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Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme., A breed apart: The non‐ribosomal peptide synthetase (NRPS) XfpS, which produces the peptides xefoampeptides A and B, can be artificially split without loss of biosynthetic activity by introducing docking domains into XfpS. In fact, in this case, the production of both peptides from the split system increased compared to product levels from the full‐length XfpS. Splitting XfpS into three subunits also led to a functional NRPS system.

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by docking domains (DDs) to synthesize well-defined peptide products. In contrast to these classical NRPSs, the subunits of rhabdopeptide/xenortide producing NRPSs can act iteratively and in different order resulting in libraries of peptide products. In order to define the structural and thermodynamic basis for their unusual interaction patterns, we determined the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterized all putative DD interactions thermodynamically for one such system. Key amino acid residues for DD interactions were identified that upon their exchange not only changed the DD affinity but also resulted in rationally predictable changes in peptide production. A simple set of ‘recognition rules’ for DD interactions was identified that also operates in other megasynthase complexes.

M.H.M. was supported by a Rubicon fellowship of the Netherlands Organization for Scientific Research (NWO;Rubicon 825.13.001). The work of R.K. was supported by the European Union’s Seventh Framework Programme(Joint Call OCEAN.2011–2: Marine microbial diversity—new insights into marine ecosystems functioning and its biotechnological potential) under the grant agreement no.287589 (Micro B3). M.C. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC)studentship (BB/J014478/1). The GSC is supported by funding from the Natural Environment Research Council(UK), the National Institute for Energy Ethics and Society(NIEeS; UK), the Gordon and Betty Moore Foundation,the National Science Foundation (NSF; US) and the US Department of Energy. The Manchester Synthetic Biology Research Centre, SYNBIOCHEM, is supported by BBSRC/Engineering and Physical Sciences Research Council(EPSRC) grant BB/M017702/1, Medema, M.H., Kottmann, R., Yilmaz, P., Cummings, M., Biggins, J.B., Blin, K., De Bruijn, I., Chooi, Y.H., Claesen, J., Coates, R.C., Cruz-Morales, P., Duddela, S., Düsterhus, S., Edwards, D.J., Fewer, D.P., Garg, N., Geiger, C., Gomez-Escribano, J.P., Greule, A., Hadjithomas, M., Haines, A.S., Helfrich, E.J.N., Hillwig, M.L., Ishida, K., Jones, A.C., Jones, C.S., Jungmann, K., Kegler, C., Kim, H.U., Kötter, P., Krug, D., Masschelein, J., Melnik, A.V., Mantovani, S.M., Monroe, E.A., Moore, M., Moss, N., Nützmann, H.-W., Pan, G., Pati, A., Petras, D., Reen, F.J., Rosconi, F., Rui, Z., Tian, Z., Tobias, N.J., Tsunematsu, Y., Wiemann, P., Wyckoff, E., Yan, X., Yim, G., Yu, F., Xie, Y., Aigle, B., Apel, A.K., Balibar, C.J., Balskus, E.P., Barona-Gómez, F., Bechthold, A., Bode, H.B., Borriss, R., Brady, S.F., Brakhage, A.A., Caffrey, P., Cheng, Y.Q., Clardy, J., Cox, R.J., De Mot, R., Donadio, S., Donia, M.S., Van Der Donk, W.A., Dorrestein, P.C., Doyle, S., Driessen, A.J.M., Ehling-Schulz, M., Entian, K.-D., Fischbach, M.A., Gerwick, L., Gerwick, W.H., Gross, H., Gust, B., Hertweck, C., Höfte, M., Jensen, S.E., Ju, J., Katz, L., Kaysser, L., Klassen, J.L., Keller, N.P., Kormanec, J., Kuipers, O.P., Kuzuyama, T., Kyrpides, N.C., Kwon, H.-J., Lautru, S., Lavigne, R., Lee, C.Y., Linquan, B., Liu, X., Liu, W., Luzhetskyy, A., Mahmud, T., Mast, Y., Méndez, C., Metsä-Ketelä, M., Micklefield, J., Mitchell, D.A., Moore, B.S., Moreira, L.M., Müller, R., Neilan, B.A., Nett, M., Nielsen, J., O'Gara, F., Oikawa, H., Osbourn, A., Osburne, M.S., Ostash, B., Payne, S.M., Pernodet, J.-L., Petricek, M., Piel, J., Ploux, O., Raaijmakers, J.M., Salas, J.A., Schmitt, E.K., Scott, B., Seipke, R.F., Shen, B., Sherman, D.H., Sivonen, K., Smanski, M.J., Sosio, M., Stegmann, E., Süssmuth, R.D., Tahlan, K., Thomas, C.M., Tang, Y., Truman, A.W., Viaud, M., Walton, J.D., Walsh, C.T., Weber, T., Van Wezel, G.P., Wilkinson, B., Willey, J.M., Wohlleben, W., Wright, G.D., Ziemert, N., Zhang, C., Zotchev, S.B., Breitling, R., Takano, E., Glöckner, F.O.

