Daniel Krug

antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters

Abstract Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

Complete genome sequence of the myxobacterium Sorangium cellulosum.

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 strains 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.

Myxovirescin A biosynthesis is directed by hybrid polyketide synthases/nonribosomal peptide synthetase, 3-hydroxy-3-methylglutaryl-CoA synthases, and trans-acting acyltransferases.

Myxococcus xanthus DK1622 is shown to be a producer of myxovirescin (antibiotic TA) antibiotics. The myxovirescin biosynthetic gene cluster spans at least 21 open reading frames (ORFs) and covers a chromosomal region of approximately 83 kb. In silico analysis of myxovirescin ORFs in conjunction with genetic studies suggests the involvement of four type I polyketide synthases (PKSs; TaI, TaL, TaO, and TaP), one major hybrid PKS/NRPS (Ta‐1), and a number of monofunctional enzymes similar to the ones involved in type II fatty‐acid biosyntesis (FAB). Whereas deletion of either taI or taL causes a dramatic drop in myxovirescin production, deletion of both genes (ΔtaIL) leads to the complete loss of myxovirescin production. These results suggest that both TaI and TaL PKSs might act in conjunction with a methyltransferase, reductases, and a monooxygenase to produce the 2‐hydroxyvaleryl–S–ACP starter that is proposed to act as the biosynthetic primer in the initial condensation reaction with glycine. Polymerization of the remaining 11 acetates required for lactone formation is directed by 12 modules of Ta‐1, TaO, and TaP megasynthetases. All modules, except for the first module of TaL, lack cognate acyltransferase (AT) domains. Furthermore, deletion of a discrete tandem AT—encoded by taV—blocks myxovirescin production; this suggests an “in trans” mode of action. To embellish the macrocycle with methyl and ethyl moieties, assembly of the myxovirescin scaffold is proposed to switch twice from PKS to 3‐hydroxy‐3‐methylglutaryl–CoA (HMG–CoA)‐like biochemistry during biosynthesis. Disruption of the S‐adenosylmethionine (SAM)‐dependent methyltransferase, TaQ, shifts production toward two novel myxovirescin analogues, designated myxovirescin Qa and myxovirescin Qc. NMR analysis of purified myxovirescin Qa revealed the loss of the methoxy carbon atom. This novel analogue lacks bioactivity against E. coli.

Discovering the Hidden Secondary Metabolome of Myxococcus xanthus: a Study of Intraspecific Diversity

ABSTRACT As a monophyletic group, the myxobacteria are known to produce a broad spectrum of secondary metabolites. However, the degree of metabolic diversity that can be found within a single species remains unexplored. The model species Myxococcus xanthus produces several metabolites also present in other myxobacterial species, but only one compound unique to M. xanthus has been found to date. Here, we compare the metabolite profiles of 98 M. xanthus strains that originate from 78 locations worldwide and include 20 centimeter-scale isolates from one location. This screen reveals a strikingly high level of intraspecific diversity in the M. xanthus secondary metabolome. The identification of 37 nonubiquitous candidate compounds greatly exceeds the small number of secondary metabolites previously known to derive from this species. These results suggest that M. xanthus may be a promising source of future natural products and that thorough intraspecific screens of other species could reveal many new compounds of interest.

Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix espanaensis

The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-p-coumaric acid is first activated before the 3-hydroxylation of its ring takes place.

Reconstitution of the Myxothiazol Biosynthetic Gene Cluster by Red/ET Recombination and Heterologous Expression in Myxococcus xanthus

ABSTRACT Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the myxothiazol biosynthetic genes in M. xanthus results in the production of myxothiazol in yields comparable to the natural producer strain.

Myxoprincomide: A Natural Product from Myxococcus xanthus Discovered by Comprehensive Analysis of the Secondary Metabolome

