Breüner, A. | bioRxiv Science

bioRxiv2022

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production

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 the 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 a control in a time-series RNA-seq experiment. We observe transcription in 542 previously un-annotated 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. Via gene-set over-representation analysis of the differentially expressed genes, we identified putatively regulated biological processes; 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.

microbiology↗

bioRxiv2022

CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis

Enhancing yield during bacterial enzyme production could have positive economic and environmental impacts. For cell factories, such improvements in yields could potentially be obtained by fine-tuning the metabolic processes and their regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two -amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Complex operons that are regulated by a wide variety of transcription factors, non-coding and structured RNAs add to the challenge of finding yield-affecting candidates. Surprisingly, we found that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation (75% of novel RNA predictions had absolute logFC > 2). Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expressed annotations, including both novel candidate and prior annotated ncRNAs, we selected 53 non-coding candidates. The targeting with CRISPRi knockdowns transcription in a genomic region on both the sense and the antisense strand. Thus, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

microbiology↗