Despite the wide availability of antibiotics, infectious diseases remain a leading cause of death worldwide.1 In the absence of new therapies, mortality rates due to untreatable infections are predicted to rise more than 10-fold by 2050. Natural products (NPs) made by cultured bacteria have been a major source of clinically useful antibiotics. In spite of decades of productivity, the use of bacteria in the search for new antibiotics was largely abandoned due to high rediscovery rates.2, 3 As only a fraction of bacterial diversity is regularly cultivated in the laboratory and just a fraction of the chemistries encoded by cultured bacteria is detected in fermentation experiments, most bacterial NPs remain hidden in the global microbiome. In an effort to access these hidden NPs, we have developed a culture-independent NP discovery platform that involves sequencing, bioinformatic analysis, and heterologous expression of biosynthetic gene clusters (BGCs) captured on DNA extracted from environmental samples (eDNA). Here, we describe the application of this platform to the discovery of the malacidins, a distinctive class of antibiotics that are commonly encoded in soil microbiomes but have never been reported in culture-based NP discovery efforts. The malacidins are active against multidrug-resistant (MDR) pathogens, sterilize MRSA skin infections in an animal wound model, and did not select for resistance under our laboratory conditions.

Numerous therapeutically relevant small molecules have been identified from the screening of natural products (NPs) produced by environmental bacteria. These discovery efforts have principally focused on culturing bacteria from natural environments rich in biodiversity. We sought to assess the biosynthetic capacity of urban soil environments using a phylogenetic analysis of conserved NP biosynthetic genes amplified directly from DNA isolated from New York City park soils. By sequencing genes involved in the biosynthesis of nonribosomal peptides and polyketides, we found that urban park soil microbiomes are both rich in biosynthetic diversity and distinct from nonurban samples in their biosynthetic gene composition. A comparison of sequences derived from New York City parks to genes involved in the biosynthesis of biomedically important NPs produced by bacteria originally collected from natural environments around the world suggests that bacteria producing these same families of clinically important antibiotics, antifungals, and anticancer agents are actually present in the soils of New York City. The identification of new bacterial NPs often centers on the systematic exploration of bacteria present in natural environments. Here, we find that the soil microbiomes found in large cities likely hold similar promise as rich unexplored sources of clinically relevant NPs.

Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of small-molecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.