Abstract
The multifunctional proteins of class 2 CRISPR systems such as Cas9, have been employed to activate cryptic biosynthetic gene clusters (BGCs) in Streptomyces, which represent a large and hidden reservoir of natural products. However, such approaches are not applicable to most Streptomyces strains with reasons to be comprehended. Inspired by the prevalence of the class 1 subtype especially the type I-E CRISPR system in Streptomyces, here we report the development of the type I-E CRISPR system into a series of transcriptional regulation tools. We further demonstrate the effectiveness of such activators in nine phylogenetically distant Streptomyces strains. Using these tools, we successfully activate 13 out of 21 BGCs and lead to the identification and characterization of one polyketide, one Ripp and three alkaloid products. Our work is expected to have a profound impact and to facilitate the discovery of numerous structurally diverse compounds from Streptomyces.
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Introduction
Microbes are renowned as prolific antibiotic producers, among them, Streptomyces are particularly important for producing approximately two-thirds of natural antibiotics1. However, the pace of discovering new antibiotics from microbes has slowed down since the late twentieth century2. With the development of second-generation sequencing technology in the new century, the volume of microbial genomic data has been rapidly expanded. The genomic data disclosed a rich repertoire of biosynthetic gene clusters (BGCs). However, further bioinformatic analysis revealed that, indeed, over 90% of putative microbial metabolites are not produced under normal laboratory culture conditions in the absence of unknown in vivo regulatory signals that trigger their biosynthesis3. Therefore, developing tools to activate cryptic BGCs has become an effective strategy for mining new natural products.
Cas9 of the class 2 CRISPR system and its homologs, such as Cas12a, versatile enzymes that contain multiple domains and exhibit both nuclease and helicase activities, have been extensively employed for genome editing in various organisms4,5,6. Their applications in Streptomyces have also been reported, which include deletion and insertion of large fragments7,8,9, and direct capture of large BGCs10,11. The class 2 CRISPR system can also be used for gene regulation. Previously, a promoter knock-in strategy was applied to drive the in situ expression of cryptic BGCs, leading to the activation of four BGCs in five Streptomyces strains12.
Gene expression regulation can also be precisely programmed in a variety of organisms, from bacteria, and yeast to humans, by CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation), which employ dCas proteins (dCas9, dCas12a), mutants of Cas9 or Cas12a lacking nuclease activity and their fusion proteins with an effector attached respectively4,13,14. The CRISPRi system targets the ORF upstream region, blocking the recruitment of RNA polymerase and attenuating its activity15, resulting in up to 99% decrease in gene expression in bacteria16. This system can be developed to target multiplex genes. Down-regulation of seven genes simultaneously in Saccharomyces cerevisiae was recently reported17. By contrast, the CRISPRa system turns on gene expression, in which the dCas protein is fused to an RNA polymerase activation ___domain, guiding the recruitment of RNA polymerases to specific genomic loci and triggering the transcription of downstream genes18. Taken together, the power of CRISPRi and CRISPRa lies in the flexibility of gRNAs designed to target any DNA sequence in close proximity to a three-nucleotide protospacer adjacent motif (PAM), as well as the ability to rapidly and precisely modulate endogenous gene expression without chromosome engineering. Although dCas9-based CRISPRi systems have demonstrated functionality in select Streptomyces strains4,19,20,21,22, the dCas9-based CRISPRa systems have typically shown restricted efficacy in the majority of Streptomyces strains, with a recent report of success only in a specific Streptomyces strain23.
Exploring the diverse CRISPR systems presents a promising avenue for developing bacterial gene regulation tools with unique characteristics. Given that the majority of prokaryotes possess indigenous CRISPR systems, it is feasible to leverage these inherent immune mechanisms for gene regulation purposes. This approach allows for the utilization of nature’s own toolkit, potentially leading to more efficient and targeted genetic manipulation strategies within bacterial hosts. A recent in-depth study revealed that most Streptomyces strains harbor the class 1 type I-E CRISPR systems24. Unlike the class 2 CRISPR system that consists of a single protein, all class 1 systems contain multiple Cas subunits, which form a complex known as Cascade (the CRISPR-associated complex for antiviral defense)25. A typical type I-E CRISPR system contains three core genes (cas1, cas2, and cas3) and Cascade genes (casA-casB-cas7-cas5-cas6)25. Cas1 and Cas2 form an alliance to capture a piece of DNA from the invader during the adaptation process. Cas3 alone is capable of unwinding DNA/DNA and RNA/DNA duplexes and cutting single-stranded DNA26, while the Cascade/Cas3 complex mediates DNA nicking in the interference process. Using the type I-A CRISPR system, different types of mutation were generated, including deletion, insertion, and point mutations27. The employment of type I-B CRISPR systems has illustrated the facile programmability and remarkable efficacy of native CRISPR in facilitating genome engineering within Clostridium tyrobutyricum28,29. Similarly, the application of type I-E CRISPR systems has unveiled the capacity to engineer a CRISPRa system endowed with unique and broadened regulatory capabilities in E. coli30,31. These discoveries suggested that the Streptomyces endogenous class 1 CRISPR systems may be developed into tools for transcriptional regulation.
