Heterologous Microcompartment Assembly in Bacillaceae: Establishing the Components Necessary for Scaffold Formation.
Heterologous Microcompartment Assembly in Bacillaceae: Establishing the Components Necessary for Scaffold Formation.
复制标题
芽孢杆菌科异源微区室组装：建立支架形成所需的成分。
DOI：
10.1021/acssynbio.9b00155
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发表时间：
2019
影响因子：
4.7
通讯作者：
Wade Y
中科院分区：
文献类型：
--
作者：
Wade Y
细菌微室(BMC)是具有合成代谢和分解代谢功能的特定生化反应的细胞器。含有异源酶途径的不同形态的工程化骨髓基质细胞已经显示出对商品化学品的高生产率，这使得骨髓基质细胞成为代谢工程的重要焦点。在嗜热菌中，获得对BMC组装和异源酶货物的掺入的控制尚未实现。在这里，我们首先通过对嗜热副杆菌中丙二醇利用(PDU)操纵子的详细生物信息学分析来解决这一问题。然后，我们在体内证明了在60℃的高温下组装天然BMCs的能力。枯草芽孢杆菌的热糖苷可使单个管状BMC组装，其平均长度为1.4mBMC；诱导表达壳操纵子20min后可组装成BMCs。此外，我们还证明了通过融合至少24个氨基酸的丙二醇利用蛋白(PDUP)的N端序列，有可能将单体超折叠GFP(MsfGFP)靶向隔间内部。本研究建立了在工业上重要的芽孢杆菌和地杆菌中建立小分子细胞工厂的可行性。由异种载货BMC生产组装而成。此外，这项研究还提供了实验证实，BMCs是在嗜热菌中产生的，这为未来研究改变天然细胞器的用途以在高温下提供新功能开辟了一条道路。
Bacterial microcompartments (BMCs) are organelles that host specific biochemical reactions for both anabolic and catabolic functions. Engineered morphologically diverse BMCs bearing heterologous enzymatic pathways have shown enhanced productivity for commodity chemicals, which makes BMCs an important focus for metabolic engineering. Gaining control of BMC assembly and incorporation of a heterologous enzymatic cargo has yet to be achieved in thermophiles. Herein, we address this by first conducting a detailed bioinformatic analysis of the propanediol utilization (pdu) operon in the thermophileParageobacillus thermoglucosidasius. We then demonstrated,in vivo, the ability to assemble the native BMCs at an elevated temperature of 60 °C. Heterologous expression of Pdu shell proteins fromP. thermoglucosidasiusinBacillus subtilisresulted in the assembly of a single tubular BMC with an average length of 1.4 μm; BMCs assembled after a 20 min induction of expression of the shell operons. Moreover, we show that it is possible to target the monomeric superfolder GFP (msfGFP) to the interior of the compartment by fusion of an N-terminal sequence of the propanediol utilization protein (PduP) of at least 24 amino acids. This study establishes the feasibility of constructing cell factories for small molecules in industrially importantBacillusandGeobacillusspp. by heterologous cargo-carrying BMC production and assembly. Additionally, the study provides experimental confirmation that BMCs are produced in thermophilic bacteria, which opens a path for future research on repurposing the native organelles to provide new functionality at elevated temperatures.