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The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature's repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody-drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide.

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.

d-Amino acids can have major effects on the structure, proteolytic stability, and bioactivity of peptides. Proteusin radical S -adenosyl methionine epimerases regioselectively install such residues in ribosomal peptides to generate peptides with the largest number of d-residues currently known in biomolecules. To study their utility in synthetic biology, we investigated the substrate tolerance and substrate-product relationships of the cyanobacterial model epimerase OspD using libraries of point mutants as well as distinct extended peptides that were fused to an N-terminal leader sequence. OspD was found to exhibit exceptional substrate promiscuity in E. coli , accepting 15 different amino acids and converting peptides with a broad range of compositions, secondary structures, and polarities. Diverse single and multiple epimerization patterns were identified that were dictated by the peptide sequence. The data suggest major potential in creating genetically encoded products previously inaccessible by synthetic biology.

The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera Xenorhabdus or Photorhabdus . In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected Xenorhabdus and Photorhabdus mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine ( fcl ) biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS 2 experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class., (Copyright © 2020, Wenski et al.; licensee Beilstein-Institut.)

Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacteria from the Bacteroidetes phylum are known producers of the chemotaxonomic relevant flexirubins. These orange pigments comprise a non-isoprenoid aryl-polyene carboxylic acid esterified with a dialkylresorcinol. Herein, we report a gene cluster from Chitinophaga pinensis encoding the biosynthesis of the polyene moiety and the biochemical characterization of a tyrosine ammonia-lyase and a 4-coumarate-CoA ligase responsible for the initiation of the polyene biosynthesis. Additionally, the flexirubin of C. pinensis was characterized by a combination of feeding experiments, high-performance liquid chromatography tandem mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry., (© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The spore-forming bacterium Paenibacillus larvae causes a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced by P. larvae are as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee, Apis mellifera carnica, with different P. larvae isolates. Honeybee larvae were reared in vitro in 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates of P. larvae, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts of P. larvae cultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate that P. larvae secretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) of P. larvae. Genome mining of P. larvae subsp. larvae BRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential of P. larvae to produce such compounds.

A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in Azoarcus sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized.

The structure of the fabclavines-unique mixtures of nonribosomally derived peptide-polyketide hybrids connected to an unusual polyamino moiety-has been solved by detailed NMR and MS methods. These compounds have been identified in two different entomopathogenic Xenorhabdus strains, thereby leading also to the identification of the fabclavine biosynthesis gene cluster. Detailed analysis of these clusters and initial mutagenesis experiments allowed the prediction of a biosynthesis pathway in which the polyamino moiety is derived from an unusual type of fatty acid synthase that is normally involved in formation of polyunsaturated fatty acids. As fabclavines show broad-spectrum activity against bacteria, fungi, and other eukaryotic cells, they might act as "protection factors" against all kinds of food competitors during the complex life cycle of Xenorhabdus, its nematode host, and their insect prey., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although sharing a certain degree of structural uniformity, natural product classes exhibit variable functionalities such as different amino acid or acyl residues. During collision induced dissociation, some natural products exhibit a conserved fragmentation pattern close to the precursor ion. The observed fragments result from a shared set of neutral losses, creating a unique fragmentation pattern, which can be used as a fingerprint for members of these natural product classes. The culture supernatants of 69 strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus were analyzed by MALDI-MS(2), and a database comprising MS(2) data from each strain was established. This database was scanned for concordant fragmentation patterns of different compounds using a customized software, focusing on relative mass differences of the fragment ions to their precursor ion. A novel group of related natural products comprising 25 different arginine-rich peptides from 16 different strains was identified due to its characteristic neutral loss fragmentation pattern, and the structures of eight compounds were elucidated. Two biosynthesis gene clusters encoding nonribosomal peptide synthetases were identified, emphasizing the possibility to identify a group of structurally and biosynthetically related natural products based on their neutral loss fragmentation pattern.

Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thirteen novel PAX (peptide-antimicrobial-Xenorhabdus) peptides were identified in Xenorhabdus nematophila HGB081. Their structures including the absolute configuration were elucidated using a combination of labeling experiments, detailed MS/MS experiments, the advanced Marfey's method, and a detailed analysis of the biosynthesis gene cluster, which was identified as well.

Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.