Guosong Zheng

One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces

The RNA-guided DNA editing technology CRISPRs (clustered regularly interspaced short palindromic repeats)/Cas9 had been used to introduce double-stranded breaks into genomes and to direct subsequent site-specific insertions/deletions or the replacement of genetic material in bacteria, such as Escherichia coli, Streptococcus pneumonia, and Lactobacillus reuteri. In this study, we established a high-efficiency CRISPR/Cas9 genome editing plasmid pKCcas9dO for use in Streptomyces genetic manipulation, which comprises a target-specific guide RNA, a codon-optimized cas9, and two homology-directed repair templates. By delivering pKCcas9dO series editing plasmids into the model strain Streptomyces coelicolor M145, through one-step intergeneric transfer, we achieved the genome editing at different levels with high efficiencies of 60%-100%, including single gene deletion, such as actII-orf4, redD, and glnR, and single large-size gene cluster deletion, such as the antibiotic biosynthetic clusters of actinorhodin (ACT) (21.3 kb), undecylprodigiosin (RED) (31.6 kb), and Ca(2+)-dependent antibiotic (82.8 kb). Furthermore, we also realized simultaneous deletions of actII-orf4 and redD, and of the ACT and RED biosynthetic gene clusters with high efficiencies of 54% and 45%, respectively. Finally, we applied this system to introduce nucleotide point mutations into the rpsL gene, which conferred the mutants with resistance to streptomycin. Notably, using this system, the time required for one round of genome modification is reduced by one-third or one-half of those for conventional methods. These results clearly indicate that the established CRISPR/Cas9 genome editing system substantially improves the genome editing efficiency compared with the currently existing methods in Streptomyces, and it has promise for application to genome modification in other Actinomyces species.

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

GlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces. In this work, evidence that GlnR is also directly involved in the regulation of antibiotic biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in antibiotic biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII. To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates antibiotic biosynthesis directly through pathway-specific regulators in Streptomyces. Our results suggest that GlnR-mediated regulation of antibiotic biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and antibiotic biosynthesis in streptomycetes.

Functional analysis of TetR-family regulator AmtRsav in Streptomyces avermitilis.

In actinomycetes, two main regulators, the OmpR-like GlnR and the TetR-type AmtR, have been identified as the central regulators for nitrogen metabolism. GlnR-mediated regulation was previously identified in different actinomycetes except for members of the genus Corynebacterium, in which AmtR plays a predominant role in nitrogen metabolism. Interestingly, some actinomycetes (e.g. Streptomyces avermitilis) harbour both glnR- and amtR-homologous genes in the chromosome. Thus, it will be interesting to determine how these two different types of regulators function together in nitrogen regulation of these strains. In this study, AmtRsav (sav_6701) in S. avermitilis, the homologue of AmtR from Corynebacterium glutamicum, was functionally characterized. We showed, by real-time reverse transcription (RT)-PCR (qPCR) in combination with electrophoretic mobility shift assays (EMSAs), that gene cluster sav_6697-6700 encoding a putative amidase, a urea carboxylase and two hypothetical proteins, respectively, and sav_6709 encoding a probable amino acid permease are under the direct control of AmtRsav. Using approaches of comparative analysis combined with site-directed DNA mutagenesis, the AmtRsav binding sites in the respective intergenic regions of sav_6700/6701 and sav_6709/6710 were defined. By genome screening coupled with EMSAs, two novel AmtRsav binding sites were identified. Taken together, AmtRsav seems to play a marginal role in regulation of nitrogen metabolism of S. avermitilis.

Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis

UNLABELLED Pristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded by SSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, from Corynebacterium glutamicum serves as a transcriptional repressor of the paa genes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion of paaR led to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue from C. glutamicum, PaaR is also involved in the suppression of paa expression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway of L-phenylglycine (L-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription of paa genes in a ΔpaaR mutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux toward L-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ΔpaaR mutant was restored by L-Phg supplementation as well as by deletion of the paaABCDE operon in the ΔpaaR mutant. Altogether, this study provides new insights into the regulation of PI biosynthesis by S. pristinaespiralis. IMPORTANCE A better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues for Streptomyces strain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription of paa genes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors, L-phenylglycine, in Streptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis in Streptomyces strains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.

