Investigating the biosynthetic potential of an Antarctic soil through metagenomics, cultivation, and heterologous expression

Abstract

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as sources of new antimicrobials. Metagenomic sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, and metagenomic library studies have yielded several novel natural products. However, accessing these compounds remains challenging. The constraints of short-read sequencing mean that the assembly of full-length BGC sequences from uncultured bacteria is nigh impossible, thus making assessment of BGC diversity difficult and downstream cloning infeasible. Conversely, metagenomic library approaches suffer from a bias towards known compounds as well as difficulties with expressing recovered BGCs. In the present work, a three-pronged approach was taken to access the biosynthetic diversity of bacteria from an Antarctic soil: A hybrid shotgun metagenome was sequenced and BGCs cloned and expressed, a novel regulatory gene-based screen for libraries was developed, and a number of isolates were obtained by culturing. Through metagenomic sequencing, many highly divergent BGCs were found in phyla such as Acidobacteriota and Verrucomicrobiota, but also the methanotrophic gammaproteobacterial order UBA7966. Sequencing of isolates obtained from the same soil indicated little overlap between the biosynthetic potential of readily cultured and uncultured bacteria. Several metagenomic BGCs were PCR-amplified, cloned and expressed in Pseudomonas and Streptomyces. While the sequencing of Streptomyces exconjugants showed that many inserts were truncated, a phenotype was observed in Pseudomonas. The library screening approach was validated in isolates, but the targets were absent in the metagenomic library used. In conclusion, the results uncover the rich diversity of BGCs from uncultured lineages present in the soil, show the potential of long-read sequencing to recover full-length BGCs from uncultured soil bacteria and demonstrate the feasibility of cloning them. However, they also indicate the necessity of refined molecular tools for successful heterologous expression of metagenomic BGCs