Activating cryptic biosynthetic gene clusters via ACTIMOT

Abstract

The mainstream strategy of genome mining relies on the homologous activation and heterologous expression of target biosynthetic gene clusters (BGCs). However, the efficiency of the current techniques available for new compound discovery hardly complements these efforts. In a recent publication in Science, Xie et al. reported their breakthrough progress in expediting the discovery of untapped chemical diversity from bacteria by establishing the leveraged know-how of ACTIMOT (Advanced Cas9-mediaTed In vivo MObilization and mulTiplication of BGCs), offering a new avenue to access the unexploited, and even unpredictable, biosynthetic potential of bacteria.

Graphical abstract

1. Introduction

Natural products are important sources of new drugs [1]. Since the onset of genome mining, the specialized chemistries of numerous cryptic natural products have been unveiled [2]. The leading strategies for genome mining consist of (1) homologous expression of the target biosynthetic gene cluster (BGC) in native species using genome editing, and (2) heterologous expression of the BGC in ideal hosts after its cloning and manipulation in model bacteria or yeast [[3], [4], [5], [6]]. However, despite the nearly three decades of extensive genome mining of natural products from bacteria [7], the traditional OSMAC (one strain, many compounds) approach and genome mining strategies have yet to meet the on-going demand for the scalable discovery of novel compounds.

The genus Streptomyces is known to possess a prolific array of bioactive natural products and drug leads [8]. However, the discovery of unknown natural products from Streptomyces species is hindered by the labor intensiveness or lack of highly efficient gene editing technologies for activating cryptic BGCs in native strains. With a focus on achieving the scalable genome mining of “unseen” Streptomyces natural products in an autologous manner, Xie et al. [9] recently published their breakthrough progress in establishing a CRISPR-Cas9-based ACTIMOT (Advanced Cas9-mediaTed In vivo MObilization and mulTiplication of BGCs) system, in which the spread process of antibiotic resistance genes (ARGs) has been artificially simulated to mobilize and multiply large genomic BGCs.

The widespread dissemination of ARGs is attributed to their mobilization by either insertion sequences or integrons, followed by their relocation onto mobile genetic elements, and ultimately their horizontal transfer into other bacterial species (Fig. 1a) [10]. This inherent mechanism represents an evolutionarily successful strategy for significantly promoting the transmission of ARGs among bacteria. Contrary to the cumbersome traditional cloning and pathway engineering of large target BGCs, ACTIMOT simply accomplishes the mobilization of chromosomal target DNA regions (TDRs) using a release plasmid (pRel) carrying the SG5 Streptomyces replicon and functional CRISPR-Cas9 elements, while the simultaneous relocation and multiplication of the TDRs are achieved using a capture plasmid (pCap) carrying a multicopy Streptomyces replicon, a bacterial artificial chromosome, a PAM cassette between the upstream and downstream homologous arms. The mobilized and relocated TDRs on the multicopy pCap are promptly amplified, leading to enhanced BGC expression in a gene dosage-dependent manner in native species (Fig. 1b). Using ACTIMOT, 39 unexploited natural compounds across four diverse classes were identified via the gene dosage effect in either native species or heterologous hosts, without any further genetic modification. With its corroborated high efficiency in mobilizing, relocating, and multiplying target BGCs both natively and heterologously, ACTIMOT has been endowed with great potential to expedite the unlocking of the vast biosynthetic potential of unknown natural products from bacteria.

Fig. 1.

Comparison of ARG spreading (a) and ACTIMOT for revealing cryptic natural products (b). MGE: mobile genetic element; ARGs: antibiotic resistance genes.

