Avermectin Production Research Articles

Potential of Streptomyces avermitilis: A Review on Avermectin Production and Its Biocidal Effect.

Secondary metabolites produced by the fermentation of Streptomyces avermitilis bacterium are powerful antiparasitic agents used in animal health, agriculture and human infection treatments. Avermectin is a macrocyclic lactone with four structural components (A1, A2, B1, B2), each of them containing a major and a minor subcomponent, out of which avermectin B1a is the most effective parasitic control compound. Avermectin B1a produces two homologue avermectins (B1 and B2) that have been used in agriculture as pesticides and antiparasitic agents, since 1985. It has a great affinity with the Cl-channels of the glutamate receptor, allowing the constant flow of Cl- ions into the nerve cells, causing a phenomenon of hyperpolarization causing death by flaccid paralysis. The purpose of this work was to gather information on the production of avermectins and their biocidal effects, with special emphasis on their role in the control of pests and phytopathogenic diseases. The literature showed that S. avermitilis is an important producer of macrocyclic lactones with biocidal properties. In addition, avermectin contributes to the control of ectoparasites and endoparasites in human health care, veterinary medicine and agriculture. Importantly, avermectin is a compound that is harmless to the host (no side effects), non-target organisms and the environment.

Influence of Cluster-Situated Regulator PteF in Filipin Biosynthetic Cluster on Avermectin Biosynthesis in Streptomyces avermitilis.

Crosstalk regulation is widespread in Streptomyces species. Elucidating the influence of a specific regulator on target biosynthetic gene clusters (BGCs) and cell metabolism is crucial for strain improvement through regulatory protein engineering. PteF and PteR are two regulators that control the biosynthesis of filipin, which competes for building blocks with avermectins in Streptomyces avermitilis. However, little is known about the effects of PteF and PteR on avermectin biosynthesis. In this study, we investigated their impact on avermectin biosynthesis and global cell metabolism. The deletion of pteF resulted in a 55.49% avermectin titer improvement, which was 23.08% higher than that observed from pteR deletion, suggesting that PteF plays a more significant role in regulating avermectin biosynthesis, while PteF hardly influences the transcription level of genes in avermectin and other polyketide BGCs. Transcriptome data revealed that PteF exhibited a global regulatory effect. Avermectin production enhancement could be attributed to the repression of the tricarboxylic acid cycle and fatty acid biosynthetic pathway, as well as the enhancement of pathways supplying acyl-CoA precursors. These findings provide new insights into the role of PteF on avermectin biosynthesis and cell metabolism, offering important clues for designing and building efficient metabolic pathways to develop high-yield avermectin-producing strains.

Characterization of a pleiotropic regulator MtrA in Streptomyces avermitilis controlling avermectin production and morphological differentiation

BackgroundThe macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated.ResultsIn this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis.ConclusionsIn this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.

Streptomyces global regulators AfsR and AfsS interact to co-regulate antibiotic production and morphological development.

Streptomyces species have a complex life cycle and are the producers of ~70% of commercial antibiotics. Global regulators AfsR and AfsS are widespread among Streptomyces and have been identified as key activators of antibiotic production in several species. However, their roles as repressors of antibiotic production are unclear; in particular, nothing is known regarding the regulatory mechanism of AfsS, despite many decades of research, because it has no DNA-binding domain. Here, we demonstrate that AfsR and AfsS negatively regulate avermectin production and morphological development in the industrially important species S. avermitilis. AfsR directly represses ave structural genes (aveA1, aveA4), cluster-situated activator gene aveR, and eight key developmental genes, whereas it directly activates afsS, aco (for autoregulator avenolide biosynthesis), and avaR1 (encoding avenolide receptor). GST pull-down, microscale thermophoresis, co-immunoprecipitation, and chromatin immunoprecipitation-quantitative PCR assays demonstrated that AfsS interacts with AfsR to co-regulate target genes involved in avermectin production and development and that this interaction requires intact AfsS repeated sequences and enhances the binding affinity of AfsR to target promoters. AfsR/AfsS interaction also occurs in model species S. coelicolor and S. roseosporus (producer of daptomycin, a cyclic lipopeptide antibiotic widely used for the treatment of human infections), suggesting that such interaction is conserved in Streptomyces species. The master developmental repressor BldD acts as a direct activator of both afsR and afsS. Deletion of afsR or afsS strongly enhances avermectin production in wild-type and industrial S. avermitilis strains. Our findings demonstrate novel regulatory roles and mechanisms of AfsR and AfsS in Streptomyces and facilitate methods for antibiotic overproduction.