In this study, we systematically explore the Streptomyces type I-E CRISPR Cascade, based on which a series of repressors/activators are designed and demonstrated to be effective in activating cryptic BGCs of the non-model Streptomyces strains. Thirteen BGCs responsible for the synthesis of various natural products, including linaridins, NRPS, and polyketides, were activated, allowing the identification and characterization of these compounds. In summary, we develop a robust toolkit that aids in precisely regulating gene expression and effectively mining natural products in Streptomyces.
Results
Identification of type I-E CRISPR systems in Streptomyces
67 complete genomic sequences of Streptomyces strains, all that is available at NCBI, as well as the sequences of seven strains isolated and sequenced in our laboratory, were retrieved for analysis (Supplementary Data 1). Sequence alignment of the most conserved Cas1, followed by PILER-CR analysis to find the striking signature of a CRISPR system in the neighborhood, the repetitive sequences formed by the reciprocal structure of the CRISPR array, uncovered type I-E CRISPR systems in a total of 25 Streptomyces strains. Among them, three strains, CFMR-7, Mg1, and SCSIO 03032, possess two systems, while some other strains harbor variants of the system, with one lacking the cas2 gene and seven possessing a casA-cas3 fusion gene. These gene clusters were grouped and displayed based on the phylogenetic distances of their hosts (Fig. 1). Next, we decided to employ a cas3-free system for gene regulation, as the cas3-free complex is expected to introduce site-specific hindrance of polymerase access while maintaining the integrity of the target DNA32. To avoid complexity, those clusters containing a casA-cas3 fusion were excluded. Drawing from the protein alignment outcomes of the Cascade (Supplementary Data 2), we chose two type I-E systems that exhibit the highest and lowest homology to the Sav Cascade among the strains in our study to provide an analysis that spans the spectrum of genetic similarity, thereby enhancing our understanding of the functional diversity and potential applications of these CRISPR systems. Thus, three type I-E CRISPR systems from Streptomyces avermitilis MA-4680, Streptomyces T1-5, and Streptomyces A-23, strains available in our laboratory, were proceeded for activity screen.
The phylogenetic tree was built based on the amino acid sequences of Cas1, the most conserved protein in the CRISPR systems. The displayed 28 type I-E CRISPR systems were found from 74 Streptomyces strains.
Developing the CRISPR-based transcriptional repressor 1.0(CTR 1.0)
To achieve stable regulation in Streptomyces, we designed a dual plasmid system to express the Cascade module and spacer plasmid separately. Once the Cascade system is integrated into the genome, subsequent editing of various pathways becomes more manageable and efficient, facilitated by the use of a smaller plasmid. To test these CRISPR systems in S. coelicolor, a strain that does not possess its own type I-E CRISPR system, the Cascade expression cassettes derived from Streptomyces T1-5, Streptomyces A-23 and Streptomyces avermitilis MA-4680, with either the kasO*p promoter and oop terminator pairs or the native promoter and terminator pairs (Fig. 2a), were integrated into the ΦC31 attB site of S. coelicolor genome, generating the strains R1-1 to R1-6 (Supplementary Table 1) presumably expressing the corresponding Cascade proteins at stable levels. Next, a pYL plasmid carrying a crRNA cassette with direct repeat (DR) sequences at both ends of the spacer was conjugated into the six S. coelicolor strains, generating the CRISPR-based transcriptional repressor 1.0 (CTR 1.0).
a Schematics of CRISPR-based transcriptional repressor 1.0 (CTR 1.0) in this work. The Cascade consists of 5 genes (casA-casB-cas7-cas5-cas6). b CrRNAs targeting the act-orf4 coding region and promoter region of the ACT BGC are shown. c Repression results for targeting the act-orf4 coding sequence using the CTR 1.0. d CrRNAs targeting the red Q gene region of the RED BGC are shown. e Repression results for targeting the redQ coding sequence using the CTR 1.0. f Repression results for targeting both the redQ and act-orf4 genes using the CTR 1.0. Control (The wild-type strain of S. coelicolor).
We next decided to screen the CTR 1.0 systems, therefore, the repression of the actII-ORF4 gene (sco5085) using a colorimetric assay was designed. The actII-ORF4 gene, located in the gene cluster responsible for the biosynthesis of actinorhodin (ACT), a characteristic blue pigment of S. coelicolor, encodes a transcription activator33. Its repression shuts down the entire BGC, leading to the color of the bacterial colony shifting from blue to brown. Five crRNAs (a1–a5), each containing the canonical 5′-AAG-3′ PAM and one of the five spacers in the actII-ORF4 region, including the promoter, the coding strand, and the non-template strand were employed for each of the six R1 strains (Fig. 2b). We observed the expected color change with three crRNAs in the R1-1 strain and four crRNAs in the R1-5 strain (Fig. 2c, S1). Among the six R1 strains, R1-5, that exhibited the best repression effect was chosen for subsequent experiments.
