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Abstract：
BACKGROUND: Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.
RESULTS: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.
CONCLUSIONS: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.
RESULTS: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.
CONCLUSIONS: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.
摘要：
背景：光生和Xenorhabdus属的细菌是活跃的，与昆虫病原线虫共生的革兰氏阴性菌。由于其复杂的生命周期，它们产生大量的特殊代谢产物（天然产物），编码在生物合成基因簇（BGC）。光纹肌和直纹肌的遗传工具很少见，仅适用于少数菌株。在过去,已经开发了几种工具用于BGC的激活和单个基因的缺失。然而,这些通常效率有限或耗时。在这些限制中，有通用的表达系统和基因组编辑工具可以促进实际工作是至关重要的。
结果：在本研究中，我们使用SEVA质粒开发了几种表达载体和CRISPR-Cpf1基因组编辑载体，用于光纹肌和Xenorhabdus的遗传操作。SEVA集合基于允许不同元件(例如,复制起点和具有插入用于不同末端应用的所需序列的能力的抗生素选择标记)的可交换性的模块化载体。最初，我们测试了不同的SEVA载体，其中包含广泛的宿主来源和三种不同的卡那霉素抗性基因，庆大霉素和氯霉素，分别。我们证明了这些载体不仅在知名代表中具有复制性，例如光纹肌TTO1，但也在其他很少描述的菌株中，如Xenorhabdussp。TS4对于我们基于CRISPR/Cpf1的系统，我们使用pSEVA231骨架不仅删除了小基因，而且删除了大部分BGC。此外，我们能够激活和重构BGC，以获得高生产效价的高价值化合物，如safracinB，抗癌药物ET-743的半合成前体。
结论：这项研究的结果提供了新的诱导型表达载体和CRISPR/CPf1编码载体，全部基于SEVA（标准欧洲载体结构）收集，这可以改善光带和Xenorhabdus的遗传操作和基因组编辑过程。
结果：在本研究中，我们使用SEVA质粒开发了几种表达载体和CRISPR-Cpf1基因组编辑载体，用于光纹肌和Xenorhabdus的遗传操作。SEVA集合基于允许不同元件(例如,复制起点和具有插入用于不同末端应用的所需序列的能力的抗生素选择标记)的可交换性的模块化载体。最初，我们测试了不同的SEVA载体，其中包含广泛的宿主来源和三种不同的卡那霉素抗性基因，庆大霉素和氯霉素，分别。我们证明了这些载体不仅在知名代表中具有复制性，例如光纹肌TTO1，但也在其他很少描述的菌株中，如Xenorhabdussp。TS4对于我们基于CRISPR/Cpf1的系统，我们使用pSEVA231骨架不仅删除了小基因，而且删除了大部分BGC。此外，我们能够激活和重构BGC，以获得高生产效价的高价值化合物，如safracinB，抗癌药物ET-743的半合成前体。
结论：这项研究的结果提供了新的诱导型表达载体和CRISPR/CPf1编码载体，全部基于SEVA（标准欧洲载体结构）收集，这可以改善光带和Xenorhabdus的遗传操作和基因组编辑过程。
