Secondary Metabolism In Streptomyces Research Articles

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with “S134-H170-D198” catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min−1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme’s activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E.Key points• A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139.• Orf2 was involved in peptide metabolism both in vitro and in vivo.• Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

The roles of SARP family regulators involved in secondary metabolism in Streptomyces.

Streptomyces species are best known for their ability to produce abundant secondary metabolites with versatile bioactivities and industrial importance. These metabolites are usually biosynthesized through metabolic pathways encoded by cluster-situated genes. These genes are also known as biosynthetic gene clusters (BGCs) of secondary metabolites. The expression of BGCs is intricately controlled by pyramidal transcriptional regulatory cascades, which include various regulators. Streptomyces antibiotic regulatory proteins (SARPs), a genus-specific family of regulators, are widely distributed and play important roles in regulating the biosynthesis of secondary metabolites in Streptomyces. Over the past decade, the biological functions of SARPs have been extensively investigated. Here, we summarized the recent advances in characterizing the roles of SARPs involved in Streptomyces secondary metabolism from the following three aspects. First, the classification and domain organization of SARPs were summarized according to their size variation. Second, we presented a detailed description of the regulatory mechanisms and modes of action of SARPs involved in secondary metabolism. Finally, the biotechnological application of SARPs was illustrated by improving the production of target secondary metabolites and discovering novel bioactive natural products. This review will help researchers to comprehensively understand the roles of SARPs in secondary metabolite biosynthesis in Streptomyces, which will contribute to building a solid foundation for their future application in synthetic biology.

An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

Impact of Nitrogen-Containing Compounds on Secondary Metabolism in Streptomyces spp.—A Source of Metabolic Engineering Strategies

Actinobacteria from the genus Streptomyces feature complex primary and secondary metabolism, developmental cycle, and ability to produce a variety of natural products. These soil bacteria are major producers of antibiotics and other bioactive compounds and have been extensively investigated due to the medical and industrial relevance of Streptomyces-derived secondary metabolites. However, the genetic toolbox for Streptomyces engineering as well as yield optimization strategies for the production of relevant metabolites are limited. On the one hand, the genetic potential of these organisms has not been fully utilized due to many “silent” or poorly expressed biosynthetic gene clusters, whose activation depends on environmental stimuli and nutrient availability. On the other hand, these GC-rich Gram-positive bacteria are difficult to manipulate, and traditional genetic manipulation strategies are time-consuming and have low efficiency. Recent studies of Streptomyces metabolism and genomes provided new insights into possibilities to overcome these challenges. In this review, advances and approaches for Streptomyces manipulations and secondary metabolite production optimization are discussed. Special focus is given to understanding the interplay between primary and secondary metabolism in Streptomyces and the supply of nitrogen-containing compounds into secondary metabolism. Existing strategies to manipulate cellular metabolism in Streptomyces are reviewed.

Multi-integrated approach for unraveling small open reading frames potentially associated with secondary metabolism in Streptomyces.

Small open reading frames (smORFs) are widely distributed in various living organisms. However, their functions remain largely unexplored. In addition, annotation and detection of smORFs are limited using existing methods and hindered by their specific properties. In this study, we systematically investigated smORFs and smORF-encoded peptides (SEPs) in Streptomyces, which are well-known bacterial producers of diverse bioactive secondary metabolites. We established a peptidogenomic workflow based on multi-integrated comprehensive database search and database-independent de novo sequencing to identify smORFs in Streptomyces xinghaiensis NRRL B-24674T (S187). In addition, we described SEPome related to the secondary metabolism, which include 68 novel SEPs and 79 common smORFs with Streptomyces coelicolor A3 (2). Functional analysis of universal smORFs revealed enrichment in biosynthetic processes, stress response, ribosomes, and nucleic acid binding. Meanwhile, 5 Cryptic smORF-encoded Peptides (CSEPs) distributed in non-annotated regions of the genome, and non-coding RNAs could encode for CSEPs. A total of 66 new RNAs, including 32 non-coding RNAs (ncRNAs) were revealed, and 4 ncRNA-encoded peptides were identified. Furthermore, an investigation of carbon metabolism showed that NagE functions in spore formation and secondary metabolism in Streptomyces. Particularly, NagE was observed to function in the biosynthesis of anti-complement agents in S. xinghaiensis, suggesting a novel role of the phosphoenolpyruvate phosphotransferase system in microbial secondary metabolism. We thus provide an effective strategy for analyzing public data sets of model strains to identify smORFs for non-model species. The ncRNAs and SEPs present rich sources for engineering streptomycetes to produce bioactive compounds. IMPORTANCE Due to their small size and special chemical features, small open reading frame (smORF)-encoding peptides (SEPs) are often neglected. However, they may play critical roles in regulating gene expression, enzyme activity, and metabolite production. Studies on bacterial microproteins have mainly focused on pathogenic bacteria, which are importance to systematically investigate SEPs in streptomycetes and are rich sources of bioactive secondary metabolites. Our study is the first to perform a global identification of smORFs in streptomycetes. We established a peptidogenomic workflow for non-model microbial strains and identified multiple novel smORFs that are potentially linked to secondary metabolism in streptomycetes. Our multi-integrated approach in this study is meaningful to improve the quality and quantity of the detected smORFs. Ultimately, the workflow we established could be extended to other organisms and would benefit the genome mining of microproteins with critical functions for regulation and engineering useful microorganisms.

