Bin Zeng

Deep sequencing analysis of transcriptomes in Aspergillus oryzae in response to salinity stress

Characterization of the changes after various stimuli is crucial to comprehend the adaptation of cells to the changing condition. Aspergillus oryzae is widely used for the industrial production of soy sauce, which always encounter changes within a complex environment, such as salinity stress. However, the protective biochemical mechanisms of A. oryzae against salinity stress are poorly understood. In this study, we successfully characterized the fermentative behavior, transcriptomic profiles, and metabolite changes of A. oryzae in response to salinity stress. The results showed that salt treatment of A. oryzae inhibited the fungal development and conidia formation. Transcriptomic analysis showed an upregulated expression of the genes related to arginine accumulation and oleic acid synthesis. The results of qRT-PCR were further confirmed by the reliability and availability of the differentially expressed genes obtained from the transcriptome analysis. Metabolomic analysis revealed that the corresponding intracellular accumulation of arginine and oleic acid were also increased in response to the salinity stress. All of the results provide a global transcriptome characterization of the salt adaptation process in A. oryzae, and offer multiple target genes for salt tolerance improvement via genetic engineering.

Genome-wide identification and expression profile analysis of the HOG gene family in Aspergillus oryzae

The High osmolarity glycerol (HOG) gene family plays crucial roles in various developmental and physiological processes in fungi, such as the permeability of cell membrane, chlamydospore formation and stress signaling. Although the function of HOG genes has been investigated in Saccharomyces cerevisiae and some filamentous fungi, a comprehensive analysis of HOG gene family has not been performed in Aspergillus oryzae, a fungi mainly used for the production of soy sauce. In this study, we identified and corrected a total of 90 HOG genes from the A. oryzae genome. According to the phylogenetic relationship, they were divided into four discrete groups (Group A–D) comprising of 16, 24, 30 and 20 proteins, respectively. Six conserved motifs and exon–intron structures were examined among all HOG proteins to reveal the diversity of AoHOG genes. Based on transcriptome technology, the expression patterns of AoHOG genes across all developmental stages was identified, suggesting that the AoHOG gene family mainly functions in the logarithmic phase of development. The expression profiles of AoHOG genes under different concentrations of salt stress indicated that AoHOG genes are extensively involved in salt stress response, with possibly different mechanisms. The genome-wide identification, evolutionary, gene structures and expression analyses of AoHOG genes provide a comprehensive overview of this gene family as well as their potential involvements in development and stress responses. Our results will facilitate further research on HOG gene family regarding their physiological and biochemical functions.

Recent Advances in Ergosterol Biosynthesis and Regulation Mechanisms in Saccharomyces cerevisiae

Ergosterol, an important component of the fungal cell membrane, is not only essential for fungal growth and development but also very important for adaptation to stress in fungi. Ergosterol is also a direct precursor for steroid drugs. The biosynthesis of ergosterol can be divided into three modules: mevalonate, farnesyl pyrophosphate (farnesyl-PP) and ergosterol biosynthesis. The regulation of ergosterol content is mainly achieved by feedback regulation of ergosterol synthase activity through transcription, translation and posttranslational modification. The synthesis of HMG-CoA, catalyzed by HMGR, is a major metabolic check point in ergosterol biosynthesis. Excessive sterols can be subsequently stored in lipid droplets or secreted into the extracellular milieu by esterification or acetylation to avoid toxic effects. As sterols are insoluble, the intracellular transport of ergosterol in cells requires transporters. In recent years, great progress has been made in understanding ergosterol biosynthesis and its regulation in Saccharomyces cerevisiae. However, few reviews have focused on these studies, especially the regulation of biosynthesis and intracellular transport. Therefore, this review summarizes recent research progress on the physiological functions, biosynthesis, regulation of biosynthesis and intracellular transportation of ergosterol in S. cerevisiae.