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.

The production of the blue pigment indigoidine has been achieved in the entomopathogenic bacterium Photorhabdus luminescens by a promoter exchange and in Escherichia coli following heterologous expression of the biosynthesis gene indC. Moreover, genes involved in the regulation of this previously "silent" biosynthesis gene cluster have been identified in P. luminescens.

Exchange of the native promoter to the arabinose-inducible promoter PBAD was established in entomopathogenic bacteria to silence and/or activate gene clusters involved in natural product biosynthesis. This allowed the "on-demand" production of GameXPeptides, xenoamicins, and the blue pigment indigoidine. The gene clusters for the novel "mevalagmapeptides" and the highly toxic xenorhabdins were identified by this approach.

In the attempt to establish a reliable real-time PCR protocol for transcriptional analysis of secondary metabolism in Sorangium cellulosum strain So ce56, a RNA extraction method and a reverse transcription protocol was developed. In order to validate chivosazol or etnangien gene cluster transcripts as good candidates to develop the real-time PCR protocol, stability measurements of the transcripts were performed proving both transcripts to be very stable. The chivosazol biosynthetic gene cluster was taken as the test case to evaluate the special problems arising from the large size of the transcripts and the high G/C-content of the encoding DNA. A set of primer pairs targeting the presumed 90 kbp chivosazol transcript at different positions was employed. The production rate of chivosazol was compared to the transcription of the operon in time course experiments revealing that during the logarithmic growth phase transcription is maximally induced and levels out during the stationary phase. Some deviations in transcript numbers could be measured depending on the primer pair used, but cross-evaluation strengthened the notion that the measured numbers reflect the whole transcript quantities and the in vivo level. Finally, a putative promoter located between chiA and chiB was examined by using the developed real-time PCR protocol.

Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA). Our earlier work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein) is primarily comprised of bZIP protein TGA2.2 and of minor amounts of a protein that cross-reacts with an antibody directed against related bZIP factor TGA2.1. As this protein was significantly smaller than recombinant TGA2.1, the origin of this protein had remained unresolved. Here we demonstrate that it corresponds to a distinct cleavage product of TGA2.1 generated during extract preparation. Overexpression of TGA2.1 led to increased levels of the TGA2.1/TGA2.2 heterodimer which was as effective with regard to enhancing the SA-inducibility of as-1 containing target gene Nt103 as corresponding amounts of the TGA2.2 homodimer. Thus, the TGA2.1 specific N-terminal domain, which had revealed transcriptional activation potential in yeast, did not show enhanced transcriptional activation in planta. TGA2.1 even had a negative effect on the SA-induced expression of the truncated CaMV 35S (-90) promoter that contains an isolated as-1-element upstream of the TATA-box. Plants expressing a TGA mutant deficient in DNA binding (TGA2.1trd) showed reduced levels of SA-inducible Nt103 expression, thus resembling plants expressing the analogous TGA2.2 derivative TGA2.2trd. In contrast to TGA2.2trd, TGA2.1trd did not reduce auxin-induced expression of Nt103 and SA-induced expression of pathogenesis related protein PR-1a, indicating that TGA2.1trd and TGA2.2trd differ in their capacity to outcompete regulatory factors involved in these regulatory pathways.

