Vajente Matteo, Clerici Riccardo, Ballerstedt Hendrik, Blank Lars M, Schmidt Sandy
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713AV, The Netherlands.
Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
ACS Synth Biol. 2024 Oct 31. doi: 10.1021/acssynbio.4c00380.
Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting nonmodel bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in nonmodel bacteria. As a test strain, we use H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO and hydrogen. However, its low electroporation efficiency hampers the high-throughput genetic engineering required to develop into an industrially relevant host organism. Thus, conjugation has often been the method of choice for introducing exogenous DNA, especially when introducing large plasmids or suicide plasmids. We first propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of nonmodel bacteria.
细菌是代谢反应的宝库，但大多数工业生物技术应用依赖于有限的一组既定宿主生物体。相比之下，采用非模式细菌生产各种目标化学品往往受到其有限的遗传可操作性以及低转化效率的阻碍。在本研究中，我们提出了一系列可采取的步骤来提高非模式细菌中的电穿孔效率。作为测试菌株，我们使用H16，一种经工程改造可利用一氧化碳和氢气生产多种产品的化能自养细菌。然而，其低电穿孔效率阻碍了将其开发成具有工业相关性的宿主生物体所需的高通量基因工程。因此，接合作用常常是引入外源DNA的首选方法，尤其是在引入大型质粒或自杀质粒时。我们首先提出一种基于天然甲基化DNA和金门组装的不依赖物种的技术，将一锅克隆和电穿孔效率提高70倍。其次，利用生物信息学工具预测防御系统并制定限制规避策略，该策略用于通过电穿孔引入自杀质粒以获得驯化菌株。结果将在非模式细菌代谢工程的背景下进行讨论。