Identification of two novel regulatory genes involved in pristinamycin biosynthesis and elucidation of the mechanism for AtrA-p-mediated regulation in Streptomyces pristinaespiralis

In this study, using a transposon-based strategy, two novel regulatory genes were identified as being involved in the biosynthesis of both pristinamycin I (PI) and II (PII) in Streptomyces pristinaespiralis, including a TetR-family regulatory gene atrA-p (SSDG_00466) and an orphan histidine kinase gene SSDG_02492. The mechanism by which AtrA-p exerted a positive role in pristinamycin production was elucidated. We showed that deletion of atrA-p resulted in a delayed production of both PI and PII as well as reduced PII production. Transcriptional analysis integrated with electrophoretic mobility shift assays (EMSAs) demonstrated that AtrA-p played a positive role in pristinamycin production by directly activating the transcription of two cluster-situated regulatory genes, spbR and papR5, which encode a γ-butyrolactone receptor protein and a TetR-family repressor, respectively. The precise AtrA-p-binding sites upstream of these two targets were determined, which allowed the identification of a relatively conserved binding motif comprising two 5-nt inverted repeats separated by a variable 5-nt sequence (5′-GGAAT-n5-ATTCC-3′) possibly required for the regulation of AtrA-like regulators in Streptomyces. Base substitutions of the AtrA-p-binding sites on the genome caused similar decreases in spbR and papR5 transcription as those observed in ∆atrA-p. Taken together, herein, a novel mechanism for AtrA-dependent regulation of antibiotic biosynthesis was revealed in S. pristinaespiralis, which is distinct from those of its homologs, AtrA-c from Streptomyces coelicolor, AtrA-g from Streptomyces griseus, and AtrA from Streptomyces roseosporus that perform their effects in antibiotic biosynthesis directly via pathway-specific activator genes or the biosynthetic structural genes.

Improvement of pristinamycin I (PI) production in Streptomyces pristinaespiralis by metabolic engineering approaches

Pristinamycin, produced by Streptomyces pristinaespiralis, which is a streptogramin-like antibiotic consisting of two chemically unrelated components: pristinamycin I (PI) and pristinamycin II (PII), shows potent activity against many antibiotic-resistant pathogens. However, so far pristinamycin production titers are still quite low, particularly those of PI. In this study, we constructed a PI single component producing strain by deleting the PII biosynthetic genes (snaE1 and snaE2). Then, two metabolic engineering approaches, including deletion of the repressor gene papR3 and chromosomal integration of an extra copy of the PI biosynthetic gene cluster (BGC), were employed to improve PI production. The final engineered strain ΔPIIΔpapR3/PI produced a maximum PI level of 132 mg/L, with an approximately 2.4-fold higher than that of the parental strain S. pristinaespiralis HCCB10218. Considering that the PI biosynthetic genes are clustered in two main regions in the 210 kb “supercluster” containing the PI and PII biosynthetic genes as well as a cryptic polyketide BGC, these two regions were cloned separately and then were successfully assembled into the PI BGC by the transformation-associated recombination (TAR) system. Collectively, the metabolic engineering approaches employed is very efficient for strain improvement in order to enhance PI titer.

The complete genome sequence of a high pristinamycin-producing strain Streptomyces pristinaespiralis HCCB10218.

Streptomyces pristinaespiralis produces the streptogramin-like antibiotic pristinamycin, which is a mixture of two structurally different components: pristinamycin I (PI) and pristinamycin II (PII). Herein, we report the complete genome sequence of a high pristinamycin-producing strain HCCB10218 (8.5 Mb) obtained by using PacBio RSII combined with Illumina HiSeq 2500 sequencing system. The genome sequence presented here provides clues for the mechanism underlying the higher pristinamycin production of HCCB10218.

CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces

Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression. Herein, the authors developed a novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor. In this system, the integrative plasmid pSET152 is used as the backbone for the expression of the dCas9/sgRNA complex and both dCas9 and sgRNAs are designed to be under the control of constitutive promoters. Using the integrative CRISPRi system, the authors achieved efficient repression of multiple genes simultaneously; the mRNA levels of four targets are reduced to 2-32% of the control. Furthermore, it is successfully employed for functional gene screening, and an orphan response regulator (RR) (encoded by SCO2013) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Collectively, this integrative CRISPRi system is very effective for multiplex gene repression in S. coelicolor, which could be extended to other Streptomyces strains for functional gene screening as well as for metabolic engineering.

