Yinhua Lu

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.

afsQ1-Q2-sigQ is a pleiotropic but conditionally required signal transduction system for both secondary metabolism and morphological development in Streptomyces coelicolor

Two-component system AfsQ1-Q2 of Streptomyces coelicolor was identified previously for its ability to stimulate actinorhodin (ACT) and undecylprodigiosin (RED) production in Streptomyces lividans. However, disruption of either afsQ1 or afsQ2 in S. coelicolor led to no detectable changes in secondary metabolite formation or morphogenesis. In this study, we reported that, when cultivated on defined minimal medium (MM) with glutamate as the sole nitrogen source, the afsQ mutant exhibited significantly decreased ACT, RED, and calcium-dependent antibiotic (CDA) production and rapid growth of aerial mycelium. In addition, we also found that deletion of sigQ, which is located upstream of afsQ1-Q2 and encodes a putative sigma factor, led to the precocious hyperproduction of these antibiotics and delayed formation of sporulating aerial mycelium in the same glutamate-based defined MM. Reverse-transcription polymerase chain reaction and egfp fusion analyses showed that the expression of sigQ was under control by afsQ. In addition, deletion of both afsQ-sigQ resulted in the phenotype identical to that of afsQ mutant. The results suggested that afsQ1-Q2 and sigQ worked together in the regulation of both antibiotic biosynthesis and morphological development, and sigQ might be responsible for antagonizing the function of AfsQ1-Q2 in S. coelicolor, however, in a medium-dependent manner. Moreover, the study showed that the medium-dependent regulation of antibiotic biosynthesis by AfsQ1-Q2-SigQ was through pathway-specific activator genes actII-ORF4, redD, and cdaR. The study provides new insights on regulation of antibiotic biosynthesis and morphological development in S. coelicolor.

Identification of two-component system AfsQ1/Q2 regulon and its cross-regulation with GlnR in Streptomyces coelicolor

The two‐component system AfsQ1/Q2 of Streptomyces coelicolor was identified in our previous work as a pleiotropic regulator for antibiotic biosynthesis and morphological differentiation under the condition of a minimal medium supplemented with 75 mM glutamate. In this work, we report the dissection of the mechanism underlying the function of AfsQ1/Q2 on antibiotic production and also the identification of the AfsQ1/Q2 regulon. The results showed that AfsQ1/Q2 stimulated antibiotic ACT, RED and CDA production directly through the pathway‐specific activator genes actII‐ORF4, redZ and cdaR respectively. In addition, expression of sigQ that encodes a sigma factor and is divergently transcribed from afsQ1 was also subject to direct regulation by AfsQ1/Q2. The precise AfsQ1 binding sites in the upstream regions of these target genes were determined by DNase I footprinting assays coupled with site‐directed DNA mutagenesis. By computational prediction and functional analysis, at least 17 new AfsQ1 targets were identified, including pstS gene encoding a high‐affinity phosphate‐binding protein and two developmental genes whiD, bldM. For the AfsQ1/Q2 regulon, an AfsQ1 binding motif comprising the sequence GTnAC‐n6‐GTnAC has been defined. Interestingly, we found from electrophoretic mobility shift assays and transcriptional analysis that AfsQ1/Q2 can also function as a repressor for nitrogen assimilation, and AfsQ1 can compete with GlnR for the promoter regions of glnA and nirB, suggesting the cross‐regulation between AfsQ1/Q2 and GlnR in nitrogen metabolism. These findings suggested that AfsQ1/Q2 is important not only for antibiotic biosynthesis but also in maintaining the metabolic homeostasis of nutrient utilization under the stress of high concentration of glutamate in S. coelicolor.

Differential regulation of antibiotic biosynthesis by DraR‐K, a novel two‐component system in Streptomyces coelicolor

A novel two‐component system (TCS) designated as DraR‐K (sco3063/sco3062) was identified to be involved in differential regulation of antibiotic biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow‐pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR‐K activates ACT but represses yCPK production directly through the pathway‐specific activator genes actII‐ORF4 and kasO, respectively, while its role on RED biosynthesis was independent of pathway‐specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at −124 to −98 nt and −24 to −1 nt relative to the respective transcription start point of actII‐ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR‐binding sequence, 5′‐AMAAWYMAKCA‐3′ (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel‐retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR‐K in primary metabolism. DraR‐K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR‐Ksav (sav_3481/sav_3480, homologue of draR‐K) in the industrial model strain S. avermitilis NRRL‐8165 led to similar abnormal antibiotic biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR‐K‐mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of antibiotic biosynthesis in Streptomyces.