The biosynthesis of undecylprodigiosin (RED), a red-pigmented antibiotic, was then used as another readout of the assay and the proof of concept for CTR 1.0. The red Q gene (sco5887) in the RED BGC encoding a positive regulatory protein of the SARPs family was chosen as the target. This protein is required for the synthesis of RED34, and its repressed expression has been reported to result in a shift in colony color from red to yellow. Four crRNAs (r1-r4) with two spacers in the promoter region (r1 and r2) and two spacers in the non-template strand (r3 and r4) were designed, aiming to repress redQ in the R1-5 strain (Fig. 2d). All four crRNAs efficiently repressed RED synthesis as observed by the colony colors (Fig. 2e), and transcriptional analysis revealed a maximum reduction of 98% of the red Q mRNA level (Supplementary Fig. 2a).
Dual-target transcriptional repression was then evaluated for CTR 1.0. Spacer r2a1(r2 and a1) repressed the synthesis of both RED and ACT, while spacer r3a4 (r3 and a4) efficiently repressed RED formation, but not as obviously for ACT accumulation (Fig. 2f). The mRNA expression levels of actII-ORF4 and red Q were significantly decreased (Supplementary Fig. 2b).
Creating the CRISPR-based transcriptional activator 1.0 (CTA 1.0)
In pursuit of advancing gene regulatory tools that are particularly amenable to the exploration of natural products in Streptomyces, we proceeded to develop the transcriptional activator tool CTA 1.0, building upon the foundation of CTR 1.0 with activation domains fused. Our aim was to exemplify its efficacy in reactivating silenced bacterial gene clusters (BGCs), thereby enhancing the potential for uncovering bioactive compounds. An activation ___domain fused to the CRISPR system can be directed to the promoter region and activates gene transcription, by recruiting RNA polymerase (RNAP) or stabilizing the RNAP initiation complex35. Two different activation domains were selected: the ω (rpoZ) ___domain, a component of RNA polymerase crucial for antibiotic production in Streptomyces kasugaensis36, and the SoxS, a transcriptional regulator belonging to the AraC family and a component of the essential bacterial soxRS redox-sensing system, which is notably upregulated in response to oxidative stress and recruits RNA polymerase to the promoter region37. Both activation domains have been demonstrated effective in Escherichia coli38. The crystal structure of the Cascade reveals that CasA caps one end of the helical arrangement39. Therefore, a construct with the CasA N-terminus fused to an activation ___domain via a linker (two or five small amino acid residues, AA or GGGGA) was designed, in which the Cascade was derived from Streptomyces avermitilis MA-4680 (Fig. 3a). The Cascade-activator expression cassette with the kasO*p promoter and oop terminator pairs was integrated into the ΦC31 attB site of Streptomyces A-14, a non-model Streptomyces isolated from the inter-root soil of the traditional Chinese medicine Ferula officinalis with rich BGCs and fast growing property (Supplementary Data 3), to form strains A1-1 to A1-4. Subsequently, the pYL-crRNA plasmid was conjugated into the four A1 strains, generating the CRISPR-based transcriptional activator 1.0 (CTA 1.0).
a Schematics of CRISPR-based transcriptional activator 1.0 (CTA 1.0) in this work. The activation ___domain was fused to the Cascade (casA-casB-cas7-cas5-cas6) via a linker at the CasA N-terminus. b CrRNAs targeting the PTM-ctg-610 promoter region of the PTM BGC are shown. c HPLC analysis for activating PTM BGC using the CTA 1.0 and CTA 2.0 in S. A-14. (★ indicates major products of the PTM BGC). d The structures of the compound PTM-a ~ c. Control (The wild-type strain of S. A-14). Source data are provided as a Source Data file.
Analysis of the Streptomyces A-14 genome sequence by antiSMASH indicates that cluster 8 is responsible for the biosynthesis of polycyclic tetramate macrolactams (PTMs) that exhibit antitumor activities40. All six coded gene products are enzymes involved in the biosynthetic pathway and show 100% similarity to previously characterized S. griseus PTM BGC. We designed three crRNAs (p1–p3) located at 80–300 bp upstream of the transcription start site (Fig. 3b). All three crRNAs appeared to activate the PTM BGC, as the HPLC profiles of the CTA 1.0 fermentation extracts displayed prominent peaks in comparison with no observable peaks in that of the wild type strain (Fig. 3c, S3). The best hit, A1-3-p3, with Cascade under kasO*p promoter and oop terminator and CasA linked to the SoxS ___domain via AA, was subjected to further LC/MS analysis. The molecular weights for the products of peak a (m/z, 510.2730), peak b (m/z, 508.2430), and peak c (m/z, 494.2584) were compared and confirmed to be identical to PTMs derived from S. griseus (Fig. 3d, S4). A comparative analysis of the linker revealed that AA is superior to GGGGA. Moreover, the activation ___domain SoxS outperformed ω (rpoZ) (Supplementary Fig. 3), which could be attributed to its interaction with the conserved σ-factor region 4 and core RNAP α-subunit41, and was used for subsequent gene cluster activation.
