Navid Adnani

Microbial Strain Prioritization Using Metabolomics Tools for the Discovery of Natural Products

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

Universal quantification of structurally diverse natural products using an evaporative light scattering detector.

A lack of good methods for absolute quantification of natural products has limited the accuracy of high-throughput screening. Many currently used methods for quantification are either too slow or not amenable to the structural diversity of natural products. Recent developments in low-temperature evaporative light scattering detectors (ELSD-LT) have overcome several historical limitations of ELSDs, including analyte decomposition and low sensitivity. Primarily, ELSDs have been used for relative quantification and detection of compounds that lack a UV chromophore. In this study, we employ an ELSD-LT for absolute quantification of natural products. Calibration curves were constructed using a weighted least-squares analysis for a diverse set of natural products and other compounds. An average calibration curve was evaluated for the “universal” quantification of natural products. Optimization of ELSD-LT hardware and parameters improved sensitivity and throughput and established the utility of ELSD-LT for quantification of large natural product libraries.

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach

With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.

Application of 3D NMR for Structure Determination of Peptide Natural Products.

Despite the advances in NMR, structure determination is often slow and constitutes a bottleneck in natural products discovery. Removal of this bottleneck would greatly improve the throughput for antibiotic discovery as well as other therapeutic areas. Overall, faster structure methods for structure determination will serve the natural products community in a broad manner. This report describes the first application of 3D NMR for elucidation of two microbially produced peptide natural products with novel structures. The methods are cost-effective and greatly improve the confidence in a proposed structure.

Draft Genome Sequence of Micromonospora sp. Strain WMMB235, a Marine Ascidian-Associated Bacterium

ABSTRACT Micromonospora sp. strain WMMB235 was isolated in 2011 off the coast of the Florida Keys, USA, from a marine ascidian as part of an ongoing drug discovery project. Analysis of the ~7.1-Mb genome provides insight into this strains biosynthetic potential, means of regulation, and response to coculturing conditions.

Complete Genome Sequence of Rhodococcus sp. Strain WMMA185, a Marine Sponge-Associated Bacterium

ABSTRACT The Rhodococcus strain WMMA185 was isolated from the marine sponge Chondrilla nucula as part of ongoing drug discovery efforts. Analysis of the 4.44-Mb genome provides information regarding interspecies interactions as pertains to regulation of secondary metabolism and natural product biosynthetic potentials.

Brackish habitat dictates cultivable Actinobacterial diversity from marine sponges

Bacterial communities associated with marine invertebrates such as sponges and ascidians have demonstrated potential as sources of bio-medically relevant small molecules. Metagenomic analysis has shown that many of these invertebrates harbor populations of Actinobacteria, many of which are cultivable. While some populations within invertebrates are transmitted vertically, others are obtained from the environment. We hypothesized that cultivable diversity from sponges living in brackish mangrove habitats have associations with Actinobacterial populations that differ from those found in clear tropical waters. In this study, we analyzed the cultivable Actinobacterial populations from sponges found in these two distinct habitats with the aim of understanding the secondary metabolite potential. Importantly, we wanted to broadly evaluate the potential differences among these groups to guide future Actinobacterial collection strategies for the purposes of drug discovery.