CRISPR-Cpf1 assisted multiplex genome editing and transcriptional repression in Streptomyces

Rapid, efficient genetic engineering of Streptomyces strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency Streptomyces genome editing tool is established based on the FnCRISPR-Cpf1 system, which is an attractive and powerful alternative to the S. pyogenes CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, FnCpf1 enables the creation of gene(s) deletion with high efficiency. Furthermore, a ddCpf1-based integrative CRISPRi platform is established for simple, multiplex transcriptional repression. Of importance, FnCpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial Streptomyces strains (e.g., S. hygroscopicus SIPI-KF) that cannot utilize the SpCRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in Streptomyces as well as other actinomycetes. ABSTRACT Streptomyces has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the Streptococcus pyogenes CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces, it has a number of limitations, including the toxicity of SpCas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci. To address these problems, in this study, we developed a high-efficiency CRISPR-Cpf1 system (from Francisella novicida) for multiplex genome editing and transcriptional repression in Streptomyces. Using an all-in-one editing plasmid with homology-directed repair (HDR), our CRISPR-Cpf1 system precisely deletes single or double genes at efficiencies of 75 to 95% in Streptomyces coelicolor. When no templates for HDR are present, random-sized DNA deletions are achieved by FnCpf1-induced double-strand break (DSB) repair by a reconstituted nonhomologous end joining (NHEJ) pathway. Furthermore, a DNase-deactivated Cpf1 (ddCpf1)-based integrative CRISPRi system is developed for robust, multiplex gene repression using a single customized crRNA array. Finally, we demonstrate that FnCpf1 and SpCas9 exhibit different suitability in tested industrial Streptomyces species and show that FnCpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain Streptomyces hygroscopicus SIPI-KF, in which SpCas9 does not work well. Collectively, FnCpf1 is a powerful and indispensable addition to the Streptomyces CRISPR toolbox. IMPORTANCE Rapid, efficient genetic engineering of Streptomyces strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency Streptomyces genome editing tool is established based on the FnCRISPR-Cpf1 system, which is an attractive and powerful alternative to the S. pyogenes CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, FnCpf1 enables the creation of gene(s) deletion with high efficiency. Furthermore, a ddCpf1-based integrative CRISPRi platform is established for simple, multiplex transcriptional repression. Of importance, FnCpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial Streptomyces strains (e.g., S. hygroscopicus SIPI-KF) that cannot utilize the SpCRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in Streptomyces as well as other actinomycetes.

Roles of two-component system AfsQ1/Q2 in regulating biosynthesis of the yellow-pigmented coelimycin P2 in Streptomyces coelicolor

We previously demonstrated that in Streptomyces coelicolor two-component system AfsQ1/Q2 activates the production of the yellow-colored coelimycin P2 (also named as yCPK) on glutamate-supplemented minimal medium, and the response regulator AfsQ1 could specifically bind to the intergenic region between two structural genes, cpkA and cpkD Here, a more in-depth investigation was performed to elucidate the mechanism underlying the role of AfsQ1/Q2 in regulating coelimycin P2 biosynthesis. Deletion of afsQ1/Q2 resulted in markedly decreased expression of the whole coelimycin P2 biosynthetic gene cluster. Electrophoretic mobility shift assays revealed that AfsQ1 bound only to the target site identified previously, but not to any other promoters in the gene cluster. Mutations of AfsQ1-binding motif only resulted in drastically reduced transcription of the cpkA/B/C operon (encoding three type I polyketide synthases) and intriguingly, led to enhanced expression of some coelimcyin P2 genes, particularly accA1 and scF These results suggested the direct role of AfsQ1/Q2 in regulating coelimycin production, which is directly mediated by the structural genes, but not the cluster-situated regulatory genes, and also implied that other unknown mechanisms may be involved in AfsQ1/Q2-mediated regulation of coelimycin P2 biosynthesis.