji fermentation.

SUBMITTER: Zhong Y

PROVIDER: S-EPMC6105210 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Genomic and transcriptomic comparison of Aspergillus oryzae strains: a case study in soy sauce koji fermentation.

Zhong Yiyi Y Lu Xi X Xing Lei L Ho Shiu Woon Allen SWA Kwan Hoi Shan HS

Journal of industrial microbiology & biotechnology 20180705 9

The filamentous fungus Aspergillus oryzae is used in soy sauce koji making due to its high productivity of hydrolytic enzymes. In this study, we compared the genomes and transcriptomes of an industrial strain RD2 and a strain with decreased fermentation performance TS2, aiming to explain their phenotypic differences at the molecular level. Under the regulation of conidiation and fermentation conditions, the enhanced hydrolytic enzyme production and flavor precursor formation in RD2 described a c ...[more]

PMID: 29978373

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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:Koji making is a pre-fermentation stage in soy sauce manufacturing that impacts final product quality. Previous studies have provided valuable insights into the microbial species present in koji. However, changes in microbial community functional potential during koji-making are not well-known, nor are the associations among microbial populations and flavoring characteristics. In the present study, we investigated the succession of microbial communities, microbial community functional potential, metabolite profiles, and associations among microbial community members/functions with metabolites during koji making using shotgun metagenomic and metabolomic analyses. Firmicutes, Proteobacteria, and Ascomycota were identified as the most abundant microbial phyla in early koji making (0-12 h). Aspergillus (fungi) and Weissella (bacteria) exhibited marked abundance increases (0.98-38.45% and 0.31-30.41%, respectively) after 48 h of fermentation. Metabolite analysis revealed that aspartic acid, lysine, methyl acetate, isovaleraldehyde, and isoamyl alcohol concentrations increased ∼7-, 9-, 5-, 49-, and 10-fold after 48 h of fermentation. Metagenomic profiling demonstrated that koji communities were dominated by genes related to carbohydrate metabolism and amino acid metabolism, but functional profiles exhibited marked shifts after 24 h of fermentation. The abundances of genes within the categories of carbohydrate and amino acid metabolism all increased during koji making, except for pyruvate metabolism, glycolysis/gluconeogenesis, and the citrate cycle. Correlational analyses indicated that Aspergillus, Lactococcus, Enterococcus, Corynebacterium, and Kocuria abundances were positively correlated with 15 amino acid concentrations (all p < 0.05), while Weissella abundances were positively correlated with concentrations of volatile flavor compounds, including eight amino acids, phenylacetaldehyde, acetic acid, 2,3-butanediol, ethyl acetate, and ethanol (p < 0.05). These results provide valuable information for understanding the microbial-associated mechanisms of flavor formation during koji making.

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.

Dataset Information

Genomic and transcriptomic comparison of Aspergillus oryzae strains: a case study in soy sauce koji fermentation.

Publications

Genomic and transcriptomic comparison of Aspergillus oryzae strains: a case study in soy sauce koji fermentation.

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