Physiological and transcriptomic analyses revealed the change of main flavor substance of Zygosaccharomyces rouxii under salt treatment.

Zygosaccharomyces rouxii was a highly salt-tolerant yeast, playing an important role in soy sauce fermentation. Previous studies reported that Z. rouxii under salt treatment produces better fermented food. However, the detailed change of main flavor substance was not clear. In this study, the physiological and transcriptomic analyses of Z. rouxii under salt treatment was investigated. The results revealed the high salt tolerance of Z. rouxii. Analysis of physiological data showed that the proportion of unsaturated fatty acids was significantly increased with the increment of salt concentrations. The analysis of organic acids showed that the content of succinic acid was significantly higher in the salt-treated Z. rouxii while oxalic acid was only identified at the 18% salt concentration-treated group. Results of volatile substances analysis showed that concentrations of 3-methyl-1-butanol and phenylethyl alcohol were significantly increased with the increment of salt concentrations. A comparison of transcriptome data showed that the genes involved in the TCA cycle and the linoleic acid synthesis process exhibited different expressions, which is consistent with the results of physiological data. This study helps to understand the change of main flavor substance of Z. rouxii under salt treatment and guide their applications in the high salt liquid state fermentation of the soy sauce.

Effects of Nitrogen and Phosphorus Limitation on Fatty Acid Contents in Aspergillus oryzae.

Aspergillus oryzae, commonly known as koji mold, has been widely used for the large-scale production of food products (sake, makgeolli, and soy sauce) and can accumulate a high level of lipids. In the present study, we showed the dynamic changes in A. oryzae mycelium growth and conidia formation under nitrogen and phosphorus nutrient stress. The fatty acid profile of A. oryzae was determined and the content of unsaturated fatty acid was found increased under nitrogen and phosphorus limitation. Oleic acid (C18:1), linoleic acid (C18:2), and γ-linolenic acid (C18:3) production were increased on five nitrogen and phosphorus limitation media, especially on nitrogen deep limitation and phosphorus limitation group, showing a 1. 2-, 1. 6-, and 2.4-fold increment, respectively, compared with the control. Transcriptomic analysis showed the expression profile of genes related to nitrogen metabolism, citrate cycle, and linoleic acid synthesis, resulting in the accumulation of unsaturated fatty acid. qRT-PCR results further confirmed the reliability and availability of the differentially expressed genes obtained from the transcriptome analysis. Our study provides a global transcriptome characterization of the nitrogen and phosphorus nutrient stress adaptation process in A. oryzae. It also revealed that the molecular mechanisms of A. oryzae respond to nitrogen and phosphorus stress. Our finding facilitates the construction of industrial strains with a nutrient-limited tolerance.

The GRAS gene family in watermelons: identification, characterization and expression analysis of different tissues and root-knot nematode infestations.

The family of GRAS plant-specific transcription factor plays diverse roles in numerous biological processes. Despite the identification and characterization of GRAS genes family in dozens of plant species, until now, GRAS members in watermelon (Citrullus lanatus) have not been investigated comprehensively. In this study, using bioinformatic analysis, we identified 37 GRAS genes in the watermelon genome (ClGRAS). These genes are classified into 10 distinct subfamilies based on previous research, and unevenly distributed on 11 chromosomes. Furthermore, a complete analysis was conducted to characterize conserved motifs and gene structures, which revealed the members within same subfamily that have analogous conserved gene structure and motif composition. Additionally, the expression pattern of ClGRAS genes was characterized in fruit flesh and rind tissues during watermelon fruit development and under red light (RL) as well as root knot nematode infestation. Finally, for verification of the availability of public transcriptome data, we also evaluated the expression levels of randomly selected four ClGRAS genes under RL and nematode infection by using qRT-PCR. The qRT-PCR results indicated that several ClGRAS genes were differentially expressed, implying their vital role in RL induction of watermelon resistance against root-knot nematodes. The results obtained in this study could be useful in improving the quality of watermelon.

Genome-wide identification of the GATA transcription factor family and their expression patterns under temperature and salt stress in Aspergillus oryzae.