Mycelial differentiation linked avermectin production in Streptomyces avermitilis studied with Raman imaging

Streptomyces avermitilis is a gram-positive bacterium that undergoes complex physiological and morphological differentiation during its life cycle, which has implications in secondary metabolite production. Avermectin, produced by S. avermitilis, is widely used as an anthelmintic and insecticidal agent. In this study, we have applied Raman microspectroscopic imaging to elucidate the correlation between production of avermectin and the morphological differentiation in S. avermitilis. We demonstrate distinctive variations in the localization of secondary metabolites at various stages of morphological differentiation. Under solid culture, avermectin was detected in the mycelia formed at the later stages of morphological differentiation (e.g., spore-bearing mycelium and spiral spore chains), but not in the early-stage substrate mycelium. On the contrary, under liquid culture condition, avermectin was found concentrated in the mycelial pellet formed at the early MII stage of differentiation. Furthermore, the chemical profiles of the mycelia were substantially different depending on the culture condition. Raman spectra corresponding to proteins, lipids, and cytochrome were observed in the mycelia irrespective of the stage of morphological differentiation, however, carotenoid was observed under solid culture condition particularly in spore-bearing mycelium and spiral spore chains. KEY POINTS: • Avermectin production is regulated during mycelial differentiation • Liquid and solid culture conditions affects mycelial differentiation • Raman microspectroscopic analysis reveals localization profiles of avermectin.

SspH, a Novel HATPase Family Regulator, Controls Antibiotic Biosynthesis in Streptomyces.

Streptomyces can produce a wealth of pharmaceutically valuable antibiotics and other bioactive compounds. Production of most antibiotics is generally low due to the rigorously controlled regulatory networks, in which global/pleiotropic and cluster-situated regulatory proteins coordinate with various intra- and extracellular signals. Thus, mining new antibiotic regulatory proteins, particularly the ones that are widespread, is essential for understanding the regulation of antibiotic biosynthesis. Here, in the biopesticide milbemycin producing strain Streptomyces bingchenggensis, a novel global/pleiotropic regulatory protein, SspH, a single domain protein containing only the HATPase domain, was identified as being involved in controlling antibiotic biosynthesis. The sspH overexpression inhibited milbemycin production by repressing the expression of milbemycin biosynthetic genes. The sspH overexpression also differentially influenced the expression of various antibiotic biosynthetic core genes. Site-directed mutagenesis revealed that the HATPase domain was essential for SspH’s function, and mutation of the conserved amino acid residues N54A and D84A led to the loss of SspH function. Moreover, cross-overexpression experiments showed that SspH and its orthologs, SCO1241 from Streptomyces coelicolor and SAVERM_07097 from Streptomyces avermitilis, shared identical functionality, and all exerted a positive effect on actinorhodin production but a negative effect on avermectin production, indicating that SspH-mediated differential control of antibiotic biosynthesis may be widespread in Streptomyces. This study extended our understanding of the regulatory network of antibiotic biosynthesis and provided effective targets for future antibiotic discovery and overproduction.

Redox-sensitive transcriptional regulator SoxR directly controls antibiotic production, development and thiol-oxidative stress response in Streptomyces avermitilis.

SummaryThe redox‐sensitive transcriptional regulator SoxR is conserved in bacteria. Its role in mediating protective response to various oxidative stresses in Escherichia coli and related enteric bacteria has been well established. However, functions and regulatory mechanisms of SoxR in filamentous Streptomyces, which produce half of known antibiotics, are unclear. We report here that SoxR pleiotropically regulates antibiotic production, morphological development, primary metabolism and thiol‐oxidative stress response in industrially important species Streptomyces avermitilis. SoxR stimulated avermectin production by directly activating ave structural genes. Four genes (sav_3956, sav_4018, sav_5665 and sav_7218) that are homologous to targets of S. coelicolor SoxR are targeted by S. avermitilis SoxR. A consensus 18‐nt SoxR‐binding site, 5′‐VSYCNVVMHNKVKDGMGB‐3′, was identified in promoter regions of sav_3956, sav_4018, sav_5665, sav_7218 and target ave genes, leading to prediction of the SoxR regulon and confirmation of 11 new targets involved in development (ftsH), oligomycin A biosynthesis (olmRI), primary metabolism (metB, sav_1623, plcA, nirB, thiG, ndh2), transport (smoE) and regulatory function (sig57, sav_7278). SoxR also directly activated three key developmental genes (amfC, whiB and ftsZ) and promoted resistance of S. avermitilis to thiol‐oxidative stress through activation of target trx and msh genes. Overexpression of soxR notably enhanced antibiotic production in S. avermitilis and S. coelicolor. Our findings expand our limited knowledge of SoxR and will facilitate improvement of methods for antibiotic overproduction in Streptomyces species.

BldD, a master developmental repressor, activates antibiotic production in two Streptomyces species.

