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. 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. 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.

中文翻译：

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Photorhabdus 和 Xenorhabdus 的遗传工具箱：基于 pSEVA 的异源表达系统和基于 CRISPR/Cpf1 的基因组编辑，用于快速天然产物分析

光杆菌属和致病杆菌属细菌是能运动的革兰氏阴性细菌，与昆虫病原线虫共生。由于其复杂的生命周期，它们产生大量由生物合成基因簇（BGC）编码的专门代谢物（天然产物）。 Photorhabdus 和 Xenorhabdus 的遗传工具很少见，仅适用于少数菌株。过去，已经开发了几种用于激活 BGC 和删除单个基因的工具。然而，这些通常效率有限或耗时。在这些限制中，必须拥有能够促进实际工作的通用表达系统和基因组编辑工具。在本研究中，我们开发了几种表达载体和 CRISPR-Cpf1 基因组编辑载体，用于使用 SEVA 质粒在 Photorhabdus 和 Xenorhabdus 中进行遗传操作。 SEVA 系列基于模块化载体，允许不同元件的交换（例如复制起点和抗生素选择标记，能够插入不同最终应用所需的序列）。最初，我们测试了不同的SEVA载体，其中包含广泛的宿主范围起源和分别针对卡那霉素、庆大霉素和氯霉素的三种不同抗性基因。我们证明这些载体不仅在众所周知的代表（例如 Photorhabdus laumondii TTO1）中具有复制性，而且在其他很少描述的菌株（如 Xenorhabdus sp）中也具有复制性。 TS4。对于我们基于 CRISPR/Cpf1 的系统，我们使用 pSEVA231 主干不仅删除小基因，还删除 BGC 的大部分。此外，我们能够激活和重构 BGC，以获得高价值化合物的高生产滴度，例如 Safracin B（抗癌药物 ET-743 的半合成前体）。本研究的结果提供了基于SEVA（标准欧洲载体架构）集合的新的诱导表达载体和CRISPR/CPf1编码载体，可以改善光杆菌属和致病杆菌属的遗传操作和基因组编辑过程。