First, Xie et al. targeted the 24 kb actinorhodin (act) BGC in the native species S. coelicolor M145, generating an enhanced high-yielding actinorhodin mutant. The improved production of actinorhodin can be largely attributed to the multiplication of the target BGC, serving as validation of the proof-of-concept of ACTIMOT and a demonstration of its feasibility and viability. Subsequently, Xie et al. optimized ACTIMOT into a single-plasmid version and confirmed its improved competence for natural product discovery. By applying single-plasmid ACTIMOT to two well-studied type strains, namely S. avidinii DSM40526 and S. armeniacus DSM19369, the authors surprisingly unlocked four classes of previously unexploited natural products featuring novel chemical characteristics and biosynthesis.

Simultaneous activation of two tail-to-tail nonribosomal peptide synthetases: The 48 kb TDR Sav11 harbors two nonribosomal peptide synthetase (NRPS) BGCs organized in a tail-to-tail pattern. The authors successfully mobilized and activated these two BGCs in the heterologous host S. albus Del14 without additional engineering. By contrast, the expression of the BGCs was highly suppressed in the native strain S. avidinii DSM40526. The activated compounds (avidistatins and avidilipopeptins) feature diverse structures, validating that ACTIMOT is an inspiring technology for the exploitation of biosynthetic pathways.

Facilitation of authentic product discovery: The 67 kb TDR Sar13 carries mop, a cryptic “ladderane-NRPS” BGC. Xie et al. successfully mobilized and relocated the mop BGC in the native species S. armeniacus DSM19369, with a 90.9 % success rate, resulting in a series of mobilipeptins with significantly enhanced yields. The technology is particularly effective in uncovering “transient” final products, as easily degraded products of mop were unexpectedly detected and identified with yield improvement at different biosynthesis stages through the use of ACTIMOT.

Unmasking of “dark matter” hidden behind unknown biosynthetic pathways: The 149 kb TDR Sav17 superficially contains a giant NRPS BGC, which ACTIMOT efficiently mobilized and relocated in native species. Further heterologous expression of Sav17 in S. albus Del14 resulted in target products with higher yields and diversity, revealing a new family of benzoxazole-containing natural products; namely, actimotins. Despite the advantages of in-depth bioinformatics analysis and gene-knockout studies, the mechanisms underlying the biosynthesis of actimotins remain to be elucidated. As a genome mining beacon, ACTIMOT has additional advantages in aiding the discovery of the unpredictable biosynthetic potential in bacteria.

Xie et al. developed ACTIMOT as a groundbreaking and highly promising technology to boost the discovery of natural products in Streptomyces by mimicking the molecular mechanisms of ARG dissemination. The exploitation of 39 previously unknown natural compounds across four distinct classes from diverse Streptomyces species has greatly expanded the application of ACTIMOT, not only in terms of mobilization and relocation of large TDRs but also in both native species and heterologous hosts.

The essence of ACTIMOT lies in the highly efficient double-strand breaks generated in the native strain as well as the high-copy replicative plasmids for the in vivo autologous mobilization and multiplication of BGCs without the need for the intermediate host (mostly Escherichia coli) required in the classical cloning and transfer strategy. CRISPR-based technologies for natural product research have rapidly advanced in recent years [11]. Xie et al. have contributed their own efficient tool, which provides key element for building ACTIMOT [9]. In combination with ACTIMOT, the rational pathway engineering (e.g. promoter exchange) of captured BGCs via Red/ET recombineering could facilitate the expression of “difficult-to-activate” BGCs in heterologous expression systems [12]. However, for the efficient mobilization and amplification of promising TDRs, the extensive off-target effects of the CRISPR-Cas system in many other bacteria cannot be ignored. By optimizing the on-target cleavage efficiency of the Cas9 nuclease and with the help of broader compatible genetic elements, the potential applications of ACTIMOT can be extended to other genetically manipulatable bacteria (e.g., rare species of Actinobacteria, Proteobacteria, and Firmicutes) regarded as storehouses of biosynthetic potential [13]. This technology paves the way for activating cryptic natural products previously referred to as “inaccessible” from bacteria and offers an innovative strategy for future biotechnological applications.

CRediT authorship contribution statement

Xiaoying Bian: Writing – review & editing, Writing – original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.