Development of the CRISPR-based transcriptional repressor/activator 2.0 (CTR/CTA 2.0)
To craft more efficient and versatile gene regulation tools capable of addressing a spectrum of application needs, including the concurrent repression and activation of genes, we have subsequently developed the transcriptional regulation tool CTR/CTA 2.0 employing Scaffold RNAs. Scaffold RNAs (each scRNA is a crRNA followed by the MS2 sequence of a bacteriophage origin) have been used in E. coli as gene regulatory tools in concert with the employment of an activation ___domain fused to the MS2 capsid protein (MCP) that specifically recognizes the MS2 sequence42. This design can be used as a transcriptional activator or repressor depending on the positioning of scRNA. When the scRNA binds to a narrow region upstream of the transcription start site, gene activation occurs. By contrast, if the scRNA binds to the coding sequence, transcriptional repression of the gene occurs35.
We decided to adopt the scRNA method for programmable transcription regulation in Streptomyces. The pYL-scRNA plasmid was designed with MS2 at the 3′ end of the crRNA and MCP protein fused to the C-terminus of the ω (rpoZ) or the SoxS ___domain via GGGGG linker. The Cascade expression box with the kasO*p promoter and oop terminator (Fig. 4a) was conjugated into S. coelicolor, S. lividans and S. A-14, respectively to form different Streptomyces chassis, followed by the introduction of various pYL-scRNA plasmids, resulting in the CRISPR based transcriptional repressor/activator 2.0 (CTR/CTA 2.0) (Fig. 4a and Supplementary Table 1).
a Schematics of CRISPR-based transcriptional repressor/activator 2.0 (CTR/CTA 2.0) in this work. The Cascade consists of 5 genes (casA-casB-cas7-cas5-cas6). The scRNA plasmid consists of a scRNA cassette with crRNA coupled to the MS2 sequence, and an activator ___domain under the control of the kasO*p promoter. b Repression results for targeting the act-orf4 coding sequence using the CTR 2.0. c Repression results for targeting the redQ coding sequence using the CTR 2.0. d Activation result for targeting the act-orf4 promoter region using the CTA 2.0. Control (For 4b and 4c, control represents the wild-type strain of S. coelicolor; For 4 d, control represents the wild-type strain of S. lividans).
To validate CTR 2.0 in S. coelicolor, we chose the same reporter genes, actII-ORF4 and red Q involved in the biosynthesis of ACT and RED respectively, and the corresponding spacers used in CTR 1.0, excluding those targeting sequences within the promoter region. Spacers a2, a4, and a5 were found able to abolish or significantly reduce the pigment accumulation likely due to lack of ACT biosynthesis and to reduce the mRNA expression levels of actII-ORF4 transcripts by 96%, 80% and 93%, respectively, compared to control strain, while spacer a3 showed no significant repression effect (Fig. 4b, S2c). Among the three effective spacers, a4 was the least effective in gene repression, which perhaps is due to its proximity to the promoter region. Spacers r3 and r4 reduced the RedQ transcript by 90% and 75%, respectively, resulting in the eyeball-distinguishable decrease of the RED pigmentation (Fig. 4c, S2d). The CTA 2.0 system was then tested in S. lividans that harbors the ACT gene cluster with the actII-ORF4 silent under normal culturing conditions43. The CTA2.0 system employing either the ω (rpoZ) or the SoxS ___domain and the crRNA targeting 86 bp upstream of the transcription start site of the actII-ORF4 gene successfully triggered the pigment accumulation of ACT (Fig. 4d).
The BGC of PTM in S. A-14 was found activatable with the CTA 2.0 system. Comparing the two CTA versions in activating the PTM cluster highlighted the efficacy of the 2.0 system. We thus decided to utilize the CTA 2.0 system to activate uncharacterized BGCs in various Streptomyces strains.
Activation of cryptic BGCs by the CTA2.0 in S. A-14
31 BGCs in the S. A-14 genome have never been characterized. They are putatively responsible for the biosynthesis of various natural products, including polyketides, NRPs, Ripps, and others. In addition to the PTM gene cluster (C8), 10 of the 31 BGCs were chosen as representatives for the validity test of CTA2.0 with expected natural products ranging from polyketide to Ripp and other uncategorized (Table 1). Among them, cluster 11, expected to produce a polyketide is of particular interest with sequences distinct from those of any cluster responsible for a previously characterized product. Three crRNAs (c1-c3) were designed to target within the region 80–300 bp upstream of the transcription start site of the first polyketides gene, ctg-949 that encodes the acyl carrier protein (Fig. 5a). LC-MS analysis revealed a prominent peak with a m/z of 638.1717 that is absent in both the wild type strain and the activated cluster-deletion strain, ΔC11 (Fig. 5b, S5). The chemical structure of the compound was delineated by nuclear magnetic resonance (NMR) spectroscopy with a scale-up sample from approximately 200 L fermentation broth (0.83 mg/L) (Fig. 5c, Supplementary Fig. 6 and Supplementary Table 2).
a CrRNAs targeting the promoter regions of ctg-949 gene are shown. b HPLC analysis for activating C11 BGC using the CTA 2.0 in S. A-14. c The structure of the compound secalonic acid. A2-4 (Activation strains with kasO*p driven the expression of the Sav Cascade and MS2-coupled SoxS ___domain); ΔC11(Strains with cluster 11 deleted in the chromosome of S. A-14, as a control); ΔC11-c1(The cluster-deletion strain with activation tool applied to target the promoter regions of ctg-949 gene, as a control); Control (The wild type strain of S. A-14). Source data are provided as a Source Data file.
