Project description:Zn (Ⅱ) and Fe (Ⅱ) are the essential metal elements for the growth of microorganisms. It illustrated that more itaconic acid was achieved for 0.25 g/L ZnSO4·2H2O and 0.40 g/L FeSO4·2H2O than the control with 0.15 g/L ZnSO4·2H2O and 0.16 g/L FeSO4·2H2O after single factor assays for Aspergillus terreus.We furhter carried out transcriptome assays to uncover molecular mechanism of the enhanced itaconic acid fermentability for A. terreus with metal ion.Therefore, our study would provide a reference metal ion concentration ratio for itaconic and other biochemicals production.
Project description:A. terreus LYT10 is an industrial strain for itaconic acid production, in which the biosynthesis of itaconic acid and glucose conversion rate were affected by temperature and initial concentration of itaconic acid in industrial production. RNA-seq was used to identify the key regulators related to tolerance mechanism toward various stress conditions.
Project description:A. terreus LYT10 is an industrial strain for itaconic acid production, in which the biosynthesis of itaconic acid and glucose conversion rate were affected by temperature and initial concentration of itaconic acid in industrial production. RNA-seq was used to identify the key regulators related to tolerance mechanism toward various stress conditions. A total of 4 samples were analzyed. The mycelia cultivated in itaconic acid production medium (IPM) on a rotary shaker at 220 rpm and 37°C for 36 h was considered as a reference. IPMs with 5 g/L and 40 g/L itaconic acid (pH 3.25) were used for high-acid culture respectively. High-temperature condition was possesed at 42°C for 36 h in IPM.
Project description:Aim: We aimed to study the differential gene expressions of the target genes encoding H+-ATPase, the mitochondrial respiratory chain complex, and the alternative oxidase (AOX) in Aspergillus terreus NRRL1960 cultivated under low dissolved oxygen Methods:The fermentation samples were collected for RNA extraction.The barcoded RNA libraries were prepared using Lexogen’s Quant-Seq 3’ mRNA seq kit (Ion Torrent, Lexogen, Vienna, Austria). DNA templates for sequencing were prepared from 200 bp v3 OT2 kit and the Ion One Touch 2 platform. Sequencing was performed on the Ion Proton by Ion Torrent Suite v5.0.4. Raw sequences form each sample were uploaded and the dapter sequences were trimmed. The reads were aligned to A. terreus NIH2624 genome references downloaded from http://fungi.ensembl.org/info/website/ftp/index.html using Star 2.4.1d. The bam index files were generated. The quantification of gene expression was performed by Htseq v0.6.0 with -f and -s options indicating bam inputs and un-stranded reads, respectively. Cuffdiff v.2.1.1 was used to estimate the transcript abundance with the -no-diff and default options to generate the differential analysis for each described comparison. Results:From the gene expression (FPKM) results, genes in Cytochrome C complex and aoxA had the high expression level compared to pma encoding H+-ATPase. However, the different gene expression analysis results shown that the target genes in the mitochondrial respiratory chain complex were predominantly down regulated as observed from the log2(fold_change) less than -1 and and the gene encoding AOX (aoxA) were down regulated. Conclusions: In this study, the transcriptomic analysis were performed for better understanding the biosynthesis pathway and the regulation of target genes in itaconic acid cluster and the ATP regeneration pathway.
Project description:We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs.
Project description:We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. To make cDNA libraries, we exploited the 2’, 3’-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion-independent endoribonucleases. Using Arabidopsis thaliana tRNA ligase, RNA fragments with 2’, 3’-cyclic phosphates were covalently attached to defined RNA linkers containing an 8-base long unique molecular identifier (UMI) sequence. Libraries prepared in this manner contain cDNA derived exclusively from RNA fragments with 2', 3'-cyclic phosphates. The UMI sequence allows for detailed quantitation. We optimized and validated 2’, 3’-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A, and RNA from uninfected and poliovirus-infected HeLa cells.
Project description:Methylorubrum extorquens AM1 is engineered to produce itaconic acid by heterologous expression of cis-aconitic acid decarboxylase. Mutation was also performed on phaR in Methylorubrum extorquens AM1, which regulate poly-beta-hydroxybutyrate accumulation, in attempt to increase carbon flux toward itaconic acid production. However, in our case, itaconic acid production by phaR mutant strain was not higher than that of the wildtype. Transcriptomic analysis was utilized in order to examine the cause for this phenomenon. RNA-seq analysis revealed that phaR mutation in the itaconic acid-producing strain might result in a complex regulatory rewiring at the gene expression level, which could cause a reduced resource flux toward ITA production. Also, RNA profiling gave a hint at the broad regulatory role of PhaR.
Project description:Aspergillus terreus is an emerging fungal pathogen in immunocompromised patients. Due to intrinsic resistance of AmB against A. terreus and acquiring resistance to azoles, alternative antifungal strategy needs investigation. Thus, we explored the activity of phytochemicals such as Shikonin, gallic acid, coumaric acid and quercetin against A. terreus. Amongst these, shikonin showed significant inhibition at MIC50;2 µg/ml, considered for proteome profiling.
Project description:Effect of mild metal ion - stress (500µM AlCl3, 500µM VCl3, 1.5mM ZnCl2) on cellular metabolism, resp. on transcription - 30min incubation in yeast Saccharomyces cerevisiae (JSwt - is a FY derivate; MATaleu2ura3trp1HIS3)