Total Synthesis and Biological Assessment of Novel Albicidins Discovered by Mass Spectrometric Networking.

Natural products represent an important source of potential novel antimicrobial drug leads. Low production by microorganisms in cell culture often delays the structural elucidation or even prevents a timely discovery. Starting from the anti-Gram-negative antibacterial compound albicidin produced by Xanthomonas albilineans, we describe a bioactivity-guided approach combined with non-targeted tandem mass spectrometry and spectral (molecular) networking for the discovery of novel antimicrobial compounds. We report eight new natural albicidin derivatives, four of which bear a ß-methoxy cyanoalanine or ß-methoxy asparagine as the central α-amino acid. We present the total synthesis of these albicidins, which facilitated the unambiguous determination of the (2 S,3 R)-stereoconfiguration which is complemented by the assessment of the stereochemistry on antibacterial activity.

Deuterium-Labeled Precursor Feeding Reveals a New pABA-Containing Meroterpenoid from the Mango Pathogen Xanthomonas citri pv. mangiferaeindicae.

A new para-aminobenzoic-acid-containing natural product from the mango pathogenic organism Xanthomonas citri pv. mangiferaeindicae is described. By means of stable isotope precursor feeding combined with nontargeted LC-MS/MS, the generated spectra were clustered and visualized in a molecular network. This led to the identification of a new member of the meroterpenoids, termed xanthomonic acid, which is composed of an isoprenylated para-aminobenzoic acid. In vitro cytotoxicity assays demonstrated activity of xanthomonic acid against several human cancer cell lines by induction of autophagy.

The Albicidin Resistance Factor AlbD Is a Serine Endopeptidase That Hydrolyzes Unusual Oligoaromatic-Type Peptides.

The para-aminobenzoic acid-containing peptide albicidin is a pathogenicity factor synthesized by Xanthomonas albilineans in infections of sugar cane. Albicidin is a nanomolar inhibitor of the bacterial DNA gyrase with a strong activity against various Gram-negative bacteria. The bacterium Pantoea dispersa expresses the hydrolase AlbD, conferring natural resistance against albicidin. We show that AlbD is a novel type of endopeptidase that catalyzes the cleavage of albicidin at a peptide backbone amide bond, thus abolishing its antimicrobial activity. Additionally, we determined the minimal cleavage motif of AlbD with substrates derived by chemical synthesis. Our results clearly identify AlbD as a unique endopeptidase that is the first member of a new subfamily of peptidases. Our findings provide the molecular basis for a natural detoxification mechanism, potentially rendering a new tool in biological chemistry approaches.

Total synthesis of albicidin: a lead structure from Xanthomonas albilineans for potent antibacterial gyrase inhibitors.

The peptide antibiotic albicidin, which is synthesized by the plant pathogenic bacterium Xanthomonas albilineans, displays remarkable antibacterial activity against various Gram-positive and Gram-negative microorganisms. The low amounts of albicidin obtainable from the producing organism or through heterologous expression are limiting factors in providing sufficient material for bioactivity profiling and structure-activity studies. Therefore, we developed a convergent total synthesis route toward albicidin. The unexpectedly difficult formation of amide bonds between the aromatic amino acids was achieved through a triphosgene-mediated coupling strategy. The herein presented synthesis of albicidin confirms the previously determined chemical structure and underlines the extraordinary antibacterial activity of this compound. The synthetic protocol will provide multigram amounts of albicidin for further profiling of its drug properties.

Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

BACKGROUND: Fungal cyclodepsipeptides (CDPs) are non-ribosomally synthesized peptides produced by a variety of filamentous fungi and are of interest to the pharmaceutical industry due to their anticancer, antimicrobial and anthelmintic bioactivities. However, both chemical synthesis and isolation of CDPs from their natural producers are limited due to high costs and comparatively low yields. These challenges might be overcome by heterologous expression of the respective CDP-synthesizing genes in a suitable fungal host. The well-established industrial fungus Aspergillus niger was recently genetically reprogrammed to overproduce the cyclodepsipeptide enniatin B in g/L scale, suggesting that it can generally serve as a high production strain for natural products such as CDPs. In this study, we thus aimed to determine whether other CDPs such as beauvericin and bassianolide can be produced with high titres in A. niger, and whether the generated expression strains can be used to synthesize new-to-nature CDP derivatives. RESULTS: The beauvericin and bassianolide synthetases were expressed under control of the tuneable Tet-on promoter, and titres of about 350-600 mg/L for bassianolide and beauvericin were achieved when using optimized feeding conditions, respectively. These are the highest concentrations ever reported for both compounds, whether isolated from natural or heterologous expression systems. We also show that the newly established Tet-on based expression strains can be used to produce new-to-nature beauvericin derivatives by precursor directed biosynthesis, including the compounds 12-hydroxyvalerate-beauvericin and bromo-beauvericin. By feeding deuterated variants of one of the necessary precursors (d-hydroxyisovalerate), we were able to purify deuterated analogues of beauvericin and bassianolide from the respective A. niger expression strains. These deuterated compounds could potentially be used as internal standards in stable isotope dilution analyses to evaluate and quantify fungal spoilage of food and feed products. CONCLUSION: In this study, we show that the product portfolio of A. niger can be expanded from enniatin to other CDPs such as beauvericin and bassianolide, as well as derivatives thereof. This illustrates the capability of A. niger to produce a range of different peptide natural products in titres high enough to become industrially relevant.

Correction to: Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

[This corrects the article DOI: 10.1186/s40694-018-0048-3.].

Molecular insights into antibiotic resistance - how a binding protein traps albicidin.

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs.

Synthesis of Albicidin Derivatives: Assessing the Role of N-terminal Acylation on the Antibacterial Activity.

The peptide antibiotic albicidin, which is synthesized by the plant pathogenic bacterium, Xanthomonas albilineans, represents the most prominent member of a new class of antibacterial gyrase inhibitors. It shows remarkable antibacterial activities against Gram-positive and Gram-negative microorganisms. Its unique structure potentially represents a new lead structure for the development of an antibacterial drug. Here we report the synthesis of 14 albicidin derivatives with structural variations at the N-terminus, primarily investigating the effects of variation of cinnamoyl, phenylpropanoyl, and benzoyl residues. Gyrase inhibition in vitro and determination of minimal inhibitory concentrations were assessed in parallel. Activities in a nanomolar range and the importance of N-acylation were demonstrated.

Synthesis and Antimicrobial Activity of Albicidin Derivatives with Variations of the Central Cyanoalanine Building Block.

To investigate the pharmacophore regions of the antibiotic albicidin, derivatives with variations on the central amino acid were synthesized. Charged as well as uncharged residues were chosen to explore the influence of charge, chirality, and steric bulk. The bioactivity of the newly synthesized derivatives was determined by a microdilution technique to obtain minimum inhibitory concentrations (MIC) values. The compounds were also tested in a cell-free system to obtain information about their ability to inhibit their primary target, DNA gyrase. It was then shown that derivatives with uncharged side chains retain antibacterial activity, whereas incorporation of charged amino acid residues decreases the antibacterial activity dramatically, possibly due to restricted cell penetration of these derivatives. From the newly synthesized derivatives, the threonine derivative shows the most promising results in both tests. The information will help to develop the features of albicidin toward more drug-like structures.

The O-Carbamoyl-Transferase Alb15 Is Responsible for the Modification of Albicidin.

Albicidin is a potent antibiotic and phytotoxin produced by Xanthomonas albilineans which targets the plant and bacterial DNA gyrase. We now report on a new albicidin derivative which is carbamoylated at the N-terminal coumaric acid by the action of the ATP-dependent O-carbamoyltransferase Alb15, present in the albicidin (alb) gene cluster. Carbamoyl-albicidin was characterized by tandem mass spectrometry from cultures of a Xanthomonas overproducer strain and the gene function confirmed by gene inactivation of alb15 in X. albilineans. Expression of alb15 in Escherichia coli and in vitro reconstitution of the carbamoyltransferase activity confirmed albicidin as the substrate. The chemical synthesis of carbamoyl-albicidin finally enabled us to assess its bioactivity by means of in vitro gyrase inhibition and antibacterial assays. Compared to albicidin, carbamoyl-albicidin showed a significantly higher inhibitory efficiency against bacterial gyrase (∼8 vs 49 nM), which identifies the carbamoyl group as an important structural feature of albicidin maturation.