GATA transcription factors (TFs) are involved in the regulation of growth processes and various environmental stresses. Although GATA TFs involved in abiotic stress in plants and some fungi have been analyzed, information regarding GATA TFs in Aspergillus oryzae is extremely poor. In this study, we identified and functionally characterized seven GATA proteins from A. oryzae 3.042 genome, including a novel AoSnf5 GATA TF with 20-residue between the Cys-X2-Cys motifs which was found in Aspergillus GATA TFs for the first time. Phylogenetic analysis indicated that these seven A. oryzae GATA TFs could be classified into six subgroups. Analysis of conserved motifs demonstrated that Aspergillus GATA TFs with similar motif compositions clustered in one subgroup, suggesting that they might possess similar genetic functions, further confirming the accuracy of the phylogenetic relationship. Furthermore, the expression patterns of seven A. oryzae GATA TFs under temperature and salt stresses indicated that A. oryzae GATA TFs were mainly responsive to high temperature and high salt stress. The protein-protein interaction network of A. oryzae GATA TFs revealed certain potentially interacting proteins. The comprehensive analysis of A. oryzae GATA TFs will be beneficial for understanding their biological function and evolutionary features and provide an important starting point to further understand the role of GATA TFs in the regulation of distinct environmental conditions in A. oryzae.

Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi.

Filamentous fungi possess the capacity to produce a wide array of secondary metabolites with diverse biological activities and structures, such as lovastatin and swainsonine. With the advent of the post-genomic era, increasing amounts of cryptic or uncharacterized secondary metabolite biosynthetic gene clusters are continually being discovered. However, owing to the longstanding lack of versatile, comparatively simple, and highly efficient genetic manipulation techniques, the broader exploration of industrially important secondary metabolites has been hampered thus far. With the emergence of CRISPR/Cas9-based genome editing technology, this dilemma may be alleviated, as this advanced technique has revolutionized genetic research and enabled the exploitation and discovery of new bioactive compounds from filamentous fungi. In this review, we introduce the CRISPR/Cas9 system in detail and summarize the latest applications of CRISPR/Cas9-mediated genome editing in filamentous fungi. We also briefly introduce the specific applications of the CRISPR/Cas9 system and CRISPRa in the improvement of secondary metabolite contents and discovery of novel biologically active compounds in filamentous fungi, with specific examples noted. Additionally, we highlight and discuss some of the challenges and deficiencies of using the CRISPR/Cas9-based genome editing technology in research on the biosynthesis of secondary metabolites as well as future application of CRISPR/Cas9 strategy in filamentous fungi are highlighted and discussed.

Transcriptomic Responses of Cordyceps militaris to Salt Treatment During Cordycepins Production.

Cordycepin is a major bioactive compound found in Cordyceps militaris (C. militaris) that exhibits a broad spectrum of biological activities. Hence, it is potentially a bioactive ingredient of pharmaceutical and cosmetic products. However, overexploitation and low productivity of natural C. militaris is a barrier to commercialization, which leads to insufficient supply to meet its existing market demands. In this study, a preliminary study of distinct concentrations of salt treatments toward C. militaris was conducted. Although the growth of C. militaris was inhibited by different salt treatments, the cordycepin production increased significantly accompanied by the increment of salt concentration. Among them, the content of cordycepin in the 7% salt-treated group was five-fold higher than that of the control group. Further transcriptome analysis of samples with four salt concentrations, coupled with Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, several differentially expressed genes (DEGs) were found. Finally, dynamic changes of the expression patterns of four genes involved in the cordycepin biosynthesis pathway were observed by the quantitative real-time PCR. Taken together, our study provides a global transcriptome characterization of the salt treatment adaptation process in C. militaris and facilitates the construction of industrial strains with a high cordycepin production and salt tolerance.

Identification and Expression Analysis of Sugar Transporter Gene Family in Aspergillus oryzae.

Sugar transporter (SUT) genes are associated with multiple physiological and biochemical processes in filamentous fungi, such as the response to various stresses. However, limited systematic analysis and functional information of SUT gene family have been available on Aspergillus oryzae (A. oryzae). To investigate the potential roles of SUTs in A. oryzae, we performed an integrative analysis of the SUT gene family in this study. Based on the conserved protein domain search, 127 putative SUT genes were identified in A. oryzae and further categorized into eight distinct subfamilies. The result of gene structure and conserved motif analysis illustrated functional similarities among the AoSUT proteins within the same subfamily. Additionally, expression profiles of the AoSUT genes at different growth stages elucidated that most of AoSUT genes have high expression levels at the stationary phase while low in the adaptive phase. Furthermore, expression profiles of AoSUT genes under salt stress showed that AoSUT genes may be closely linked to salt tolerance and involved in sophisticated transcriptional process. The protein-protein interaction network of AoSUT propounded some potentially interacting proteins. A comprehensive overview of the AoSUT gene family will offer new insights into the structural and functional features as well as facilitate further research on the roles of AoSUT genes in response to abiotic stresses.