Based on the availability of the PAM region in the gap areas and an optimal 80–300 bp distance from the transcription start site of genes, additional spacers were designed to perform in situ activation of other genes in cluster 11. Fermentation followed by HPLC assays of these mutant strains suggested that a gamma-glutamyltransferase, a phosphatase of the PhoX family, another acyl carrier protein and a flavoprotein of the FixA family, encoded by ctg1_937, ctg1_938, ctg1_954 and ctg1_972 respectively may be involved in the biosynthesis (Supplementary Fig. 7 and Supplementary Table 3. Interestingly, a gene cluster responsible for the biosynthesis of secalonic acid and structurally related compounds has been recently identified in the fungi and biochemically characterized44,45 (Supplementary Fig. 8 and Supplementary Tables 4, 5). The composition and organization of the S. A-14 gene cluster 11 suggested that this bacterium employs a synthetic route and mechanism distinct from the fungi.
Genomic sequencing and further analysis via PRISM and BAGEL4 on cluster 14 unveiled a linaridin BGC with only 33% similarity to that responsible for the synthesis of legonaridin46. It is noteworthy that the precursor peptide is indeed different from legonaridin as a member of the rare linaridin class. Three crRNAs (c4-c6) located at 80–300 bp upstream of the transcription start site of the first gene in the cluster, C14-ctg-1306 were designed (Fig. 6a). In the HPLC elution profile, a prominent peak was observed and absent in the negative control (Fig. 6b). The MALDI-TOF analysis of supernatant extracts revealed a mass of [M + Na]+ = 2197.9540 Da, consistent with the high-resolution electrospray ionization mass spectrometry (MS), which detected a mass of [M + H]+ = 2175.1531 Da (Supplementary Fig. 9). The discrepancy between the observed mass and the predicted sequence (Fig. 6c) suggested additional modifications.
a CrRNAs targeting the promoter region of the C14-ctg-1306 gene are shown. b HPLC analysis for activating the C14 BGC using the CTA 2.0 in S. A-14. c The structure of the compound C14-d. A2-4 (Activation strains with kasO*p driven the expression of the Sav Cascade and MS2-coupled SoxS ___domain); Control (The wild type strain of S. A-14). Source data are provided as a Source Data file.
Strain A2-4 was employed to activate eight unexplored cryptic BGCs, clusters 4, 13, 17, 22, 24, 25, 26, and 28, some of which are highly homologous to previously known BGCs47,48,49,50. Six of them, clusters 4, 13, 22, 24, 25, and 28, when activated, resulted in notable peaks in the fermentation extracts (Supplementary Fig. 10a), with some, clusters 4, 22, 24, and 28 finding the expected molecular weights of the natural products by LC-MS (Supplementary Fig. 10b–d).
Activation of multiple gene clusters by CTA2.0 in diverse Streptomyces
To demonstrate the general applicability of the CTA2.0 tool to a broad range of Streptomyces strains, we attempted to activate nine uncharacterized BGCs of different classes, including four polyketides, two NRPs, one Ripp, and two other uncategorized BGCs. Seven Streptomyces strains, namely S. 539, S. T2-10, S. T2-4, S. A16, S. W121, S. albus J1704, and Nh. DSM 44494 was subjected to the assay. One or more crRNAs located 80–300 bp upstream of the promoter transcription start site of the core gene were designed for each BGC. For six out of the total of nine gene clusters (Supplementary Tables 6–14), C6.1-S. 539, C12-S. A16, C21-S. W121, C24-S. W121, C22-S. albus J1704 and C11-Nh. DSM 44494, we observed remarkable peaks in the fermentation extracts (Supplementary Figs. 11–14a), with most of them, except C21-S. W121, matching exactly with the expected molecular weights by MS (Supplementary Figs. 11–14b).
Cluster 11 in Nocardiopsis halophila DSM 44494, isolated from extremophilic environments, exhibits a 52% similarity to the pyrrolizidine alkaloids (PAs) BGC (pyrrolizixenamide A) in Xenorhabdus51, was readily activated while the promoter region of the core gene ctg-16_96 was targeted with CTA2.0 (Fig. 7a). HPLC analysis revealed that the engineered strain produced a prominent peak and a couple of minor peaks that were not present in the fermentation broth of the control strain (Fig. 7b). High-resolution electrospray ionization mass spectrometry detected masses of [M + H]+ = 181.0961, 197.0896 and 320.1593 Da (Supplementary Fig. 15), consistent with the structures of legonmycin C, D and E (0.02 mg/L, 0.04 mg/L and 0.09 mg/L, respectively) as determined by NMR (Fig. 7c, Supplementary Figs. 16–18 and Supplementary Tables 15–17). Taken together, our CTA tools are well suited for in-situ activation of individual genes and/or large BGCs in the model, non-model, and extremophilic Streptomyces, and are expected to facilitate the exploration of hidden natural products from this rich source.
a CrRNA targeting the promoter region of the C11-ctg1-16_96 gene are shown. b HPLC analysis for activating C11 BGC using the CTA 2.0. c The structure of the compound C11-Legonmycin C-E. A2-4 (Activation strains with kasO*p driven the expression of the Sav Cascade and MS2-coupled SoxS ___domain); Control (The wild type strain of Nh. DSM 44494). Source data are provided as a Source Data file.