Bacteria are important sources of therapeutically relevant natural products. Early established bacterial producers include the streptomycetes, pseudomonads, and bacilli, but the list has recently expanded to include further sources, such as the myxobacteria. Access to whole genome sequences has indicated that among bacteria producing natural products through the action of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) the number of known compounds is remarkably lower than the genetic capacity of the organism for secondary-metabolite biosynthesis. 6] Thus, identification of new compound classes and their assignment to biosynthetic gene clusters is a crucial step in the discovery of novel natural products. Here, we report the discovery and structural elucidation of myxoprincomide (1), a novel NRPS/PKS natural product from Myxococcus xanthus DK1622, by combining methods of targeted mutagenesis, liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), and statistical data evaluation (Figure 1). Myxoprincomide (1), a linear peptide bearing some unusual residues, is produced by the NRPS/PKS biosynthetic machinery encoded by the mxp (MXAN_3779) gene locus. Moreover, by correlating additional very-low-abundance natural products to biosynthetic pathways in DK1622 we demonstrate how a comprehensive “metabolome-mining” approach can complement genomemining strategies in the discovery of secondary metabolites. The myxobacterial strain DK1622, considered a model organism for the study of myxobacterial social motility and multicellular differentiation, was not recognized as a producer of secondary metabolites until its genome was sequenced. However, interest in resolving its secondary metabolome has increased since bioinformatic analysis revealed it to contain 18 biosynthetic gene clusters encoding NRPS, PKS, and NRPS/PKS hybrid systems. To date only five compound classes derived from NRPS and PKS biosynthetic machineries have been characterized and correlated to their gene clusters in DK1622 (see Figure 1 in the Supporting Information). Intriguingly, evaluation at the transcriptomic and proteomic levels asserts that the remaining 13 unassigned pathways are active under standard conditions for the cultivation of DK1622. 15] We reasoned that the low abundance of the corresponding compounds may have previously prevented their detection, and thus set out to find these compounds by utilizing advanced analytical methods. The available DK1622 genome sequence facilitated the construction of a targeted mutant library including knockouts of every secondary-metabolite biosynthetic gene cluster (Table 1 in the Supporting Information). As the process of finding the possibly subtle differences between wild-type and mutant secondary-metabolite profiles by manual comparison of LC-MS data is frequently tedious, error-prone, and low in sensitivity, we sought to implement statistical tools in order to expedite our LC-MS data evaluation. In preparation for the comprehensive statistical analysis, mutant and wildtype strains were grown in small-scale fermentation in quadruplicate, replicate extracts were analyzed by LCHRMS, and data were pretreated by using a compoundfinding algorithm, resulting in the definition of > 1000 molecular features per sample. In order to identify molecular features specifically missing in culture extracts from DK1622 mutant strains, we applied principal-component analysis (PCA) to the preprocessed LC-MS datasets [*] M. Sc. N. S. Cortina, Dr. D. Krug, Dr. A. Plaza, Dipl.-Chem. O. Revermann, Prof. Dr. R. M ller Abteilung Mikrobielle Naturstoffe, Helmholtz-Institut f r Pharmazeutische Forschung Saarland (HIPS), Helmholtz-Zentrum f r Infektionsforschung and Institut f r Pharmazeutische Biotechnologie, Universit t des Saarlandes Campus, Geb ude C2.3, 66123 Saarbr cken (Deutschland) E-mail: rom@helmholtz-hzi.de Homepage: http://www.helmholtz-hzi.de/hips [] These authors contributed equally to this work.

Efficient mining of myxobacterial metabolite profiles enabled by liquid chromatography–electrospray ionisation-time-of-flight mass spectrometry and compound-based principal component analysis

Bacteria producing secondary metabolites are an important source of natural products with highly diverse structures and biological activities. Developing methods to efficiently mine procaryotic secondary metabolomes for the presence of potentially novel natural products is therefore of considerable interest. Modern mass spectrometry-coupled liquid chromatography can effectively capture microbial metabolic diversity with ever improving sensitivity and accuracy. In addition, computational and statistical tools increasingly enable the targeted analysis and exploration of information-rich LC-MS datasets. In this article, we describe the use of such techniques for the characterization of myxobacterial secondary metabolomes. Using accurate mass data from high-resolution ESI-TOF measurements, target screening has facilitated the rapid identification of known myxobacterial metabolites in extracts from nine Myxococcus species. Furthermore, principal component analysis (PCA), implementing an advanced compound-based bucketing approach, readily revealed the presence of further compounds which contribute to variation among the metabolite profiles under investigation. The generation of molecular formulae for putative novel compounds with high confidence due to evaluation of both exact mass position and isotopic pattern, is exemplified as an important key for de-replication and prioritization of candidates for further characterization.

Chapter 3. Discovering natural products from myxobacteria with emphasis on rare producer strains in combination with improved analytical methods.

Myxobacteria produce a range of structurally novel natural products which exhibit unusual or unique modes of action, attracting significant interest from both the academic and drug discovery communities. Efforts to discover new strains with the potential to biosynthesize novel molecules have revealed that myxobacterial diversity and natural products are far from exhausted. We describe here a general, nonselective approach to unearth further myxobacterial strains, in order to mine them for compounds with potential as medicines. Sample collection from locations world-wide has shown that environments which exhibit significant biological complexity yield the highest probability of isolating novel myxobacterial strains. Here, we illustrate the details of simple and efficient strain purification techniques, which lead systematically to the identification of new and promising myxobacteria. Compound identification is then facilitated by molecular biological approaches, coupled with sophisticated high resolution mass spectrometry, statistical analysis, and bioassays.

Improving natural products identification through targeted LC-MS/MS in an untargeted secondary metabolomics workflow.

Tandem mass spectrometry is a widely applied and highly sensitive technique for the discovery and characterization of microbial natural products such as secondary metabolites from myxobacteria. Here, a data mining workflow based on MS/MS precursor lists targeting only signals related to bacterial metabolism is established using LC-MS data of crude extracts from shaking flask fermentations. The devised method is not biased toward specific compound classes or structural features and is capable of increasing the information content of LC-MS/MS analyses by directing fragmentation events to signals of interest. The approach is thus contrary to typical auto-MS(2) setups where precursor ions are usually selected according to signal intensity, which is regarded as a drawback for metabolite discovery applications when samples contain many overlapping signals and the most intense signals do not necessarily represent compounds of interest. In line with this, the method described here achieves improved MS/MS scan coverage for low-abundance precursor ions not captured by auto-MS(2) experiments and thereby facilitates the search for new secondary metabolites in complex biological samples. To underpin the effectiveness of the approach, the identification and structure elucidation of two new myxobacterial secondary metabolite classes is reported.