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

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.

Enhanced production of trans-cinnamic acid in Photorhabdus luminescens with homolog expression and deletion strategies.

AIM: This study aimed to overproduce industrially relevant and safe bio-compound trans-cinnamic acid (tCA) from Photorhabdus luminescens with deletion strategies and homologous expression strategies that had not been applied before for tCA production. METHODS AND RESULTS: The overproduction of the industrially relevant compound tCA was successfully performed in P. luminescens by deleting stlB (TTO1ΔstlB) encoding a cinnamic acid CoA ligase in the isopropylstilbene pathway and the hcaE insertion (knockout) mutation (hcaE::cat) in the phenylpropionate catabolic pathway, responsible for tCA degradation. A double mutant of both stlB deletion and hcaE insertion mutation (TTO1DM ΔstlB-hcaE::cat) was also generated. These deletion strategies and the phenylalanine ammonium lyase-producing (PI-PAL from Photorhabdus luminescens) plasmid, pBAD30C, carrying stlA (homologous expression mutants) are utilized together in the same strain using different media, a variety of cultivation conditions, and efficient anion exchange resin (Amberlite IRA402) for enhanced tCA synthesis. At the end of the 120-h shake flask cultivation, the maximum tCA production was recorded as 1281 mg l-1 in the TTO1pBAD30C mutant cultivated in TB medium, with the IRA402 resin keeping 793 mg l-1 and the remaining 488 mg l-1 found in the supernatant. CONCLUSION: TCA production was successfully achieved with homologous expression, coupled with deletion and insertion strategies. 1281 mg l-1is the highest tCA concentration that achieved by bacterial tCA production in flask cultivation, according to our knowledge.

A taste of a toxin paradise: Xenorhabdus and Photorhabdus bacterial secondary metabolites against Aedes aegypti larvae and eggs.

Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria are promising sources of effective compounds with important biological activities. This study investigated the effects of cell-free supernatants of X. szentirmaii, X. cabanillasii and P. kayaii against Ae. aegypti eggs and larvae and identified the bioactive larvicidal compound in X. szentirmaii using The EasyPACId method. Among the three tested bacterial species, X. cabanillasii exhibited the highest (96%) egg hatching inhibition and larvicidal activity (100% mortality), whereas P. kayaii was the least effective species in our study. EasyPACId method revealed that bioactive larvicidal compound in the bacterial supernatant was fabclavine. Fabclavines obtained from promoter exchange mutants of different bacterial species such as X. cabanillasii, X. budapestensis, X. indica, X. szentirmaii, X. hominckii and X. stockiae were effective against mosquito larvae. Results show that these bacterial metabolites have potential to be used in integrated pest management (IPM) programmes of mosquitoes.

Investigation of the Odilorhabdin Biosynthetic Gene Cluster Using NRPS Engineering.

The recently identified natural product NOSO-95A from entomopathogenic Xenorhabdus bacteria, derived from a biosynthetic gene cluster (BGC) encoding a non-ribosomal peptide synthetase (NRPS), was the first member of the odilorhabdin class of antibiotics. This class exhibits broad-spectrum antibiotic activity and inspired the development of the synthetic derivative NOSO-502, which holds potential as a new clinical drug by breaking antibiotic resistance. While the mode of action of odilorhabdins was broadly investigated, their biosynthesis pathway remained poorly understood. Here we describe the heterologous production of NOSO-95A in Escherichia coli after refactoring the complete BGC. Since the production titer was low, NRPS engineering was applied to uncover the underlying biosynthetic principles. For this, modules of the odilorhabdin NRPS fused to other synthetases were co-expressed with candidate hydroxylases encoded in the BGC allowing the characterization of the biosynthesis of three unusual amino acids and leading to the identification of a prodrug-activation mechanism by deacylation. Our work demonstrates the application of NRPS engineering as a blueprint to mechanistically elucidate large or toxic NRPS and provides the basis to generate novel odilorhabdin analogues with improved properties in the future.

Type S Non-Ribosomal Peptide Synthetases for the Rapid Generation of Tailormade Peptide Libraries.

