Recent advances in activating silent biosynthetic gene clusters in bacteria

Abstract

The explosion of microbial genome sequences has shown that bacteria harbor an immense, largely untapped potential for the biosynthesis of diverse natural products, which have traditionally served as an important source of pharmaceutical compounds. Most of the biosynthetic genes that can be detected bioinformatically are only weakly expressed, or not at all, under standard laboratory growth conditions. Herein we review three recent approaches that have been developed for inducing these so-called silent biosynthetic gene clusters: insertion of constitutively active promoters using CRISPR-Cas9, high-throughput elicitor screening for identification of small molecule inducers, and reporter-guided mutant selection for creation of overproducing strains. Together with strategies implemented previously, these approaches promise to unleash the products of silent gene clusters for years to come.

Transcription-factor centric genome mining strategy for discovery of diverse unprecedented RiPP gene clusters

“…It is important to understand the complex regulation of secondary metabolites in bacteria, especially in Streptomyces [47,48]. It has been long known that most of the gene clusters in Streptomyces are silent under normal laboratory conditions [49,50].…”

Cebulantin, a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity‐Coupled HiTES

“…After a productive period spanning the late 1930s and the early 1960s, during which most of the current antibiotic scaffolds were identified, the pace of discovery has slowed down significantly . Today we know that one of the culprits is a feature that is inherent to microbial genomes: most biosynthetic gene clusters (BGCs)—the sets of genes responsible for the biogenesis of a natural product—are not expressed or, at best, sparingly expressed under standard growth conditions in the laboratory . Consequently, their products are not interrogated in routine bioactivity assays.…”

“…These so‐called silent or cryptic BGCs outnumber constitutively expressed ones by a factor of 5–10 and represent a treasure trove of new natural products, and possibly antibiotics. To access the products of silent BGCs, several approaches have been developed . A key drawback of nearly all available methods is that they rely on genetic or complex molecular biology manipulations and/or do not report on the bioactivity of cryptic metabolites.…”

Cebulantin, a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity‐Coupled HiTES

“…[1,2] Today we know that one of the culprits is af eature that is inherent to microbial genomes:m ost biosynthetic gene clusters (BGCs)-the sets of genes responsible for the biogenesis of an atural product-are not expressed or, at best, sparingly expressed under standard growth conditions in the laboratory. [3][4][5] Consequently,t heir products are not interrogated in routine bioactivity assays.T hese so-called silent or cryptic BGCs outnumber constitutively expressed ones by afactor of 5-10 and represent at reasure trove of new natural products, and possibly antibiotics.T oa ccess the products of silent BGCs,s everal approaches have been developed. [4][5][6][7][8][9][10] Ak ey drawback of nearly all available methods is that they rely on genetic or complex molecular biology manipulations and/or do not report on the bioactivity of cryptic metabolites.T hese aspects slow down the pace and throughput by which bioactive,c ryptic metabolites can be found.…”

“…[3][4][5] Consequently,t heir products are not interrogated in routine bioactivity assays.T hese so-called silent or cryptic BGCs outnumber constitutively expressed ones by afactor of 5-10 and represent at reasure trove of new natural products, and possibly antibiotics.T oa ccess the products of silent BGCs,s everal approaches have been developed. [4][5][6][7][8][9][10] Ak ey drawback of nearly all available methods is that they rely on genetic or complex molecular biology manipulations and/or do not report on the bioactivity of cryptic metabolites.T hese aspects slow down the pace and throughput by which bioactive,c ryptic metabolites can be found. Herein, we combine high-throughput elicitor screening (HiTES) [11] with biological activity assays for the facile,genetics-free discovery of cryptic metabolites with the desired biological properties.…”