Key Laboratory for Bio-resources and Eco-Environment of the Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, Chengdu, 610064, People's Republic of China.
Microb Cell Fact. 2023 Aug 27;22(1):163. doi: 10.1186/s12934-023-02177-0.
Global transcription machinery engineering (gTME) is an effective approach employed in strain engineering to rewire gene expression and reshape cellular metabolic fluxes at the transcriptional level.
In this study, we utilized gTME to engineer the positive transcription factor, DegU, in the regulation network of major alkaline protease, AprE, in Bacillus pumilus. To validate its functionality when incorporated into the chromosome, we performed several experiments. First, three negative transcription factors, SinR, Hpr, and AbrB, were deleted to promote AprE synthesis. Second, several hyper-active DegU mutants, designated as DegU(hy), were selected using the fluorescence colorimetric method with the host of the Bacillus subtilis ΔdegSU mutant. Third, we integrated a screened degU(L113F) sequence into the chromosome of the Δhpr mutant of B. pumilus SCU11 to replace the original degU gene using a CRISPR/Cas9 system. Finally, based on transcriptomic and molecular dynamic analysis, we interpreted the possible mechanism of high-yielding and found that the strain produced alkaline proteases 2.7 times higher than that of the control strain (B. pumilus SCU11) in LB medium.
Our findings serve as a proof-of-concept that tuning the global regulator is feasible and crucial for improving the production performance of B. pumilus. Additionally, our study established a paradigm for gene function research in strains that are difficult to handle.
全局转录机器工程（gTME）是一种在菌株工程中用于重新布线基因表达并在转录水平重塑细胞代谢通量的有效方法。
在这项研究中，我们利用 gTME 工程化了芽孢杆菌中主要碱性蛋白酶 AprE 调控网络中的正转录因子 DegU。为了验证其整合到染色体中的功能，我们进行了几项实验。首先，删除了三个负转录因子 SinR、Hpr 和 AbrB，以促进 AprE 的合成。其次，使用枯草芽孢杆菌 ΔdegSU 突变体作为宿主，使用荧光比色法选择了几个超活 DegU 突变体，命名为 DegU(hy)。第三，我们使用 CRISPR/Cas9 系统将筛选出的 degU(L113F)序列整合到芽孢杆菌 SCU11 的 Δhpr 突变体的染色体中，以取代原始的 degU 基因。最后，基于转录组学和分子动力学分析，我们解释了高产的可能机制，发现该菌株在 LB 培养基中的碱性蛋白酶产量比对照菌株（芽孢杆菌 SCU11）高 2.7 倍。
我们的研究结果证明了调节全局调节剂是可行的，对于提高芽孢杆菌的生产性能至关重要。此外，我们的研究为难以处理的菌株中的基因功能研究建立了范例。
