PDF(Pubmed)
Abstract：
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：全球转录机械工程（gTME）是一种用于菌株工程的有效方法，可在转录水平上重新连接基因表达并重塑细胞代谢通量。
结果：在这项研究中，我们利用gTME来改造正转录因子，DegU,在主要碱性蛋白酶的调节网络中，四月,在短小芽孢杆菌中。为了验证其整合到染色体中时的功能，我们做了几个实验。首先,三个负转录因子，SinR,Hpr,和Abrb,被删除以促进AprE合成。第二,几个超活跃的DegU突变体，指定为DegU(HY)，使用荧光比色法与枯草芽孢杆菌ΔdegSU突变体的宿主进行选择。第三，我们使用CRISPR/Cas9系统将筛选的degU（L113F）序列整合到短小芽孢杆菌SCU11的Δhpr突变体的染色体中，以替换原始的degU基因。最后,基于转录组学和分子动态分析，我们解释了高产的可能机制，发现该菌株产生的碱性蛋白酶比对照菌株高2.7倍(B.短小SCU11）在LB培养基中。
结论：我们的发现是一个概念证明，即调整全球监管机构对于提高短小芽孢杆菌的生产性能是可行和至关重要的。此外,我们的研究为难以处理的菌株的基因功能研究建立了范例。
结果：在这项研究中，我们利用gTME来改造正转录因子，DegU,在主要碱性蛋白酶的调节网络中，四月,在短小芽孢杆菌中。为了验证其整合到染色体中时的功能，我们做了几个实验。首先,三个负转录因子，SinR,Hpr,和Abrb,被删除以促进AprE合成。第二,几个超活跃的DegU突变体，指定为DegU(HY)，使用荧光比色法与枯草芽孢杆菌ΔdegSU突变体的宿主进行选择。第三，我们使用CRISPR/Cas9系统将筛选的degU（L113F）序列整合到短小芽孢杆菌SCU11的Δhpr突变体的染色体中，以替换原始的degU基因。最后,基于转录组学和分子动态分析，我们解释了高产的可能机制，发现该菌株产生的碱性蛋白酶比对照菌株高2.7倍(B.短小SCU11）在LB培养基中。
结论：我们的发现是一个概念证明，即调整全球监管机构对于提高短小芽孢杆菌的生产性能是可行和至关重要的。此外,我们的研究为难以处理的菌株的基因功能研究建立了范例。