Discussion
Streptomyces are ubiquitous Gram-positive bacteria and are known as a treasure trove of structurally diverse natural products, many of which have the potential to be developed into therapeutic compounds. CRISPR systems have been used as valuable tools for genetic manipulation in model Streptomyces strains. However, when they are applied to most of the non-model strains, such exogenous systems face limitations due to cellular toxicity and the intricate metabolic milieu of these bacteria. Given the prevalence of type I-E CRISPR systems in Streptomyces, our study focused on the development of gene regulatory tools employing such endogenous systems. These endogenous CRISPR systems possess notable advantages over prevalent CRISPR systems like Cas9 or Cas12a, such as enhanced compatibility, extensive distribution, and operational simplicity, given that there is no requirement for the delivery of foreign Cas genes52,53. To ascertain the relative efficacy of the dCas9 and the I-E CRISPR-derived Cascade system, we also assessed the proteotoxicity of Cas9, dCas9 and Cascade within various Streptomyces strains (Supplementary Fig. 19), under identical conditions, the conjugative transfer efficiencies followed the order: Cas9 < dCas9 < Cascade. This observation implies that the Cascade system, which originates from Streptomyces, may be better adapted for applications within Streptomyces species.
Upon systematic mining and comparative analysis, we identified and developed effective gene regulatory tools derived from the type I-E CRISPR system from Streptomyces avermitilis MA-4680. These tools were further demonstrated to be effective for the activation of various BGCs in model and non-model strains, including extremophilic Streptomyces strains. Streptomyces adapt to survival challenges posed by extreme environmental factors by producing distinct profiles of natural products. Our methods advance in overcoming the constraints associated with traditional screening techniques and opening avenues for the discovery of previously untapped bioactive molecules in extreme environments. Despite the fact that the CTR and CTA systems developed in this study utilize two distinct plasmids, the integration of the Cascade system simplifies subsequent gene editing to merely the introduction of the crRNA expression plasmid, thereby enhancing the efficiency of the conjugation process. This refinement allows for a more streamlined approach to genetic manipulation, reducing the complexity and increasing the potential for successful gene editing outcomes. It is widely recognized that a single Streptomyces strain typically harbors multiple natural product biosynthetic pathways. Therefore, with the Cascade system integrated just once, subsequent editing of various pathways becomes more manageable and efficient, facilitated by the use of a smaller plasmid. This approach actually simplifies the genetic manipulation process, making it an advantageous strategy for Streptomyces gene regulation.
Activation of Cluster 11 in Streptomyces A-14 with our tools leads to the production of secalonic acid, a tetrahydroxanthone dimer, by a bacterial strain. Tetrahydroxanthone dimers were previously isolated from fungi and shown to inhibit the growth of Helicobacter pylori, over 30-fold more potent than clinically used metronidazole44,45. Bioinformatics analysis revealed this gene cluster with the core polyketide biosynthesis gene exhibiting only 24% identity to the fungal counterpart44, implying a different biosynthetic route and mechanism to be further characterized.
The tools reported in this study are modular, allowing substitution of various components for different purposes, e.g., introducing the gain of function SoxS mutants, R93A and S101A, to boost the activation effect50. The increasing number of subclasses within class I CRISPR systems and their wide distribution in different bacterial phyla4 may allow quick expansion and advance of the toolbox by adopting new components to the systems.
The distance of the spacer binding site in relation to the promoter is critical for the activation effect, as previously observed for the application of Cas9 in E. coli35. Noteworthy, our systems appeared to be less restricted and effective in a broader range of 80–300 bases upstream of the gene transcription start site, compared to 50–90 bases for Cas9 in E. coli35. The requirement of specific spatial orientation of gRNA binding was observed for the Cas9 system, working effectively with gRNA binding to the non-template strand in Streptomyces23, whereas our system demonstrated better flexibility, being effective with gRNA binding to both template and non-template strands, perhaps due to a larger complex and a longer interdomain linker employed.
In summary, we demonstrated the versatility of the Streptomyces type I-E CRISPR system in gene regulation, encompassing both repression and activation. Robust gene regulation systems for Streptomyces successfully constructed in this study led to the discovery of various natural products. Our work rationalizes strategies utilizing endogenous CRISPR systems and promises to catalyze further development of similar strategies in other prokaryotes for various purposes, including but not restricted to the mining of natural products.
