Studien zur Biosynthese des Hormaomycins

In der vorliegenden Arbeit werden Experimente zur Aufklärung der Biosynthese, Untersuchungen zum evolutionären Ursprung der Adenylierungsdomänen und Studien zur Flexibilität der Biosyntheseenzyme des bakteriellen Peptids Hormaomycin präsentiert. Hormaomycin wird von Streptomyces griseoflavus W-384 synthetisiert und besitzt mehrere interessante biologische Aktivitäten. Einzigartig ist die Struktur des Hormaomycins; es ist ein zyklisches Peptidlacton mit acht Bausteinen, von denen sieben nicht-proteinogenen Ursprungs sind. Einige dieser Bausteine, wie (3-Nitrocyclopropyl)alanin [(3-Ncp)Ala], 4-(Z-Propenyl)prolin [(4-Pe)Pro] und 5-Chlor-N-hydroxy-pyrrol-2-carbonsäure [Chpca] sind in der Natur bisher einzigartig. Vorläufer-dirigierte Biosynthese-experimente haben gezeigt, dass die Biosynthesemaschinerie von Hormaomycin ungewöhnlich flexibel ist. In der Arbeitsgruppe Piel konnte in Vorarbeiten der Biosynthesegencluster des Hormaomycins isoliert und sequenziert werden. Die Analyse der Sequenz zeigte, dass Hormaomycin von einer nichtribosomalen Peptidsynthetase (NRPS) synthetisiert wird. Im ersten Teil der vorliegenden Arbeit wurden die Biosynthesen der Vorstufen durch heterologe Expressionen von Biosynthesegenen teilweise aufgeklärt. Durch bioinformatische Analysen wurden zwei Gene mit unbekannter Funktion, hrmI und hrmJ, dem postulierten Biosyntheseweg von (3-Ncp)Ala zugeordnet. Beide Enzyme haben keine eng verwandten Homologe. Eine Fütterung von (3-Ncp)Ala zu einer Knock-Out-Mutante von hrmI führte zu einer Komplementierung der Hormaomycinproduktion, was die Zugehörigkeit des Enzyms zu diesem Biosyntheseweg beweist. HrmJ, eine α-Ketoglutarat-abhängige Oxygenase, katalysiert vermutlich den ersten Schritt des Biosyntheseweges. Das Enzym konnte in hohen Ausbeuten in E. coli exprimiert und isoliert werden und bildete in Enzymassays aus Lysin ein neues Produkt. Massenspektrometrische Messungen geben erste Hinweise darauf, dass es sich hierbei um ein hydroxyliertes Lysin mit terminaler Oximfunktion handelt. HrmI ist putativ eine Oxidase und könnte zu einer neuen Enzymfamilie gehören. (4-Pe)Pro wird, analog zur Biosynthese des Lincomycins, über Tyrosin und L-Dihydroxyphenylalanin (L-DOPA) synthetisiert. HrmF, eine L-DOPA-Dioxygenase, katalysiert die Öffnung des Catecholrings zu einem instabilen, gelb gefärbten Pyrrolinintermediat. HrmF wurde in dieser Arbeit kinetisch charakterisiert. Die Ergebnisse zeigen, dass das Enzym eine fast 160-fach höhere katalytische Aktivität als sein Homolog aus der Lincomycinbiosynthese, LmbB1, besitzt. Das Enzym ist aufgrund seiner hohen Aktivität, der sehr losen Substratspezifität und hohen Expressionsausbeuten ein guter Kandidat für chemoenzymatische Syntheseverfahren. Die Startereinheit der Hormaomycinbiosynthese, Chpca, wird aufgrund von bioinformatischen Voraussagen aus Prolin, kovalent gebunden an HrmL, ein freistehendes Peptidylcarrierprotein (PCP), synthetisiert. Im Rahmen dieser Arbeit wurden die Acyl-CoA-Synthetase HrmK und HrmL heterolog exprimiert und charakterisiert. Es konnte gezeigt werden, dass Prolin vom Enzym sehr selektiv adenyliert wird. Für HrmL wurde massenspektrometrisch die 4´-Phosphopanthetheinylierung nachgewiesen. Der zweite Teil der Arbeit beschäftigt sich mit der Charakterisierung der Adenylierungsdomänen (A-Domänen) der Hormaomycin-NRPS. Alle sieben A-Domänen wurden mit dem MbtH-artigen Protein HrmR coexprimiert und isoliert. Für den Komplex wurde ein stöchiometrisches Verhältnis von 1:1 bestimmt. Die Proteine wurden in einem massenspektrometrisch basierten Assay umfassend charakterisiert und zur Eliminierung von falsch positiven Resultaten mit FPLC weiter aufgereinigt. Für HrmO3A und HrmP1A wurde eine selektive Aktivierung von (β-Me)Phe nachgewiesen. HrmO1A und HrmO4A aktivieren (3-Ncp)Ala fast quantitativ und Leucin am zweithäufigsten [jeweils etwa 10% im Vergleich zu (3-Ncp)Ala]. HrmO2A adenyliert L-Threonin sehr selektiv, und HrmP3A zeigt eine fast quantitative Umsetzung für (4-Pe)Pro. HrmP3A kann zusätzlich ein Prolinderivat mit einer Ethinylkette aktivieren. Dieses Ergebnis ist sehr vielversprechend im Hinblick auf die Mutasynthese eines Hormaomycinderivates mit Alkinylseitenkette. Dieses Derivat könnte in Pull-down-Experimenten zur Auffindung des molekularen