Co-Expression of Transcriptional Regulators and Housekeeping Genes in Streptomyces spp.: A Strategy to Optimize Metabolite Production.

The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance. Streptomyces spp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites in Streptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains of Streptomyces spp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistant Streptomyces strains (mutations known to enhance secondary metabolism in Streptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains' metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulate Streptomyces metabolism and explore their wide secondary metabolites production potential.

Effect of PAS-LuxR Family Regulators on the Secondary Metabolism of Streptomyces.

With the development of sequencing technology and further scientific research, an increasing number of biosynthetic gene clusters associated with secondary Streptomyces metabolites have been identified and characterized. The encoded genes of a family of regulators designated as PAS-LuxR are gradually being discovered in some biosynthetic gene clusters of polyene macrolide, aminoglycoside, and amino acid analogues. PAS-LuxR family regulators affect secondary Streptomyces metabolites by interacting with other family regulators to regulate the transcription of the target genes in the gene cluster. This paper provides a review of the structure, function, regulatory mechanism, and application of these regulators to provide more information on the regulation of secondary metabolite biosynthesis in Streptomyces, and promote the application of PAS-LuxR family regulators in industrial breeding and other directions.

AdpA, a developmental regulator, promotes\xa0\u03b5-poly-l-lysine biosynthesis in Streptomycesalbulus

BackgroundAdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-l-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.ResultsIn this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomycesalbulus NK660 by heterologously expressing adpA from S.neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S.albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S.albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S.albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF–MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S.albulus.ConclusionsHere, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

Evaluation of copper uptake in individual spores of Streptomyces coelicolor and endogenic nanoparticles formation to modulate the secondary metabolism.

Copper modulates secondary metabolism in Streptomyces. Although the cytosolic copper concentration is controlled by several chaperones and transporters, the formation of copper nanoparticles (NPs) and its relation to the antibiotic production has never been established in the model Streptomyces coelicolor. In this work, state-of-the-art analytical tools are used to evaluate the incorporation of copper in individual spores of S. coelicolor at different exposure concentrations (40, 80, and 160 μM Cu). Among them, the use of single cell-inductively coupled plasma-mass spectrometry revealed incorporation levels in the range of 2 to 2.5 fg/spore (median) increasing up to 4.75 fg/spore at the upper exposure concentrations. The copper storage within the spores in the form of NPs was evaluated using a combination of single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The obtained data confirmed the presence of NPs in the range of 8 to 40 (mean size 21 nm) inside S. coelicolor spores. The presence of the NPs was correlated with the actinorhodin production in liquid non-sporulating cultures amended with up to 80 μM Cu. However, further increase to 160 μM Cu, yielded to a significant decrease in antibiotic production. Secondary metabolism is activated under stressful conditions and cytosolic copper seems to be one of the signals triggering antibiotic production. Particularly, NP formation might contribute to modulate the secondary metabolism and prevent for copper toxicity. This work describes, for first time, the formation of endogenous copper NPs in S. coelicolor and reveals their correlation with the secondary metabolism.

System-Level Analysis of Transcriptional and Translational Regulatory Elements in Streptomyces griseus.

Bacteria belonging to Streptomyces have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in Streptomyces, system-level information remains limited. In this study, we integrated four multi-omics datasets from Streptomyces griseus NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3′-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5′-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in Streptomyces.

Activation of Secondary Metabolism in Red Soil-Derived Streptomycetes via Co-Culture with Mycolic Acid-Containing Bacteria.

Our previous research has demonstrated a promising capacity of streptomycetes isolated from red soils to produce novel secondary metabolites, most of which, however, remain to be explored. Co-culturing with mycolic acid-containing bacteria (MACB) has been used successfully in activating the secondary metabolism in Streptomyces. Here, we co-cultured 44 strains of red soil-derived streptomycetes with four MACB of different species in a pairwise manner and analyzed the secondary metabolites. The results revealed that each of the MACB strains induced changes in the metabolite profiles of 35–40 streptomycetes tested, of which 12–14 streptomycetes produced “new” metabolites that were not detected in the pure cultures. Moreover, some of the co-cultures showed additional or enhanced antimicrobial activity compared to the pure cultures, indicating that co-culture may activate the production of bioactive compounds. From the co-culture-induced metabolites, we identified 49 putative new compounds. Taking the co-culture of Streptomyces sp. FXJ1.264 and Mycobacterium sp. HX09-1 as a case, we further explored the underlying mechanism of co-culture activation and found that it most likely relied on direct physical contact between the two living bacteria. Overall, our results verify co-culture with MACB as an effective approach to discover novel natural products from red soil-derived streptomycetes.

