I. Introduction and Scope 4589 II. NMR Methods for Structural Determination 4591 A. Carbohydrate Structural Elements and Classical NMR Methods 4591 1. Number of Sugar Residues 4592 2. Constituent Monosaccharides 4592 3. Anomeric Configuration 4593 4. Linkage and Sequence 4594 5. Position of Appended Groups 4594 6. Classical NMR Methods 4594 B. New NMR Methods 4596 C. Computer-Assisted Structural Analysis 4598 D. NMR Spectroscopy of Mixtures 4599 III. Large and Unusual Oligosaccharides 4600 IV. Polysaccharides 4603 V. NMR Analysis of Small Sample Amounts 4604 A. NMR Probes and Sample Tubes 4605 B. Examples of Structural Analysis with Small Sample Amounts 4606 VI. Conclusions 4610 VII. Acknowledgments 4610 VIII. References 4610 † This paper is dedicated to the late Professor R. U. Lemieux in memory of an always exiting and stimulating collaboration on structural studies of oligosaccharides.Jens Ø. Duus received his M.Sc. degree in Chemical Engineering from the Technical University of Denmark in 1989. Following this, he continued his graduate study with Professor Klaus Bock at the Carlsberg Laboratory and was granted his Ph.D. degree from the Technical University of Denmark in 1993. During his graduate study he was twice a Visiting Scientist at Dupont Experimental Station in Wilmington, DE working with Dr. S. Sabesan. From 1993 to 1994 he was a Research Fellow in Chemistry at the Beckman Institute at the California Institute of Technology working with Professor Harry B. Gray. After returning to the Carlsberg Laboratory, he was appointed Senior Scientist. His main fields of research are structure and dynamics of biomolecules with emphasis on carbohydrates using NMR and fluroescence.

Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodical gene deletions to identify supporting enzymes for key synthases one cluster at a time. In this study, we design and apply a DNA expression array for Aspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association-based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions. A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A. We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.aspergilli | natural products | secondary metabolism | polyketide synthases N o other group of biochemical compounds holds as much promise for drug development as the secondary (nongrowth associated) metabolites (SMs). A review from 2012 (1) found that for small-molecule pharmaceuticals, 68% of the anticancer agents and 52% of the antiinfective agents are natural products, or derived from natural products. The fact that SMs are often synthesized as polymer backbones that are subsequently diversified greatly via the actions of tailoring enzymes sets the stage for combinatorial biochemistry (2), because their biosynthesis is modular.Major groups of SMs include polyketides (PKs) consisting of -CH 2 -(C = O)-units, ribosomal and nonribosomomal peptides (NRPs), and terpenoids made from C 5 isoprene units. These polymer backbones are, with the exception of ribosomal peptides, made by synthases or synthetases and are modified by a plethora of tailoring enzymes, including (de)hydratases, oxygenases, hydrolases, methylases, and others.In fungi, these biosynthetic genes of secondary metabolism are organized in discrete clusters around the synthase genes. Although quite accurate algorithms are available for identification of possible SM biosynthetic genes, particularly PK synthases (PKSs), NRP synthetases (NRPSs), and dimethylallyl tryptophan synthases (DMATSs) (3, 4), the assignment and prediction of the members of the individual clusters solely from the genome sequence have not been accurate. Relevant protein domains can be predicted for some of the genes (e....

Secondary metabolites in filamentous fungi constitute a rich source of bioactive molecules. We have deduced the genetic and biosynthetic pathway of the antibiotic yanuthone D from Aspergillus niger. Our analyses show that yanuthone D is a meroterpenoid derived from the polyketide 6-methylsalicylic acid (6-MSA). Yanuthone D formation depends on a cluster composed of ten genes including yanA and yanI, which encode a 6-MSA polyketide synthase and a previously undescribed O-mevalon transferase, respectively. In addition, several branching points in the pathway were discovered, revealing five yanuthones (F, G, H, I, and J). Furthermore, we have identified another compound (yanuthone X1) that defines a class of yanuthones that depend on several enzymatic activities encoded by genes in the yan cluster but that are not derived from 6-MSA.

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.

On a global research expedition, over 500 bacterial strains inhibitory towards pathogenic bacteria were isolated. Three hundred of the antibacterial strains were assigned to the Vibrionaceae family. The purpose of the present study was to investigate the phylogeny and bioactivity of five Vibrionaceae strains with pronounced antibacterial activity. These were identified as Vibrio coralliilyticus (two strains), V. neptunius (two strains), and Photobacterium halotolerans (one strain) on the basis of housekeeping gene sequences. The two related V. coralliilyticus and V. neptunius strains were isolated from distant oceanic regions. Chemotyping by LC-UV/MS underlined genetic relationships by showing highly similar metabolite profiles for each of the two V. coralliilyticus and V. neptunius strains, respectively, but a unique profile for P. halotolerans. Bioassay-guided fractionation identified two known antibiotics as being responsible for the antibacterial activity; andrimid (from V. coralliilyticus) and holomycin (from P. halotolerans). Despite the isolation of already known antibiotics, our findings show that marine Vibrionaceae are a resource of antibacterial compounds and may have potential for future natural product discovery.