Characterization of a novel two-component regulatory system involved in the regulation of both actinorhodin and a type I polyketide in Streptomyces coelicolor

To seek more information on function of two-component regulatory systems (TCSs) in Streptomycescoelicolor, a dozen TCS-knockout mutants were generated, and phenotype changes were determined. One TCS (SCO5403/5404)-deleted mutant with phenotype change was obtained. Here, we report the characterization of this novel TCS, designated as RapA1/A2 (regulation of both actinorhodin and a type I polyketide), using genetic and proteomic approaches. Although growth and morphological analyses showed no difference between the knockout mutant and wild-type strain M145, a visible decrease of the production of actinorhodin (Act) was observed in rapA1/A2 mutant. The decrease can be restored by introducing rapA1/A2 genes on an integrative vector. A 2D-gel based proteomic analysis showed that knockout of rapA1/A2 resulted in reduced expression of a putative 3-oxoacyl-[acyl-carrier protein] reductase that is part of a biosynthetic cluster for a cryptic type I polyketide. Further reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed that expression levels of several biosynthetic genes and the respective pathway-specific regulatory genes actII-ORF4 and kasO for these two clusters were all down-regulated in the rapA1/A2 mutant, compared to M145. Taken together, the results demonstrated that RapA1/A2 may serve as a positive regulator for biosynthesis of both Act and the uncharacterized polyketide in S. coelicolor, and the effects exerted by RapA1/A2 were dependent on the pathway-specific regulatory genes.

Characterization of a negative regulator AveI for avermectin biosynthesis in Streptomyces avermitilis NRRL8165

A transcriptional activator for actinorhodin biosynthesis, AtrA, was previously characterized in Streptomyces coelicolor A3(2), and an orthologue of atrA, named aveI, is identified in the Streptomyces avermitilis NRRL8165 genome (Uguru et al., Mol Microbiol, 58:131–150, 2005). In this study, genetic and functional characterization of aveI gene was reported. Deletion of aveI gene led to increased biosynthesis of avermectin B1a by about 16-fold. The increased synthesis of avermectin B1a was suppressed by complementation with either aveI gene or its orthologue gene atrA from S. coelicolor, suggesting AveI and AtrA shared the similar functionality and were negative regulators for avermectin biosynthesis in S. avermitilis. However, when aveI was introduced into S. coelicolor on a multi-copy plasmid, the production of actinorhodin was significantly increased, indicating that aveI had a positive effect on actinorhodin biosynthesis in S. coelicolor, the same as its orthologue atrA. Electrophoretic mobility shift assays revealed AveI can bind specifically to the promoter region of actII-ORF4 in vitro but not that of aveR. Although its mechanism still needs to be defined, the species-differential regulation by the same regulator may represent an example of the evolutional strategy that enables bacteria to adapt the existing molecular machinery to a variety of functionalities for growth and survival.

A stepwise increase in pristinamycin II biosynthesis by Streptomyces pristinaespiralis through combinatorial metabolic engineering

Pristinamycin, which is a streptogramin antibiotic produced by Streptomyces pristinaespiralis, contains two chemically unrelated compounds, pristinamycin I (PI) and pristinamycin II (PII). Semi-synthetic derivatives of PI and PII have been approved for use in human medicine to treat a broad range of drug-resistant pathogens. In this study, we design and implement a combinatorial metabolic engineering strategy for improving PII production. First, an extra copy of the PII biosynthetic gene cluster, which was assembled using a modified Gibson assembly method for cloning large DNA fragments with high GC contents, was introduced into a high-producing strain S. pristinaespiralis HCCB10218. This duplication of the PII biosynthetic gene cluster resulted in a maximum increase in PII titer by 45%. Second, all seven cluster-situated regulatory genes (from papR1 to papR6 and spbR) were systematically manipulated. Higher PII titers were achieved by deleting either one of the two repressor genes papR3 or papR5 in combination with overexpression of both activator genes papR4 and papR6, and the resulting strains ∆papR3+R4R6 and ∆papR5+R4R6 showed maximum increases in PII production by 99% and 75%, respectively. A combination of the above two different approaches was employed. Integration of the assembled PII gene cluster (BAC-F1F15) into ∆papR5+R4R6 led to the highest PII titer improvement, which was approximately 1.5-fold higher than the parental strain. By adding the macroreticular resin, which can separate pristinamycin in situ and thereby lessen end-product feedback inhibition and toxic effects, PII titers of the final engineered strain ∆papR5+R4R6/BAC-F1F15 reached 1.13 and 1.16g/L in the Erlenmeyer flask and 5-L bioreactor, respectively, with 5.13- and 5.26-fold improvements over the parental strain. Taken together, this combinatorial strategy is an efficient method to optimize PII biosynthesis of S. pristinaespiralis and may be extended to other industrially used streptomycetes for strain improvement.

