Section for Computational and RNA Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Novozymes A/S, Copenhagen, Denmark.
Microb Cell Fact. 2022 Jul 2;21(1):131. doi: 10.1186/s12934-022-01861-x.
Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients.
In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2-threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar.
We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.
枯草芽孢杆菌是一种革兰氏阳性细菌，被用作蛋白质生产的细胞工厂。在过去的几十年中，生产菌株的持续优化提高了酶（如淀粉酶）的产量，并使商业应用成为可能。然而，目前的产量仍然明显低于基于可用碳源的理论可能产量。在其自然环境中，枯草芽孢杆菌可以通过分化为可移动的细胞来应对不利的生长条件，这些细胞利用鞭毛游向可用的营养物质。
在这项研究中，我们分析了枯草芽孢杆菌α-淀粉酶生产菌株在 5 天发酵过程中不同时间点的现有转录组数据。我们观察到，fla/che 操纵子的基因，这些基因对于鞭毛组装和运动是必不可少的，随着时间的推移而差异表达。为了研究flagella 操纵子的表达是否影响产量，我们使用 sgRNA 靶向 flgE、fliR 和 flhG 基因对 fla/che 操纵子进行了 CRISPR-dCas9 敲低。敲低导致运动能力受到抑制，α-淀粉酶产量显著增加 2-3 倍。此外，用红霉素抗性基因替换 flgE（鞭毛钩组装所必需的），并在其后添加转录终止子，使α-淀粉酶产量增加了约 30%。CRISPR-dCas9 敲低以及 flgE 缺失菌株中α-淀粉酶的转录水平没有改变，但所有操作都破坏了细胞在琼脂上游泳的能力。
我们证明，在枯草芽孢杆菌α-淀粉酶生产菌株中，无论是通过 CRISPR-dCas9 敲低操纵子还是用红霉素抗性基因替换 flgE 并在其后添加转录终止子来破坏 flagella，都可以增加小规模发酵中α-淀粉酶的产量。
