CRISPR-BASED MANIPULATION OF TRANSCRIPTIONAL REGULATORS FOR SILENT ANTIBIOTIC BGC ACTIVATION IN STREPTOMYCES

Abstract

Most biosynthetic gene clusters (BGCs) contained in Streptomyces genomes are not expressed under commonly used laboratory conditions, and thus represent an enormous and largely unexplored source of new antibiotic scaffolds. Promoter replacement, regulator overexpression and heterologous expression have all been shown to be effective conventional activation strategies, but they are limited by operation complexity, limited scalability and the lack of understanding of the native regulatory architecture. New approaches have emerged along with the discovery of CRISPR-based tools that manipulate transcription without physically altering BGC structure – these tools act in trans to rewire the regulatory networks that control the expression of BGCs. In this review we will discuss the shift from conventional gene editing to programmable transcriptional regulation in Streptomyces, focusing on the use of CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and on the engineering of both pathway-specific and global regulators by CRISPR. We review evidence showing that the capability to activate silent pathways without chromosomal engineering can be achieved by CRISPRi to repress endogenous repressors or CRISPRa to activate biosynthetic operons, and that targeting pleiotropic regulators like AdpA, DasR, and Bld-family proteins can enable coordinated activation across multiple BGCs. Recent progress with endogenous Type I-E Cascade systems has allowed activation of thirteen cryptic BGCs to be achieved in non-model strains and new scaffold RNA architectures have been developed to allow both activation and repression from the same complex. The translational potential of these approaches is highlighted by case studies such as jadomycin B and auroramycin, and the recently activated linaridin pathways. We conclude that the challenge has shifted from the tool to understanding the regulatory network: the power of CRISPR based activation is now becoming constrained not by the number of programmable effectors available, but by what is known regarding the nodes to target.