A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Abstract

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or "warhead") found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

Development of a Transcription Factor-Based Lactam Biosensor

“…org/index.php), 58 and many more are continually being discovered through large-scale metagenomics sequencing efforts. 59,60 We used the BRENDA enzyme/ligand database, filtering with "Enzyme, Ligand" and "exact") to confirm the existence of catabolism pathways and organisms that carry such pathways.…”

Isolation and Identification of Three New 5-Alkenyl-3,3(2H)-furanones from Two Streptomyces species using a Genomic Screening Approach

“…Eco86 were analyzed by genome scanning technique as described previously by Zazopoulos et al [2]. The DNA and protein sequences that comprise E-837, E-492 and E-975 gene clusters are deposited in GeneBank under accession numbers DQ272520 (E-837) and DQ272521, DQ272522 (two contigs from the E-492/E-975 producer).…”

“…The genomics of secondary metabolite biosynthesis has recently evolved to the point where analysis of the genome of an organism can define its secondary metabolic capabilities. A genome scanning technique has been developed in our laboratories to greatly reduce the amount of sequencing required to define this capability [2,3]. This approach not only ascertains the potential of a producing organism, but it provides the scientist with a handle to identify, isolate and structurally define a specific metabolite.…”

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

“…While the pursuit of the idea of a microbial paclitaxel source, providing for an inexhaustible supply of this antineoplastic blockbuster and novel taxanes, is being hampered by obtaining disappointingly low yields, the active ingredient is still mainly derived via chemical semisynthesis from the advanced taxoid, 10-deacetylbaccatin III, readily available from the needles of the European yew tree, Taxus baccata, being a renewable source and, to a lesser extent, by means of plant cell culture methods (Leistner 2005;Frense 2007). Nonetheless, the exciting progress that has been made in the elucidation of biosynthetic route leading to paclitaxel in planta due to fundamental works of Croteau and his co-workers (Walker and Croteau 2001;Jennewein et al 2004a, b;Croteau 2005;DeJong et al 2006;Nims et al 2006), as well as the recent advances in microbial genomics (Zazopoulos et al 2003;Stephanopoulos et al 2004;Keller et al 2005;van Lanen and Shen 2006) and combinatorial biosynthesis (Floss 2006;Nguyen 2006;KleinMarcuschamer et al 2007), might still revive and boost the interest in endophytic paclitaxel synthesizers.…”

“…Whole-genome sequence mining (Lautru et al 2005) and genome scanning as an alternative approach, providing an efficient way to discover natural product biosynthetic gene clusters without having the complete genome sequence (Zazopoulos et al 2003); advances in microbial cell fermentation technology (Zengler et al 2005;Weuster-Botzl et al 2007) and metagenomics as a valuable alternative offering a cultivation-independent approach (Schloss and Handelsman 2005); ample successes in heterologous expression and metabolic engineering (among many others: Alper et al 2005;Schmidt et al 2005;Wenzel et al 2005;DeJong et al 2006;Julsing et al 2006;Lindahl et al 2006;Nims et al 2006;Ro et al 2006), the latter being in fact perceived as a progenitor of functional genomics and systems biology (Stephanopoulos et al 2004;Tyo et al 2007) Á to name only a few highlights.…”