Bacterial natural products in general, and non-ribosomally synthesized peptides in particular, are structurally diverse and provide us with a broad range of pharmaceutically relevant bioactivities. Yet, traditional natural product research suffers from rediscovering the same scaffolds and has been stigmatized as inefficient, time-, labour- and cost-intensive. Combinatorial chemistry, on the other hand, can produce new molecules in greater numbers, cheaper and in less time than traditional natural product discovery, but also fails to meet current medical needs due to the limited biologically relevant chemical space that can be addressed. Consequently, methods for the high throughput generation of new natural products would offer a new approach to identifying novel bioactive chemical entities for the hit to lead phase of drug discovery programs. As a follow-up to our previously published proof-of-principle study on generating bipartite type S non-ribosomal peptide synthetases (NRPSs), we now envisaged the de novo generation of non-ribosomal peptides (NRPs) on an unreached scale. Using synthetic zippers, we split NRPSs in up to three subunits and rapidly generated different bi- and tripartite NRPS libraries to produce 49 peptides, peptide derivatives, and de novo peptides at good titres up to 145 mg L-1 . A further advantage of type S NRPSs not only is the possibility to easily expand the created libraries by re-using previously created type S NRPS, but that functions of individual domains as well as domain-domain interactions can be studied and assigned rapidly.

Phurealipids, produced by the entomopathogenic bacteria, Photorhabdus, mimic juvenile hormone to suppress insect immunity and immature development.

Phurealipids (Photorhabdus urea lipids) are synthesized from Photorhabdus bacteria that are symbiotic to entomopathogenic nematodes. Their chemical structures are similar to that of juvenile hormone (JH) and have been suspected to mimic JH signaling in immunity and the development of insects. This study investigated the physiological roles of phurealipids with respect to their contribution to bacterial pathogenicity using four natural (HB13, HB69, HB416, and HB421) and one derivative (HB27) compound. First, phurealipids like JH suppressed insect immune responses. Overall, phurealipids showed JH like immunosuppressive behavior in a lepidopteran insect Spodoptera exigua larvae. More specifically, phurealipids significantly suppressed the hemocyte spreading behavior which is a key immune response upon immune challenge. Interestingly, the methyl urea derivatives (HB13, HB27, and HB69) were more potent than the unmethylated forms (HB416 and HB421). The inhibitory activity of phurealipids prevented the cellular immune response measured by hemocytic nodule formation in response to the bacterial challenge. Phurealipids also suppressed the expression of cecropin and gallerimycin, which are two highly inducible antimicrobial peptides, in S. exigua upon immune challenge. The immunosuppressive activity of the phurealipids significantly enhanced the bacterial pathogenicity of Bacillus thuringiensis against S. exigua. Second, phurealipids like JH prevented insect metamorphosis. Especially, the methylated urea derivatives of the phurealipids showed the JH-like function by inducing the expression of S. exigua Kr-h1, a transcriptional factor. At the pupal stage, exhibiting the lowest expression of Kr-h1, phurealipid treatments elevated the expression level of Kr-h1 and delayed the pupa-to-adult metamorphosis. These results suggest that phurealipids play crucial roles in Photorhabdus pathogenicity by suppressing host immune defenses and delaying host metamorphosis.

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Genome Mining Enabled by Biosynthetic Characterization Uncovers a Class of Benzoxazolinate-Containing Natural Products in Diverse Bacteria.

Benzoxazolinate is a rare bis-heterocyclic moiety that interacts with proteins and DNA and confers extraordinary bioactivities on natural products, such as C-1027. However, the biosynthetic gene responsible for the key cyclization step of benzoxazolinate remains unclear. Herein, we show a putative acyl AMP-ligase responsible for the last cyclization step. We used the enzyme as a probe for genome mining and discovered that the orphan benzobactin gene cluster in entomopathogenic bacteria prevails across Proteobacteria and Firmicutes. It turns out that Pseudomonas chlororaphis produces various benzobactins, whose biosynthesis is highlighted by a synergistic effect of two unclustered genes encoding enzymes on boosting benzobactin production; the formation of non-proteinogenic 2-hydroxymethylserine by a serine hydroxymethyltransferase; and the types I and II NRPS architecture for structural diversity. Our findings reveal the biosynthetic potential of a widespread benzobactin gene cluster.

Climate-specific biosynthetic gene clusters in populations of a lichen-forming fungus.

Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.

Activation, Structure, Biosynthesis and Bioactivity of Glidobactin-like Proteasome Inhibitors from Photorhabdus laumondii.

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.

An Unconventional Melanin Biosynthesis Pathway in Ustilago maydis.

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well-established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4, and pks5), a cytochrome P450 monooxygenase (cyp4), and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimers to generate orsellinic acid (OA), which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL), also derived from Pks3/Pks4 heterodimers to form larger molecules, including novel coumarin derivatives. Our findings suggest that U. maydis synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or l-3,4-dihydroxyphenylalanine (l-DOPA). Along with these observations, this work also provides insight into the mechanisms of polyketide synthases in this filamentous fungus.IMPORTANCE The fungus Ustilago maydis represents one of the major threats to maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, such as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multidomain enzymes and their evolution in the fungal kingdom.