Targets für Hormaomycin eingesetzt werden. HrmP2A aktiviert in vitro bevorzugt Valin, was der bioinformatischen Voraussage entspricht. Allerdings wird in vivo Isoleucin von der NRPS inkorporiert. Die Gründe für diese Diskrepanz sind nicht bekannt. Die Sequenzen der Hormaomycin A-Domänen zeigen in einigen Fällen eine weitgehende Übereinstimmung am N- und C-Terminus (jeweils etwa 200 Aminosäuren). Das legt einen möglichen Austausch der zentralen DNA-Abschnitte, und damit der Substratspezifität, durch Rekombination während der Evolution nahe. Um diese Hypothese experimentell zu überprüfen, wurden fünf rekombinante A-Domänen konstruiert, die alle die N-und C-terminale Sequenz von HrmO3A trugen. Die zentralen Abschnitte wurden einerseits drei A-Domänen aus der Hormaomycin-NRPS andererseits zwei A-Domänen der NRPS des calcium dependent antibiotic (CDA) aus Streptomyces coelicolor A3(2), entnommen. Alle rekombinanten Enzyme wurden stabil in E. coli mit HrmR coexprimiert. Die Hormaomycinfusionen waren aktiv und zeigten in etwa die gleiche Substratspezifität und Aktivität wie die nativen, die zentralen Abschnitte enthaltenden, A-Domänen. Die Ergebnisse dieser Experimente geben Einblicke in die Evolution der Hormaomycin-A-Domänen und zeigen neue Strategien für die kombinatorische Biosynthese von nichtribosomalen Peptiden auf. Im dritten Teil wurden die ersten bekannten, natürlich gebildeten Analoga des Hormaomycins aus einem Extrakt eines Hormaomycin-Überproduktionsstammes isoliert und deren Strukturen anschließend aufgeklärt. Hormaomycin A1 ist ein Deschlorohormaomycin, wogegen in Hormaomycin A2 Valin statt Isoleucin in das Peptid eingebaut wurde. Hormaomycin A3 und A4 tragen jeweils ein Leucin, Hormaomycin A5 zwei Leucine statt (3-Ncp)Ala im Molekül. Der Einbau der proteinogenen Aminosäuren in Hormaomycin A2-5 korrespondiert sehr gut mit den Ergebnissen der A-Domänentests und demonstriert die Anwendbarkeit dieser Tests für zukünftige Mutasyntheseexperimente. Die Aktivitäten der isolierten Analoga werden weitere Einblicke in die Struktur-Wirkungsbeziehungen von Hormaomycin geben.Studies on the biosynthesis of hormaomycin Presented here are the results carried out on the elucidation of the biosynthesis, evolutionary investigations on the adenylation domains and studies on the flexibility of the biosynthetic machinery of the bacterial peptide hormaomycin. Hormaomycin, produced by Streptomyces griseoflavus W-384, bears several interesting biological activities. In addition, hormaomycin has a unique and interesting structure; it is a cyclic peptide lactone with eight building blocks, seven of which are non-proteinogenic. Some of these building blocks are unprecedented, such as (3-nitrocyclopropyl)alanine [(3-Ncp)Ala], 4-(Z-propenyl)proline [(4-Pe)Pro] and 5-chloro-N-hydroxypyrrole-2-carboxylic acid [Chpca]. Previous precursor-directed biosynthetic experiments showed an unusually flexible biosynthetic machinery. In the Piel group, the putative biosynthetic gene cluster of hormaomycin was isolated and sequenced. Analysis of the sequence revealed that hormaomycin is assembled by a nonribosomal peptide synthetase (NRPS). In the first part of this work, the biosyntheses of the unique building blocks were partially elucidated with heterologous expressions of various biosynthetic genes in E. coli. Bioinformatic analyses could assign two gene candidates with unknown function, hrmI and hrmJ, to the (3-Ncp)Ala biosynthetic pathway. For both enzymes no close homologs are known. Feeding of (3-Ncp)Ala to a knock-out mutant of hrmI could complement hormaomycin production, proving that HrmI belongs to this precursor pathway. HrmJ, a α-ketoglutarate-dependent oxygenase, is hypothezised to catalyze the first step of the (3-Ncp)Ala pathway. The enzyme was heterologously expressed in E. coli and purified in high yields. Enzymatic assays with HrmJ and lysine produced a new product that was detected by mass spectrometry. The results provide evidence for a hydroxylated lysine with a terminal oxime moiety. HrmI is a putative oxidase that could belong to a new enzyme family. (4-Pe)Pro was shown to be synthesized from tyrosine and dihydroxypenylalanine (L-DOPA), analogous to the lincomycin pathway. HrmF, a L-DOPA dioxygenase, catalyzes ring opening of the catechol leading to formation