easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria

Bode, Edna; Daniela, Assmann; Happel, Petra; Meyer, E.; Münch, Karin; Rössel, Nicole; Bode, Helge B.

doi:10.21769/bioprotoc.4709

Cited by 6 publications

(2 citation statements)

References 25 publications

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“…Finally, 50 µl of the cell suspension was plated on a selection plate with kanamycin and arabinose. Successful editing approaches were confirmed by colony PCR [ 45 ].…”

Section: Methodsmentioning

confidence: 99%

Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

Rill,

Zhao,

Bode

2024

Microb Cell Fact

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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.

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“…Finally, 50 µl of the cell suspension was plated on a selection plate with kanamycin and arabinose. Successful editing approaches were confirmed by colony PCR [ 45 ].…”

Section: Methodsmentioning

confidence: 99%

Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

Rill,

Zhao,

Bode

2024

Microb Cell Fact

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“…In addition, a previously generated ∆hfq mutant, in which the gene encoding Hfq (an RNA chaperone mediating mRNA/sRNA interaction) was deleted, revealed a deprived ability to produce XRDs (Figure S3). 11 In the easyPACiD (easy Promoter Activated Compound Identification) approach 11,12 , the ∆hfq mutant can be used for the selective production of specialized metabolites using promoter-exchange. Therefore, we exchanged the natural promoter of NRPS xrdA with an arabinose inducible promoter, resulting in strain ∆hfq PBAD_xrdA (Table S3).…”

Section: Identification Of the Xrd Mt Xrdmmentioning

confidence: 99%

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

Su,

Huber,

Westphalen

et al. 2024

Preprint

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Xenorhabdins (XRDs) are produced by Xenorhabdus species and are members of the dithiopyrrolone (DTP) class of natural products that have potent antibacterial, antifungal and anticancer activity. The amide moiety of their DTP core can be methylated or not to fine-tune the bioactivity properties. However, the enzyme responsible for the amide N-methylation remained elusive. Here, we identified and characterized the amide methyltransferase XrdM that is encoded nearly 600 kb away from the XRD gene cluster using proteomic analysis, methyltransferase candidate screening, gene deletion, and allied approaches. In addition, crystallographic analysis and site-directed mutagenesis proved that XrdM is completely distinct from the recently reported DTP methyltransferase DtpM, and that both have been tailored in a species-specific manner for DTP biosynthesis in Gram-negative/positive organisms. Our study expands the limited knowledge of post-NRPS amide methylation in DTP biosynthesis and reveals the evolution of two structurally completely different enzymes for the same reaction in different organisms.

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Natural products from Xenorhabdus and Photorhabdus show promise as biolarvicides against Aedes albopictus

Touray,

Ulug,

Gulsen

et al. 2024

Pest Management Science

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BACKGROUNDIn the perpetual struggle to manage mosquito populations, there has been increasing demand for the development of biopesticides to supplant/complement current products. The insecticidal potential of Xenorhabdus and Photorhabdus has long been recognized and is of interest for the control of important mosquitoes like Aedes albopictus which vectors over 20 different arboviruses of global public health concern.RESULTSThe larvicidal effects of cell‐free supernatants, cell growth cultures and cell mass of an extensive list of Xenorhabdus and Photorhabdus spp. was investigated. They were quite effective against Ae. albopictus causing larval mortality ranging between 52–100%. Three Photorhabdus spp. and 13 Xenorhabdus spp. release larvicidal compounds in cell‐free supernatants. Cell growth culture of all tested species exhibited larvicidal activity, except for Xenorhabdus sp. TS4. Twenty‐one Xenorhabdus and Photorhabdus bacterial cells (pellet) exhibited oral toxicity (59–91%) against exposed larvae. The effect of bacterial supernatants on the mosquito eggs were also assessed. Bacterial supernatants inhibited the hatching of mosquito eggs; when unhatched eggs were transferred to clean water, they all hatched. Using the easyPACId approach, the larvicidal compounds in bacterial supernatant were identified as fabclavine from X. szentirmaii and xencoumacin from X. nematophila (causing 98 and 70% mortality, respectively, after 48 h). Xenorhabdus cabanillasii and X. hominickii fabclavines were as effective as commercial Bacillus thuringiensis subsp. israelensis and spinosad products within 5 days post‐application (dpa).CONCLUSIONFabclavine and xenocoumacin can be developed into novel biolarvicides, can be used as a model to synthesize other compounds or/and can be combined with other commercial biolarvicides.This article is protected by copyright. All rights reserved.

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easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria

Cited by 6 publications

References 25 publications

Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

Natural products from Xenorhabdus and Photorhabdus show promise as biolarvicides against Aedes albopictus

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