A rapid and efficient strategy to identify and recover biosynthetic gene clusters from soil metagenomes

Abstract

Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes. Key points • An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery. • A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore. • Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids.

Long-Read Metagenome-Assembled Genomes Improve Identification of Novel Complete Biosynthetic Gene Clusters in a Complex Microbial Activated Sludge Ecosystem

“…Laborious attempts to culture these populations should be preceded by in situ screening, as many genes may be silent under laboratory or growth conditions ( 7 ). Exciting developments in the extraction and expression of BGCs from metagenomes suggests a potential high-throughput approach for product characterization ( 25 , 65 ), though product detection remains challenging. Applying metatranscriptomics to narrow down potential targets highly expressed in situ could increase the chances of success.…”

Bacterias extremótrofas y extremófilas resistentes a radiación UV-B aisladas de la laguna de Salinas, un humedal altoandino del Perú

“…Nuestro estudio representa una contribución al entendimiento de la biodiversidad microbiana presente en la laguna de la Salinas. No obstante, debemos reconocer que este trabajo solo comprende bacterias cultivables, lo que implica que nuestro conocimiento de la biodiversidad es parcial, por lo que es fundamental destacar la relevancia de los enfoques independientes de cultivo, basados en tecnologías de secuenciación de nueva generación que permiten un análisis más profundo de la diversidad microbiana a través de enfoques metagenómicos (Arya y Ravindra, 2020;Dubey et al, 2022) y también posibilitan la búsqueda de genes de interés ecológico y biotecnológico (Alonso-Reyes et al, 2020;Negri et al, 2022;Rascovan et al, 2016). En entornos análogos a la laguna de Salinas, como en el salar de Huasco, Maras y Acos, mediante estos métodos se ha logrado reportar comunidades muy diversas y una dominancia de los phyla Proteobacteria, Bacteroidetes, Balneolaeota y Actinobacteria (Castelán-Sánchez et al, 2019;Castro-Severyn et al, 2021).…”

Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications

“…On the other hand, the high microbial diversity found in the environmental metagenome and the limitations of short reads by Illumina (Chen, Chiang, et al, 2021 ) do not yield the long contigs necessary for good genome mining, and a low biosynthetic potential was detected in this microbial community. BGC identification in environmental samples requires an efficient method of BGC identification such as hybrid‐assembled contigs (long‐ and short‐read sequencing) (Chen, Chiang, et al, 2021 ; Negri et al, 2022 ).…”