Cross-talk between an orphan response regulator and a noncognate histidine kinase in Streptomyces coelicolor

Two-component systems (TCSs), typically consisting of a histidine kinase (HK) and a cognate response regulator (RR), are the most common signaling systems in bacteria. Besides paired genes encoding TCSs, there also exists unpaired HKs and orphan RRs. In Streptomyces coelicolor, 13 orphan RRs have been annotated. Because of lack of cognate HKs, little is known as yet about the regulation of orphan RRs. Bioinformatic analysis revealed that several orphan RRs had high amino acid sequence identities with RRs from typical TCSs in S. coelicolor. Among them, the orphan RR SCO3818 and RR SCO0204, which paired with HK SCO0203, showed the highest identity (65%), suggesting that the two RRs might both be under the regulation of SCO0203. Following studies showed that SCO0203 could phosphorylate not only SCO0204 but also SCO3818. Deletion of either sco0203 or sco3818 led to enhanced production of blue-pigmented antibiotic actinorhodin, which indicated a functional correlation between SCO0203 and SCO3818. These results suggested that SCO3818 might be regulated by SCO0203. This is the first report describing the regulation of an orphan RR by an HK. Moreover, this is also the first identification of cross-talk between different TCS components in S. coelicolor.

An Orphan Histidine Kinase, OhkA, Regulates Both Secondary Metabolism and Morphological Differentiation in Streptomyces coelicolor

We report here the physiological and genetic characterization of an orphan histidine kinase (HK) (OhkA, SCO1596) in Streptomyces coelicolor and its homolog (OhkAsav, SAV_6741) in Streptomyces avermitilis. The physiological analysis showed that the ohkA mutant of S. coelicolor exhibits impaired aerial mycelium formation and sporulation and overproduction of multiple antibiotics on mannitol-soy flour (MS) medium, especially actinorhodin (ACT) and calcium-dependent antibiotic (CDA), and disruption of ohkAsav in S. avermitilis also led to the similar phenotypes of impaired morphological differentiation and significantly increased oligomycin A production. DNA microarray analysis combined with real-time reverse transcription-PCR (RT-PCR) and RNA dot blot assay in the S. coelicolor ohkA deletion mutant confirmed the physiological results by showing the upregulation of genes involved in the biosynthesis of ACT, CDA, undecylprodigiosin (RED), a yellow type I polyketide (CPK, SCO6273-6289), and a sesquiterpene antibiotic, albaflavenone (SCO5222-5223). The results also suggested that the increased production of ACT and RED in the mutant could be partly ascribed to the enhanced precursor malonyl coenzyme A (malonyl-CoA) supply through increased transcription of genes encoding acetyl-CoA carboxylase (ACCase). Interestingly, DNA microarray analysis also showed that deletion of ohkA greatly downregulated the transcription of chpABCDEFGH genes essential for aerial mycelium formation by S. coelicolor on MS medium but significantly increased transcription of ramS/C/R, which is responsible for SapB formation and regulation and is normally absent on MS medium. Moreover, many other genes involved in development, such as bldM/N, whiG/H/I, ssgA/B/E/G/R, and whiE, were also significantly downregulated upon ohkA deletion. The results clearly demonstrated that OhkA is an important global regulator for both morphological differentiation and secondary metabolism in S. coelicolor and S. avermitilis.

Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer, Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions

Background: Orphan response transcription factor GlnR regulates nitrogen metabolism in important actinomycetes. Results: GlnR has no typical “phosphorylation pocket,” where the only conserved Asp is unphosphorylated but is essential for functional homodimerization. Conclusion: Actinomycete GlnR is an atypical response regulator functioning as a homodimer. Significance: Conserved Asp-focused charge interactions of actinomycete GlnR are probably the mechanism that stabilizes the homodimer for physiological function. The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp50 residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called “phosphorylation pocket” is not conserved, with its space being occupied by an Arg52 from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp50 with the highly conserved Arg52 and Thr9 in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.