title: 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.
authors.affiliation: Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Rashel V
authors.initials: RV
authors.lastname: Grindberg
authors.forename: Thomas
authors.initials: T
authors.lastname: Ishoey
authors.forename: Dumitru
authors.initials: D
authors.lastname: Brinza
authors.forename: Eduardo
authors.initials: E
authors.lastname: Esquenazi
authors.forename: R Cameron
authors.initials: RC
authors.lastname: Coates
authors.forename: Wei-ting
authors.initials: WT
authors.lastname: Liu
authors.forename: Lena
authors.initials: L
authors.lastname: Gerwick
authors.forename: Pieter C
authors.initials: PC
authors.lastname: Dorrestein
authors.forename: Pavel
authors.initials: P
authors.lastname: Pevzner
authors.forename: Roger
authors.initials: R
authors.lastname: Lasken
authors.forename: William H
authors.initials: WH
authors.lastname: Gerwick
mesh_terms: D001427:Bacterial Toxins / Q000096:biosynthesis*; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial; D005810:Multigene Family; D059010:Single-Cell Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001427:Bacterial Toxins