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 α-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%. We discuss how the findings of this study can be further investigated to elucidate potential direct and indirect mechanisms on yield changes.

中文翻译：

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用于提高枯草芽孢杆菌异源 α-淀粉酶产量的 CRISPRi 筛选

提高细菌酶生产过程中的产量可能会产生积极的经济和环境影响。对于细胞工厂来说，产量的提高可能可以通过微调候选基因的代谢过程及其调节机制来实现。为了寻找这样的候选者，我们对两种产生 α-淀粉酶的芽孢杆菌菌株进行了 RNA 测序，并预测了数百个假定的新型非编码转录区域。受多种转录因子、非编码和结构化 RNA 调控的复杂操纵子增加了寻找影响产量的候选物的挑战。令人惊讶的是，我们发现非编码基因组区域在发酵过程中按比例经历了最高的表达变化（75％的新RNA预测的绝对logFC>2）。由于这些类别的 RNA 也尚未得到充分研究，我们针对相应的基因组区域进行 CRIPSRi 敲低，以测试对产量的任何潜在影响。从差异表达的注释中，包括新的候选和先前注释的 ncRNA，我们选择了 53 个非编码候选。CRISPRi 的靶向可敲低基因组区域有义链和反义链上的转录。因此，CRISPRi 实验无法将产量变化的原因与有义或反义破坏联系起来。尽管如此，我们在几个实例中观察到酶产量发生了剧烈变化。针对 skfA-skfH 操纵子 3' UTR 的推定反义 RNA 的基因组区域的敲低导致产量增加 21%。相比之下，针对细胞色素 c 氧化酶亚基 1 (ctaD)、sigma 因子 sigH 和未表征基因 yhfT 的推定反义 RNA 的基因组区域的敲低，产量降低了 31% 至 43%。我们讨论如何进一步研究本研究的结果，以阐明产量变化的潜在直接和间接机制。