Genome-wide Identification and Expression Analysis of the TRK Gene Family in Aspergillus oryzae

Aspergillus oryzae is a traditional production strain in China for brewing wine, edible soy sauce, fermented soya beans and so on. It can be well adapted to salt stress, low pH and alcohol stress, and the TRK gene family plays an important role in maintaining stable osmotic balance in vivo. In this study, the members of TRK gene family in Aspergillus oryzae were identified and analyzed on the whole genome level by means of bioinformatics. The phylogenetic tree, gene structure and motifs were analyzed. The results showed that five TRK gene family members were identified in Aspergillus oryzae, and the expression analysis showed that four of the TRK genes increased with the increase of salt concentration. The results of this study will lay a theoretical foundation for the cloning of the members of the TRK gene family and the identification of their functions. It will also provide reference information for studying the cationic transporter and osmotic pressure regulation mechanism of Aspergillus oryzae.

Transcriptome analysis of different growth stages of Aspergillus oryzae reveals dynamic changes of distinct classes of genes during growth

BackgroundThe gene expression profile and metabolic pathways of Aspergillus oryzae underlying the anatomical and morphological differentiation across different growth stages have not been fully characterized. The rapid development of next-generation sequencing technologies provides advanced knowledge of the genomic organization of A. oryzae.ResultsIn this study, we characterized the growth and development of A. oryzae at different growth stages, including the adaptive phase, logarithmic phase, and stationary phase. Our results revealed that A. oryzae undergoes physiological and morphological differentiation across the different stages. RNA-seq was employed to analyze the three stages of A. oryzae, which generated more than 27 million high-quality reads per sample. The analysis of differential gene expression showed more genes expressed differentially upon transition from the adaptive phase to the logarithmic and stationary phases, while relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase. GO classification of the differentially expressed genes among different growth stages revealed that most of these genes were enriched for single-organism process, metabolic process, and catalytic activity. These genes were then subjected to a clustering analysis. The results showed that the cluster with the majority of genes with increased expression upon transition from the adaptive phase to the logarithmic phase, and steady expression from the logarithmic phase to the stationary phase was mainly involved in the carbohydrate and amino acid metabolism.ConclusionOur results provide a foundation for identifying developmentally important genes and understanding the biological processes across various growth stages.

Genome-wide identification of the fatty acid desaturases gene family in four Aspergillus species and their expression profile in Aspergillus oryzae

Fatty acid desaturases play a key role in producing polyunsaturated fatty acids by converting single bonds to double bonds. In the present study, a total of 13, 12, 8 and 8 candidate fatty acid desaturases genes were identified in the Aspergillus oryzae, Aspergillus flavus, Aspergillus fumigatus and Aspergillus nidulans genomes through database searches, which were classified into five different subfamilies based on phylogenetic analysis. Furthermore, a comprehensive analysis was performed to characterize conserved motifs and gene structures, which could provide an intuitive comprehension to learn the relationship between structure and functions of the fatty acid desaturases genes in different Aspergillus species. In addition, the expression pattern of 13 fatty acid desaturases genes of A. oryzae was tested in different growth stages and under salt stress treatment. The results revealed that the fatty acid desaturases genes in A. oryzae were highly expressed in adaptive phase growth and up-regulated under salt stress treatment. This study provided a better understanding of the evolution and functions of the fatty acid desaturases gene family in the four Aspergillus species, and would be useful for seeking methods to improve the production of unsaturated fatty acids and enhance efforts for the genetic improvement of strains to adapt to the complex surrounding environment.