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect-specific production of the new GameXPeptides E-H (5-8) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions.

An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide "xenotetrapeptide" from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D-amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2 O.

Gibberellin (GA) 20-oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C-20 methyl group of a diterpene carboxylic acid precursor (e.g. GA 12 ) to form an alcohol (in our case GA 15 -open lactone) and an aldehyde (GA 24 ). The aldehyde is either oxidised to a tricarboxylic acid (GA 25 ) or, with loss of carbon-20 and lactonisation, to C 19 -GA (GA 9 ). This branching is interesting to study, because C 19 -GA derivatives function as plant hormones in different tissues, whereas the C 20 -GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20-oxidase from immature seeds of Cucurbita maxima L., which produces mainly C-20 carboxylic acids, with a 20-oxidase from Marah macrocarpus immature seeds, which forms predominantly C 19 -GAS. The cDNAs encoding these two very similar 20-oxidases were digested with restriction endonucleases Van91I, BclI, and BsaWI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1-construct was obtained by exchanging nt 303-809 of the Cucurbita cDNA with the homologous DNA from the Marah 20-oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810-992, nt 993-end, nt 303-992, nt 810-end, and nt 311-end were exchanged, respectively. All constructs were cloned in a pUC 18 vector and functionally expressed in E. coli NM522 cells. GA 20-oxidase activity was detectable in cell-lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA 12 was found with pCMl and pCM3, one-tenth of this conversion was observed with pCM5 and pCM6, and one-hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C 19 -end product, GA 9 , nor the C 20 -end product, GA 25 , was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6, GA 9 accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C 19 -GAs in this order. Although GA24 is the immediate precursor, much less end products were formed by using this substrate.

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k(cat) = 153 s(-1), 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.

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.

Isovaleryl-CoA (IV-CoA) is usually derived from the degradation of leucine by using the Bkd (branched-chain keto acid dehydrogenase) complex. We have previously identified an alternative pathway for IV-CoA formation in myxobacteria that branches from the well-known mevalonate-dependent isoprenoid biosynthesis pathway. We identified 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus, which is induced in mutants with impaired leucine degradation (e.g., bkd(-)) or during myxobacterial fruiting-body formation. Here, we show that the proteins required for leucine degradation are also involved in the alternative IV-CoA biosynthesis pathway through the efficient catalysis of the reverse reactions. Moreover, we conducted a global gene-expression experiment and compared vegetative wild-type cells with bkd mutants, and identified a five-gene operon that is highly up-regulated in bkd mutants and contains mvaS and other genes that are directly involved in the alternative pathway. Based on our experiments, we assigned roles to the genes required for the formation of IV-CoA from HMG-CoA. Additionally, several genes involved in outer-membrane biosynthesis and a plethora of genes encoding regulatory proteins were decreased in expression levels in the bkd(-) mutant; this explains the complex phenotype of bkd mutants including a lack of adhesion in developmental submerse culture.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation. Originally found in promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack. Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid response protein (SARP) bind specifically to as-1. Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP. Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable. Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2, 4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents. This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations. However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment.

SummaryMotile predatory Myxobacteria are producers of multiple secondary metabolites and, on starvation, undergo concerted cellular differentiation to form multicellular fruiting bodies. These abilities demand myxobacterial genomes to encode sophisticated regulatory networks that are not satisfactorily understood. Here, we present two bacterial enhancer binding proteins (bEBPs) encoded in Myxococcus xanthus acting as direct regulators of secondary metabolites intriguingly exhibiting activating and inhibitory effects. Elucidation of a regulon for each bEBP enabled us to unravel their role in myxococcal development, predation, and motility. Interestingly, both bEBPs are able to interact by forming a hetero-oligomeric complex. Our findings represent an alternative mode of operation of bEBPs, which are currently thought to enhance promoter activity by acting as homo-oligomers. Furthermore, a direct link between secondary metabolite gene expression and predation, motility, and cellular development could be shown for the first time.