Transcriptomic impact of PrsA over-expression during fed-batch Bacillus subtilis alpha-amylase fermentation
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ABSTRACT:
PROVIDER: PRJNA783567 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17038042.fastq.gz | Fastqsanger.gz | |||
| SRR17038043.fastq.gz | Fastqsanger.gz | |||
| SRR17038044.fastq.gz | Fastqsanger.gz | |||
| SRR17038045.fastq.gz | Fastqsanger.gz | |||
| SRR17038046.fastq.gz | Fastqsanger.gz |
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Project description:The production of alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperon aids the enzyme folding and thus reduces stress. Stress pathways are complex and intertwined in regulation of a variety of cellular mechanism; for instance, PrsA over-expression leads to reduced cell growth. To capture an overview over the impacted mechanisms, we analyze the transcriptomic changes during fed-batch AMY fermentation between a PrsA over-expressing strain and a control in a time-series RNA-seq experiment (n=3, 6 timepoints). We found evidence of transcription in 542 previously un-annotated regions, of which 234 had significant changes in transcription levels (Overall FDR 0.01). The pairwise comparison of up-/downregulated genes and their over-representation in biological processes (FDR 0.01) suggest that the PrsA over-expression might alter ATP biosynthesis activity, amino acid metabolism, and cell wall stability. An additional investigation of the protein-protein association network highlights potential cell motility signaling. Overall, these highlighted mechanisms could potentially further reduce secretion stress or detrimental aspects of PrsA over-expression during AMY production.
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Project description:The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes via gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.
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Project description:The gene aml encoding alpha-amylase in Streptomyces lividans was cloned in the multicopy plasmid pIJ486, generating plasmid pAMI11. Plasmid pAMI11 and pIJ486 were propagated in S. lividans TK21 to obtain S. lividans TK21(pAMI11) and its isogenic strain S. lividans TK21(pIJ486). Transcriptional profiling of the bacterium that overproduces alpha-amylase mainly resulted in the upregulation of genes involved in the biogenesis and function of ribosomes, together with the upregulation of the genes involved in the redox processes, the ABC transporters, the central carbon, aminoacid and purine /pyrimidine metabolism. Moreover, some genes involved in oxidative stress were upregulated. The number of genes downregulated was much lower than the upregulated ones. Therefore, bacteria respond by favouring alpha-amylase overproduction that apparently does not cause damage to the cell. All microarray analyses were performed with RNA samples obtained from three independent cultures grown under identical conditions. The cDNA obtained from each RNA preparation of the alpha-amylase overproducer strain was hybridised with the cDNA obtained from the equivalent RNA preparation of the isogenic strain (S. lividans TK21 [pIJ486]).
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