Dual phenazine gene clusters enable diversification during biosynthesis.

Biosynthetic gene clusters (BGCs) bridging genotype and phenotype continuously evolve through gene mutations and recombinations to generate chemical diversity. Phenazine BGCs are widespread in bacteria, and the biosynthetic mechanisms of the formation of the phenazine structural core have been illuminated in the last decade. However, little is known about the complex phenazine core-modification machinery. Here, we report the diversity-oriented modifications of the phenazine core through two distinct BGCs in the entomopathogenic bacterium Xenorhabdus szentirmaii, which lives in symbiosis with nematodes. A previously unidentified aldehyde intermediate, which can be modified by multiple enzymatic and non-enzymatic reactions, is a common intermediate bridging the pathways encoded by these BGCs. Evaluation of the antibiotic activity of the resulting phenazine derivatives suggests a highly effective strategy to convert Gram-positive specific phenazines into broad-spectrum antibiotics, which might help the bacteria-nematode complex to maintain its special environmental niche.

Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditis entomopathogenic nematodes.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1T, Q614T and PB68.1T, isolated from the digestive system of Heterorhabditis entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family Morganellacea, and to the genus Photorhabdus. Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1T is closely related to Photorhabdus akhursti, that Q614T is closely related to Photorhabdus heterorhabditis, and that PB68.1T is closely related to Photorhabdus australis. In silico genomic comparisons confirm these observations: BA1T and P. akhursti 15138T share 68.8 % digital DNA-DNA hybridization (dDDH), Q614T and P. heterorhabditis SF41T share 75.4 % dDDH, and PB68.1T and P. australis DSM 17609T share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described Photorhabdus species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1T as a new species within the genus Photorhabdus, Q614T as a new subspecies within P. heterorhabditis, and PB68.1T as a new subspecies within P. australis. Hence, the following names are proposed for these strains: Photorhabdus aegyptia sp. nov. with the type strain BA1T(=DSM 111180T=CCOS 1943T=LMG 31957T), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614T (=DSM 111144T=CCOS 1944T=LMG 31959T) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1T (=DSM 111145T=CCOS 1942T). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM17609T as the type strain (currently classified as P. australis).

Competition and Co-existence of Two Photorhabdus Symbionts with a Nematode Host.

Photorhabdus spp. (Enterobacteriales: Morganellaceae) occur exclusively as symbionts of Heterorhabditis nematodes for which they provide numerous services, including killing insects and providing nutrition and defence within the cadavers. Unusually, two species (Photorhabdus cinerea and Photorhabdus temperata) associate with a single population of Heterorhabditis downesi at a dune grassland site. Building on previous work, we investigated competition between these two Photorhabdus species both at the regional (between insects) and local (within insect) level by trait comparison and co-culture experiments. There was no difference between the species with respect to supporting nematode reproduction and protection of cadavers against invertebrate scavengers, but P. cinerea was superior to P. temperata in several traits: faster growth rate, greater antibacterial and antifungal activity and colonisation of a higher proportion of nematodes in co-culture. Moreover, where both bacterial symbionts colonised single nematode infective juveniles, P. cinerea tended to dominate in numbers. Differences between Photorhabdus species were detected in the suite of secondary metabolites produced: P. temperata produced several compounds not produced by P. cinerea including anthraquinone pigments. Bioluminescence emitted by P. temperata also tended to be brighter than that from P. cinerea. Bioluminescence and pigmentation may protect cadavers against scavengers that rely on sight. We conclude that while P. cinerea may show greater local level (within-cadaver) competitive success, co-existence of the two Photorhabdus species in the spatially heterogeneous environment of the dunes is favoured by differing specialisations in defence of the cadaver against differing locally important threats.

Antifungal activity of different Xenorhabdus and Photorhabdus species against various fungal phytopathogens and identification of the antifungal compounds from X. szentirmaii.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter-activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. KEY POINTS: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified.

Structure and biosynthesis of deoxy-polyamine in Xenorhabdus bovienii.

Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.

Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Correction: Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

[This corrects the article DOI: 10.1371/journal.pgen.1007511.].

Synthetic Zippers as an Enabling Tool for Engineering of Non-Ribosomal Peptide Synthetases*.

Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge. Here we describe a cloning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L-1 . Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits.

Cooperation between a T†Domain and a Minimal C-Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS.

Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/C DDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of "designer" NRPS from proteins that normally do not interact. The multiprotein "peptide-antimicrobial-Xenorhabdus" (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small C DD of PaxA cooperates with its preceding thiolation (T1 ) domain to bind the N DD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.