Methods
Strains, materials, and reagents
Streptomyces coelicolor A3(2), Streptomyces lividans 1326, E. coli DH5α and E. coli ET12567/pUZ8002 were gifted from Professor Huimin Zhao (University of Illinois, Urbana, IL). The plasmid pYES and pCRISPomyces-2 were gifted from Professor Huimin Zhao (University of Illinois, Urbana, IL). The plasmid pWHU2653 was gifted from Professor Yuhui Sun (SIBS, Chinese Academy of Sciences). S. A-14 and Nh. DSM44494 were excavated by our laboratory. Reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, United States), Oxoid (Basingstoke, Hampshire, United Kingdom), and Sangon Biotech Co., Ltd. (Shanghai, China). All restriction endonucleases, as well as T4 DNA ligase and DNA polymerase, were purchased from New England Biolabs (Beverly, MA, United States). SYBR® Green PCR Master Mix was purchased from Applied Biosystems (San Francisco, CA, United States). The DNA isolation and purification kits were purchased from TIANGEN (Beijing, China). All primers were synthesized by Tsingke (Beijing, China). E. coli strains were grown in Luria Bertani (LB) broth, containing 100 µg/mL ampicillin or 50 µg/mL apramycin as needed. Streptomyces strains were grown in MYG liquid medium (10 g/L malt extract, 4 g/L yeast extract, and 4 g/L glucose), or MS agar medium (20 g/L D-mannitol, 20 g/L soybean meal, and 20 g/L agar) at 30 °C unless otherwise indicated. 2 × YT medium-containing 1% yeast extract, 1% tryptone, and 0.5% NaCl (pH 7.0) was used for strain washing and spore germination before conjugation. M-ISP4 (5 g/L Soybean meal, 5 g/L D-Mannitol, 5 g/L Starch soluble, 2 g/L Tryptone, 1 g/L Yeast extract, 1 g/L NaCl, 2 g/L (NH4)2SO4, 1 g/L K2HPO4, 2 g/L CaCO3, 20 g/L Agar, 0.1%Trace element which contain 1 g/L FeSO4, MnCl2 and ZnSO4) was used for conjugation. R2YE agar medium (103 g/L Sucrose, 0.25 g/L K2SO4, 10.12 g/L MgCl2.6H2O, 10 g/L Glucose, 0.1 g/L Casamino acid, 5 g/L yeast extract, 0.5%KH2PO4, 5 M CaCl2.2H2O, 20% L-proline, 5.73% TES buffer, 0.2 ml Trace element solution, and 20 g/L agar) was used for phenotypic analysis.
Plasmid construction and conjugation
The Cascade or Cascade-activator expression cassette was cloned into the vector pYES54 and assembled via Gibson assembly55. The pYL-crRNA plasmid and pYL-scRNA plasmid with MS2 at the 3′end of the crRNA and MCP protein fused to the C-terminus of the activation ___domain were constructed based on pYL vector9 by Golden Gate assembly.
Streptomyces codon-optimized MCP was chemically synthesized by Genewiz (Suzhou, Jiangsu, China), and activation domains, including ω and SoxS were amplified from purified genomic DNA of S. griseus. The presence of the correct plasmids was confirmed by sequencing (Tsingke, Beijing, China). All primers used in this study are listed in Supplementary Data 4. E. coli ET12567/pUZ8002 cells were transformed with the plasmids to be conjugated and selected on LB agar plates supplemented with 50 ug/mL apramycin, 50 ug/mL kanamycin and 25 µg/mL chloramphenicol. These transformants were then used as the donors for conjugative transfer of the assembled plasmids to Streptomyces strains. M-ISP4 solid medium containing 25 mM MgCl2 and 2 × YT liquid medium were used for conjugation. Exconjugants were transferred to M-ISP4 medium containing 25 μg/ml apramycin and 25 μg/ml nalidixic acid and were cultured at 30 °C56. Streptomyces exconjugants were picked and re-streaked on ISP2 plates supplemented with 50 µg/mL apramycin and grown for two days.
qPCR analysis
The total RNA of Streptomyces cultivations under MYG medium after 72 h was extracted using the BACTERIAL Total RNA Isolation Mini Kit (Bio-Tek, USA). Reverse transcription was carried out using the First-strand cDNA Synthesis Kit (Bio-Rad, Carlsbad, CA, USA). Real-time PCR was performed with SYBR® Green PCR Master Mix on the 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Primers were designed by the online tool provided by Integrated DNA Technologies (https://www.idtdna.com/scitools/Applications/RealTimePCR/). 10 μL of 2 × SYBR Green Mix, 1 μL of cDNA, 1 μL of each primer at a concentration of 10 pmol/µL, and 7 µl of ddH2O were mixed gently in each well of the Applied Biosystems® MicroAmp® Optical 96-Well Reaction Plate. Reactions were performed using the following program: 2 min at 50 °C, 10 min at 95 °C for one cycle, followed by 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C for 40 cycles with a final cycle of 10 min at 72 °C. The endogenous gene hrdB, encoding RNA polymerase sigma factor, was used as the internal control for gene regulation. The expression levels of other candidate genes were normalized to the expression of the control. Data were analyzed using SDS2.4 software (Applied Biosystems, Carlsbad, CA, USA).