Highly efficient improvement of Monascus pigment production by accelerating starch hydrolysis in Monascus ruber CICC41233

To investigate the relationship between starch hydrolysis and Monascus pigments (MPs) production, the α-amylase gene (AOamyA) from Aspergillus oryzae was heterologously expressed in Monascus ruber CICC41233, and we obtained a positive transformant named Monascus ruber Amy9. In M. ruber Amy9, the α-amylase activities were 6.65- and 4.26-fold higher at 72xa0h and 144xa0h, respectively, than those in the parent strain with the glucose as solo carbon medium. Surprisingly, in the MPs fermentation medium with rice powder as solo material, M. ruber Amy9 completely degraded starch at 48xa0h, while 43.93 and 7.29xa0mg/mL starch remained at 48 and 144xa0h, respectively, in the parent strain. Monascus ruber Amy9 accelerated starch hydrolysis, which enhanced biomass and also increased total MPs by 132% after 144xa0h. Compared with M. ruber CICC41233, the relative gene expression levels, as determined by a quantitative real-time polymerase chain reaction analysis, of acl2 encoding ATP-citrate lyase subunit 2, pks encoding polyketide synthase, and fasB encoding the fatty acid synthase beta subunit increased by 33.14, 145.18, and 32.15%, respectively, after 144xa0h in M. ruber Amy9. The up-regulated expression of these key genes in MPs synthesis contributed to the large increase in MPs production. This interesting work provided us with a new idea and a new target for the study of the MPs production.

The acyl-CoA binding protein affects Monascus pigment production in Monascus ruber CICC41233

The present study verified whether acyl-coenzyme A (acyl-CoA)-binding protein (ACBP) affected the production of Monascus pigments (MPs) in Monascus ruber CICC41233 (MrACBP). Phylogenetic analysis revealed that the cloned Mracbp gene, which encoded the MrACBP protein, exhibited the closest match (99% confidence level) to the gene from Penicilliopsis zonata. The MrACBP and maltose-binding protein (MBP) were simultaneously expressed in Escherichia coli Rosetta DE3 in the form of a fusion protein. The microscale thermophoresis binding assay revealed that the purified MBP–MrACBP exhibited a higher affinity for myristoyl-CoA (Kdxa0=xa088.16xa0nM) than for palmitoyl-CoA (Kdxa0=xa0136.07xa0nM) and octanoyl-CoA (Kdxa0=xa0270.9xa0nM). Further, the Mracbp gene was homologously overexpressed in M. ruber CICC41233, and a positive transformant M. ruber ACBP5 was isolated. The fatty acid myristic acid in M. ruber ACBP5 was lower than that in the parent strain M. ruber CICC41233. However, when compared with the parent strain, the production of total MPs, water-soluble pigment, and ethanol-soluble pigment in M. ruber ACBP5 increased by 11.67, 9.80, and 12.70%, respectively, after 6xa0days. The relative gene expression level, as determined by a quantitative real-time polymerase chain reaction analysis, of the key genes acbp, pks, mppr1, fasA, and fasB increased by 4.03-, 3.58-, 1.67-, 2.11-, and 2.62-fold after 6xa0days. These data demonstrate the binding preference of MrACBP for myristoyl-CoA, and its influence on MPs production.

Improvement in xylooligosaccharides production by knockout of the β-xyl1 gene in Trichoderma orientalis EU7-22

The goal of this study was to enhance the production of xylooligosaccharides (XOs) and reduce the production of xylose. We investigated β-xylosidases, which were key enzymes in the hydrolysis of xylan into xylose, in Trichoderma orientalis EU7-22. The binary vector pUR5750G/bxl::hph was constructed to knock out the β-xyl1 gene (encoding β-xylosidases) in T. orientalis EU7-22 by homologous integration, producing the mutant strain T. orientalis Bxyl-1. Xylanase activity for strain Bxyl-1 was 452.42xa0IU/mL, which increased by only 0.07% compared to that of parental strain EU7-22, whereas β-xylosidase activity was 0.06xa0IU/mL, representing a 91.89% decrease. When xylanase (200xa0IU/g xylan), produced by T. orientalis EU7-22 and T. orientalis Bxyl-1, was used to hydrolyze beechwood xylan, in contrast to the parental strain, the XOs were enhanced by 83.27%, whereas xylose decreased by 45.80% after 36xa0h in T. orientalis Bxyl-1. Based on these results, T. orientalis Bxyl-1 has great potential for application in the production of XOs from lignocellulosic biomass.