Genetically engineered rpsL merodiploidy impacts secondary metabolism and antibiotic resistance in Streptomyces.

Certain point mutations within gene for ribosomal protein S12, rpsL, are known to dramatically change physiological traits of bacteria, most prominently antibiotic resistance and production of various metabolites. The rpsL mutants are usually searched among spontaneous mutants resistant to aminoglycoside antibiotics, such as streptomycin or paromomycin. The shortcomings of traditional selection are as follows: random rpsL mutants may carry undesired genome alterations; many rpsL mutations cannot be isolated because they are either not associated with increased antibiotic resistance or non-viable in the absence of intact rpsLWT gene. Introduction of mutant rpsL alleles in the rpsLWT background can be used to circumvent these obstacles. Here we take the latter approach and report the generation and properties of a set of stable rpsL merodiploids for Streptomyces albus J1074. We identified several rpsL alleles that enhance endogenous and heterologous antibiotic production by this strain and show that rpsLWTrpsLK88E merodiploid displays increased streptomycin resistance. We further tested several promising rpsL alleles in two more strains, Streptomyces cyanogenus S136 and Streptomyces ghanaensis ATCC14672. In S136, plasmid-borne rpsLK88E+P91S and rpsLK88R led to elevated landomycin production; no changes were detected for ATCC14672 merodiploids. Our data outline the prospects for and limitations to rpsL merodiploids as a tool for rapid enhancement of secondary metabolism in Streptomyces.

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

SummaryButenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

Growth, Differentiation and Secondary Metabolism in Streptomyces

Growth, Differentiation and Secondary Metabolism in Streptomyces

Critical review of fermentation and extraction of anti-Vibrio compounds from Streptomyces

A single Streptomyces strain often have the potential to produce more than one bioactive compound. Fermentation parameters include media compositions, temperature and pH, have great impact on the secondary metabolism of Streptomyces and subsequently on production of different microbial products. This review aims to consolidate the studies on the cultivation parameters used to enhance the production of secondary metabolite with anti-Vibrio activity from a single Streptomyces strain. In turn, this review sheds light on the possible alterations of the cultivation parameters to obtain desired anti-Vibrio compounds from Streptomyces sp. Furthermore, the bioactive compounds with anti-Vibrio activity identified from Streptomyces sp. were demonstrated to exhibit immense values for future antibacterial agent developments.

Identification of a secondary metabolism-responsive promoter by proteomics for over-production of natamycin in Streptomyces.

Streptomyces is currently the main producer of microbial pharmaceuticals from its secondary metabolites as natural products. It will be more beneficial if the promoters, which are particularly strong during the secondary metabolism of Streptomyces, are used to drive the efficient production of desired natural products with the coordination of bacterial growth. Here, in an industrial natamycin producer Streptomyces chattanoogensis L10, a strong promoter groESp was identified for this purpose based on the comparative proteomic analysis of the primary and secondary metabolism. With a constitutive promoter ermEp* as a control, the activity of groESp was weak in the primary metabolism, but about sixfold higher than ermEp* in the secondary metabolism, when the representative antibiotic natamycin was highly produced. Furthermore, when ScnRII, a pathway-specific positive regulator in natamycin biosynthesis, was expressed under groESp, the productivity of natamycin was about 20% higher in the secondary metabolism than that from ermEp*, but had no discrimination in the early 2 days. Thus, we showed that proteomics is an effective alternative way to identify promoters for the high yield of natamycin in S. chattanoogensis, and this strategy can be widely adaptable to other Streptomyces species for the full development of secondary metabolites with promising bioactivities.

Dual regulation between the two-component system PhoRP and AdpA regulates antibiotic production in Streptomyces.

Antibiotic production during secondary metabolism in Streptomyces spp. is elaborately controlled by multiple environmental signals and intracellular cascades. These include the two-component system PhoRP responding to phosphate starvation and a conserved signaling pathway mediated by the pleiotropic regulator AdpA. However, little information exists about how these two pathways work together for secondary metabolite production of Streptomyces. Herein, we report the dual regulation from the phosphate starvation-responsive regulator PhoP and AdpA on atrA promoter (atrAp) for the production of daptomycin, an antibiotic produced by Streptomyces roseosporus. We found that PhoP directly binds to atrAp, positively regulates atrA expression and thus daptomycin production. We also observed positive auto-regulation of phoRP expression during fermentation for daptomycin production. Moreover, partial overlap between PhoP- and AdpA-binding sites on atrAp was observed, which results in partial competitive binding between these two regulators. This partial overlapping and competition between PhoP and AdpA was further confirmed by mutations and binding assays. In summary, our findings have revealed dual regulation of PhoP and AdpA on the same promoter for antibiotic production in Streptomyces. This mechanism would be beneficial to further environment-responsive fermentation optimization for antibiotic production.