Fermentation and HPLC-MS analysis
Exconjugants were picked and inoculated in 50 ml of MYG supplemented with apramycin and grown to high density (usually 2-3 days) in 250 ml shake flasks at 30 °C. For the fermentation of PTM compounds, aliquots of the seed culture were spread for confluence over Petri plates containing approximately 25–30 ml of MYG solid agar medium, and the plates were incubated for 7 days at 30 °C40. For other compounds, fermentation cultures were incubated at 30 °C with constant shaking (220 rpm) for seven days, with a ratio of 1:25. To isolate PTM compounds, agar, and the adherent cells were stored at 80 C for 24 h, after which the frozen cultures were thawed completely and the released liquids were collected. The liquids were extracted by ethyl acetate with a ratio of 1:1 twice, concentrated 1000-fold, and subjected to HPLC analysis. To isolate potential Ripps compounds, the supernatant of the fermentation was extracted by n-butanol with a ratio of 1:1 twice, concentrated 1000-fold, and subjected to HPLC analysis. To isolate potential other compounds, the supernatant of the fermentation was extracted by ethyl acetate with a ratio of 1:1 twice, concentrated 1000-fold, and subjected to HPLC analysis.
HPLC was performed on an HPLC system (Shimadzu, LC-20a) with a Shimadzu C18 reverse-phase column (4.6 × 250 mm, 5 μm) at room temperature. The HPLC parameters were as follows: solvent A, 0.1% trifluoroacetate in water; solvent B, 0.1% trifluoroacetate in acetonitrile; gradient, 10% B for 10 min, to 100% B in 30 min, maintain at 100% B for 10 min, return to 10% B in 1 min and finally maintain at 10% B for 20 min; flow rate 1 mL/min; detection by UV spectroscopy at 280/215 nm. The mass spectrometry system was operated using a drying temperature of 350 °C, a nebulizer pressure of 35 psi., a drying gas flow of 8.5 L/min, and a capillary voltage of 4500 V.
Structure elucidation of the compounds
Compounds were purified by reverse-phase HPLC using a Shimadzu C18 reverse-phase column (4.6 × 250 mm, 5 μm), on an HPLC system (Shimadzu, LC-20a) with the HPLC conditions described above. The high-resolution MS was performed on a Q Exactive HF (Q Exactive™ HF/UltiMate™ 3000 RSLC nano) using electron spray ionization (ESI) in positive or negative mode (Thermo Fisher Scientific, USA). All spectroscopic experiments were carried out on a Bruker AVANCE III 400, 500, or 600 MHz spectrometer (Bruker Biospin AG, Switzerland). The chemical shift values (δ) were given in ppm with the residual signals of CDCl3 as the internal standard, and coupling constants (J) in Hz. The structures were determined based on the extensive analysis of 1D and 2D-NMR spectroscopic data.
Statistics & reproducibility
Experiments were performed using at least three biological replicates per condition tested unless otherwise indicated. Data for the column chart showed are reported as bars showing mean, with error bars showing standard deviation. Compound production experiments were performed in biological duplicates.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
67 Streptomyces genome information were collected online from NCBI, the complete genome sequence of Streptomyces A-14 is available with the BioProject ID PRJNA1083101. The complete genome sequence of Streptomyces T1-5, Streptomyces T2-4, Streptomyces T2-10, Streptomyces A-7, Streptomyces A-16, and Streptomyces A-23 have been submitted to the Genome Sequence Archive (GSA) under the accession code CRA016553. The RNA-seq dataset is available in NCBI under accession code PRJNA1168667. The data needed to evaluate the conclusions in this study have been deposited in Figshare. Sourced data are available in Figure Share: https://figshare.com/articles/online_resource/Sourced_data_in_NCOMMS-24-27794A/27170487. The associated mass spectrometry data are available in Figure share: https://figshare.com/articles/online_resource/MASS_SPECTROMETRY-NCOMMS-24-27794A/27170583. Source data for the main and other Supplementary Figs. are provided in this paper. The data supporting the findings of this study are available within the article, Supplementary, or Source data files. Source data are provided in this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (32471492, 32071426, to Y.L.), the National Key Research and Development Program of China (2018YFA0903300, to Y.L.), the Haihe Laboratory of Sustainable Chemical Transformations for financial support (24HHWCSS00006, to Y.L.), and the Key-Area Research and Development Program of Guangdong Province (2020B0303070002, to Y.L.). We thank Prof. Min Yin (Yunnan University) for providing strain Nocardiopsis halophila DSM 44494.
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Y.L. designed the experiments. Y.Z., Q.Z., and H.W. performed the experiments and analyzed the data. C.K., S.G., and Y.Y. determined the chemical structures of the compounds. H.L. performed the strain construction. Y.L. Y.Z., and Q.Z. wrote the manuscript.
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Zhou, Q., Zhao, Y., Ke, C. et al. Repurposing endogenous type I-E CRISPR-Cas systems for natural product discovery in Streptomyces. Nat Commun 15, 9833 (2024). https://doi.org/10.1038/s41467-024-54196-z
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DOI: https://doi.org/10.1038/s41467-024-54196-z