of a unstable, yellow pyrroline intermediate. In this work, HrmF was kinetically characterized and shown that the enzyme has catalytic efficiency of around 160 times higher than its homolog from the lincomycin pathway, LmbB1. The high activity, very relaxed substrate specificity and high protein yields make HrmF a good candidate for chemoenzymatic synthetic utility. The starter unit of hormaomycin biosynthesis, Chpca, was proposed to be synthezised from proline covalently bound to HrmL, a peptidyl carrier protein (PCP) through bioinformatic analyses. In this work, HrmK and HrmL were heterologously expressed and purified. The Acyl-CoA synthetase HrmK was characterized in a mass spectrometry based assay. Proline was shown to be activated very selectively and quantitatively in the assay. Analogs of proline could be activated only in trace amounts. For HrmL, 4´-phosphopanthetheinylation was observed by mass spectrometry. The second part of this work deals with the characterization of the adenylation domains (A domains) of the hormaomycin NRPS. All seven A domains could be heterologously expressed and isolated. It was shown that all A domains need HrmR, a MbtH-like protein, for their catalytic activities. Hence, HrmR was coexpressed with the adenylation domains. For the protein complex, a stochiometric ratio of 1:1 was determined. The proteins were comprehehensively characterized in a mass spectrometry-based assay and further purified by FPLC to remove false positive activities. For HrmO3A and HrmP1A, (β-Me)Phe was shown to be the native substrates. HrmO1A and HrmO4A activate (3-Ncp)Ala to a great extent; the second preferred substrate was shown to be leucine [around 10% compared to (3-Ncp)Ala]. HrmO2A adenylates threonine very selectively and HrmP3A shows a high turnover for (4-Pe)Pro. HrmP3A can additionally activate a proline derivative with an ethinyl side chain. This result is very promising in terms of mutasynthetic experiments, to yield a hormaomycin derivative with an alkinyl side chain. This hormaomycin could then be used in pull-down experiments for the identification of the molecular target of hormaomycin. HrmP2A was shown to activate valine in vitro, which fits to the bioinformatic prediction. However, in vivo, isoleucine is incorporated into hormaomycin. This discrepancy has yet to be elucidated. Multiple hormaomycin A domains exhibit high sequence identity at their N- and C-termini (around 200 amino acids in each case). This suggested an exchange of the central DNA stretches of the domains might have occurred during evolution, and thus an exchange of substrate specificity via recombinatoric events. To test this hypothesis experimentally, five recombinant A domains were constructed, all of them having the same sequence of HrmO3A at their N- and C-termini. Three central DNA stretches originated from hormaomycin A domains on the one hand while the other two were taken from adenylation domains of the NRPS of the “calcium-dependent antibiotic” (CDA) from Streptomyces coelicolor A3(2). All five recombinant enzymes were coexpressed with HrmR in E. coli. The hormaomycin fusions were active and showed virtually the same substrate specificity and enzymatic turnover as the native central-domain containing enzymes. The results of this study give new insights into the evolution of the hormaomycin adenylation domains and point to a combinatorial exchange of substrate specificities. This study could lead to new, evolution-based strategies for nonribosomal peptide combinatorial biosynthesis. In the third part, the first known natural hormaomycin analogs were isolated from an extract of a hormaomycin overproducing strain and their structures were subsequently elucidated. Hormaomycin A1 is a dechlorohormaomycin, while in hormaomycin A2, valine was shown to be incorporated instead of isoleucine. Hormaomycin A3 and A4 have one leucine each and Hormaomycin A5 harbors two leucines instead of (3-Ncp)Ala. The incorporation of the proteinogenic amino acids into hormaomycin A2-5 corresponded well with the the results of the A domain assays and demonstrates the applicability of these tests for mutasynthetic experiments in the future. Furthermore, the bioactivities of the isolated analogs will give further insights into the structure-activity relationships of hormaomycin