A Novel AdpA Homologue Negatively Regulates Morphological Differentiation in Streptomyces xiamenensis 318.

The pleiotropic transcriptional regulator AdpA positively controls morphological differentiation and regulates secondary metabolism in most Streptomyces species. Streptomyces xiamenensis 318 has a linear chromosome 5.96 Mb in size. How AdpA affects secondary metabolism and morphological differentiation in such a naturally minimized genomic background is unknown. Here, we demonstrated that AdpA Sx , an AdpA orthologue in S. xiamenensis, negatively regulates cell growth and sporulation and bidirectionally regulates the biosynthesis of xiamenmycin and polycyclic tetramate macrolactams (PTMs) in S. xiamenensis 318. Overexpression of the adpASx gene in S. xiamenensis 318 had negative effects on morphological differentiation and resulted in reduced transcription of putative ssgA, ftsZ, ftsH, amfC, whiB, wblA1, wblA2, wblE, and a gene encoding sporulation-associated protein (sxim_29740), whereas the transcription of putative bldD and bldA genes was upregulated. Overexpression of adpASx led to significantly enhanced production of xiamenmycin but had detrimental effects on the production of PTMs. As expected, the transcriptional level of the xim gene cluster was upregulated, whereas the PTM gene cluster was downregulated. Moreover, AdpA Sx negatively regulated the transcription of its own gene. Electrophoretic mobility shift assays revealed that AdpA Sx can bind the promoter regions of structural genes of both the xim and PTM gene clusters as well as to the promoter regions of genes potentially involved in the cell growth and differentiation of S. xiamenensis 318. We report that an AdpA homologue has negative effects on morphological differentiation in S. xiamenensis 318, a finding confirmed when AdpA Sx was introduced into the heterologous host Streptomyces lividans TK24.IMPORTANCE AdpA is a key regulator of secondary metabolism and morphological differentiation in Streptomyces species. However, AdpA had not been reported to negatively regulate morphological differentiation. Here, we characterized the regulatory role of AdpA Sx in Streptomyces xiamenensis 318, which has a naturally streamlined genome. In this strain, AdpA Sx negatively regulated cell growth and morphological differentiation by directly controlling genes associated with these functions. AdpA Sx also bidirectionally controlled the biosynthesis of xiamenmycin and PTMs by directly regulating their gene clusters rather than through other regulators. Our findings provide additional evidence for the versatility of AdpA in regulating morphological differentiation and secondary metabolism in Streptomyces.

Transcriptome-Based Identification of a Strong Promoter for Hyper-production of Natamycin in Streptomyces.

Streptomyces are famed producers of secondary metabolites with diverse bioactivities and structures. However, biosynthesis of natural products will consume vast precursors from primary metabolism, and some secondary metabolites are toxic to the hosts. To overcome this circumstance and over-produce secondary metabolites, one of the strategies is to over-express biosynthetic genes under strong promoters specifically expressed during secondary metabolism. For this purpose, here based on Microarray and eGFP reporter assays, we obtained a promoter thlM4p, whose activity was undetectable in the first 2 days of fermentation, but sevenfold higher than the strong promoter ermE*p in the following days. Moreover, when the positive regulator gene scnRII was driven from thlM4p, natamycin yield increased 30% compared to ermE*p. Therefore, we provide a new way to identify promoters, which is silenced during primary metabolism while strongly expressed under secondary metabolism of Streptomyces.

Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces

Pirins are evolutionarily conserved iron-containing proteins that are found in all kingdoms of life, and have been implicated in diverse molecular processes, mostly associated with cellular stress. In the present study, we started from the evidence that the insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by ΦC31 Att/Int system-based vectors in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 resulted in marked effects on central carbon and energy metabolism gene expression, high sensitivity to oxidative injury and repression of polyketide antibiotic production. By using integrated transcriptomic, proteomic and metabolite profiling, together with genetic complementation, we here show that most of these effects could be traced to the inability of the pirA-defective strain to modulate beta-oxidation pathway, leading to an unbalanced supply of precursor monomers for polyketide biosynthesis. Indeed, in silico protein-protein interaction modeling and in vitro experimental validation allowed us to demonstrate that PirA is a novel redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the first committed step of the beta-oxidation pathway.