Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

Abstract

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing

“…Recent articles (Marcy et al, 2007;Woyke et al, 2010;Youssef et al, 2011;Blainey et al, 2011;Grindberg et al, 2011) illustrate the challenges of fragment assembly in SCS. Chitsaz et al (2011) introduced the E + V-SC assembler, combining a modified EULER-SR with a modified Velvet, and achieved a significant improvement in fragment assembly for SCS data.…”

Current Approaches for Enhanced Expression of Secondary Metabolites as Bioactive Compounds in Plants for Agronomic and Human Health Purposes

“…Interest in bioactive compounds was not purely academic but was rather prompted by their great utility as dyes, polymers, fi bers, glues, oils, waxes, fl avoring agents, perfumes, and drugs. Recognition of the biological properties of myriad natural products has supported the current focus of this fi eld, the search for new drugs, antibiotics, insecticides, and herbicides [Grindberg et al, 2011]. Terpenes, phenols, fl avanoids, alkaloids, sterols, waxes, fats, tannins, sugars, gums, suberins, resin acids and carotenoids are among the many classes of compounds known as secondary [Gottlieb, 1990;Grindberg et al, 2007;Nunnery et al, 2010].…”

Marine Peptide Secondary Metabolites

“…Apratoxin A prevents the cotranslational translocation of proteins destined for the secretory pathway (Liu, Law, and Luesch, 2009). The putative biosynthetic apratoxin A gene cluster was reported using a single-cell genome amplification (Grindberg et al, 2011).…”