Function-related replacement of bacterial siderophore pathways

© The Author(s) 2018. Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co-occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters

Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high-throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid– dependent and –independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other G subunits, we here set out to rationally design G16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gq/G11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold– based inhibitors, provided that these inhibitors mimic the interaction of FR with G proteins harboring engineered FR-binding sites

A community resource for paired genomic and metabolomic data mining

Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.Peer reviewe

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data

Marine-derived myxobacteria of the suborder Nannocystineae: An underexplored source of structurally intriguing and biologically active metabolites

Myxobacteria are famous for their ability to produce most intriguing secondary metabolites. Till recently, only terrestrial myxobacteria were in the focus of research. In this review, however, we discuss marine-derived myxobacteria, which are particularly interesting due to their relatively recent discovery and due to the fact that their very existence was called into question. The to-date-explored members of these halophilic or halotolerant myxobacteria are all grouped into the suborder Nannocystineae. Few of them were chemically investigated revealing around 11 structural types belonging to the polyketide, non-ribosomal peptide, hybrids thereof or terpenoid class of secondary metabolites. A most unusual structural type is represented by salimabromide from Enhygromyxa salina. In silico analyses were carried out on the available genome sequences of four bacterial members of the Nannocystineae, revealing the biosynthetic potential of these bacteria

Manipulation of Regulatory Genes Reveals Complexity and Fidelity in Hormaomycin Biosynthesis

Hormaomycin (HRM) is a structurally remarkable peptide produced by Streptomyces griseoflavus W-384 that acts as a Streptomyces signaling metabolite and exhibits potent antibiotic activity against coryneform actinomycetes. HRM biosynthetic studies have been hampered by inconsistent and low production. To enhance fermentation titers, the role of its cluster-encoded regulatory genes was investigated. Extra copies of the putative regulators hrmA and hrmB were introduced into the wild-type strain, resulting in an increase of HRM production and its analogs up to 135-fold. For the HrmB overproducer, six bioactive analogs were isolated and characterized. This study demonstrates that HrmA and HrmB are positive regulators in HRM biosynthesis. A third gene, hrmH, was identified as encoding a protein capable of shifting the metabolic profile of HRM and its derivatives. Its manipulation resulted in the generation of an additional HRM analog

Cyclopropane-Containing Fatty Acids from the Marine Bacterium Labrenzia sp. 011 with Antimicrobial and GPR84 Activity

Bacteria of the family Rhodobacteraceae are widespread in marine environments and known to colonize surfaces, such as those of e.g., oysters and shells. The marine bacterium Labrenzia sp. 011 is here investigated and it was found to produce two cyclopropane-containing medium-chain fatty acids (1, 2), which inhibit the growth of a range of bacteria and fungi, most effectively that of a causative agent of Roseovarius oyster disease (ROD), Pseudoroseovarius crassostreae DSM 16950. Additionally, compound 2 acts as a potent partial, β-arrestin-biased agonist at the medium-chain fatty acid-activated orphan G-protein coupled receptor GPR84, which is highly expressed on immune cells. The genome of Labrenzia sp. 011 was sequenced and bioinformatically compared with those of other Labrenzia spp. This analysis revealed several cyclopropane fatty acid synthases (CFAS) conserved in all Labrenzia strains analyzed and a putative gene cluster encoding for two distinct CFASs is proposed as the biosynthetic origin of 1 and 2