BldD generally functions as a repressor controlling morphological development of Streptomyces. In this work, evidences that BldD also activates antibiotic production are provided. In Streptomyces roseosporus (which produces daptomycin widely used for treatment of human infections), deletion of bldD notably reduced daptomycin production, but enhanced sporulation. BldD stimulated daptomycin production by directly activating transcription of dpt structural genes and dptR3 (which encodes an indirect activator of daptomycin production), and repressed its own gene. BldD-binding sites on promoter regions of dptE, dptR3, and bldD were all found to contain BldD box-like sequences, facilitating prediction of new BldD targets. Two Streptomyces global regulatory genes, adpA and afsR, were confirmed to be directly activated by BldD. The protein AfsR was shown to act as an activator of daptomycin production, but a repressor of development. BldD directly represses nine key developmental genes. In Streptomyces avermitilis (which produces effective anthelmintic agents avermectins), BldD homolog (BldDsav) directly activates avermectin production through ave structural genes and cluster-situated activator gene aveR. This is the first report that BldD activates antibiotic biosynthesis both directly and via a cascade mechanism. BldD homologs are widely distributed among Streptomyces, our findings suggest that BldD may activate antibiotic production in other Streptomyces species.

AveI, an AtrA homolog of Streptomyces avermitilis, controls avermectin and oligomycin production, melanogenesis, and morphological differentiation.

Streptomyces avermitilis is well known as the producer of anthelmintic agent avermectins, which are widely used in agriculture, veterinary medicine, and human medicine. aveI encodes a TetR-family regulator, which is the homolog of AtrA. It was reported that deletion of aveI caused enhanced avermectin production. In this study, we investigated the regulatory function of the AveI in S. avermitilis. By binding to the 15-nt palindromic sequence in the promoter regions, AveI directly regulates at least 35 genes. AveI represses avermectin production by directly regulating the transcription of the cluster-situated regulator gene aveR and structural genes aveA1, aveA3, and aveD. AveI represses oligomycin production by repressing the CSR gene olmRII and structural genes olmC. AveI activates melanin biosynthesis by activating the expression of melC1C2 operon. AveI activates morphological differentiation by activating the expression of ssgR and ssgD genes, repressing the expression of wblI gene. Besides, AveI regulates many genes involved in primary metabolism, including substrates transport, the metabolism of amino acids, lipids, and carbohydrates. Therefore, AveI functions as a global regulator in S. avermitilis, controls not only secondary metabolism and morphological differentiation, but also primary metabolism.

Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis.

The bacterium Streptomyces avermitilis is an industrial-scale producer of avermectins, which are important anthelmintic agents widely used in agriculture, veterinary medicine, and human medicine. During the avermectin fermentation process, S. avermitilis is exposed to organic peroxides generated by aerobic respiration. We investigated the role of MarR-family transcriptional regulator OhrR in oxidative stress response and avermectin production in S. avermitilis. The S. avermitilis genome encodes two organic hydroperoxide resistance proteins: OhrB1 and OhrB2. OhrB2 is the major resistance protein in organic peroxide stress responses. In the absence of organic peroxide, the reduced form of OhrR represses the expression of ohrB2 gene by binding to the OhrR box in the promoter region. In the presence of organic peroxide, the oxidized form of OhrR dissociates from the OhrR box and the expression of ohrB2 is increased by derepression. OhrR also acts as a repressor to regulate its own expression. An ohrR-deletion mutant (termed DohrR) displayed enhanced avermectin production. Our findings demonstrate that OhrR in S. avermitilis represses avermectin production by regulating the expression of pathway-specific regulatory gene aveR. OhrR also plays a regulatory role in glycolysis and the pentose phosphate (PP) pathway by negatively controlling the expression of pykA2 and ctaB/tkt2-tal2-zwf2-opcA2-pgl.

SAV4189, a MarR-Family Regulator in Streptomyces avermitilis, Activates Avermectin Biosynthesis.

The bacterial species Streptomyces avermitilis is an important industrial producer of avermectins, which are widely utilized as effective anthelmintic and insecticidal drugs. We used gene deletion, complementation, and overexpression experiments to identify SAV4189, a MarR-family transcriptional regulator (MFR) in this species, as an activator of avermectin biosynthesis. SAV4189 indirectly stimulated avermectin production by altering expression of cluster-situated activator gene aveR, and directly repressed the transcription of its own gene (sav_4189) and adjacent cotranscribed gene sav_4190 (which encodes an unknown transmembrane efflux protein). A consensus 13-bp palindromic sequence, 5′-TTGCCYKHRSCAA-3′ (Y = T/C; K = T/G; H = A/C/T; R = A/G; S = C/G), was found within the SAV4189-binding sites of its own promoter region, and shown to be essential for binding. The SAV4189 regulon was thus predicted based on bioinformatic analysis. Night new identified SAV4189 targets are involved in transcriptional regulation, primary metabolism, secondary metabolism, and stress response, reflecting a pleiotropic role of SAV4189. sav_4190, the important target gene of SAV4189, exerted a negative effect on avermectin production. sav_4189 overexpression and sav_4190 deletion in S. avermitilis wild-type and industrial strains significantly increased avermectin production. SAV4189 homologs are widespread in other Streptomyces species. sav_4189 overexpression in the model species S. coelicolor also enhanced antibiotic production. The strategy of increasing yield of important antibiotics by engineering of SAV4189 homologs and target gene may potentially be extended to other industrial Streptomyces species. In addition, SAV4189 bound and responded to exogenous antibiotics hygromycin B and thiostrepton to modulate its DNA-binding activity and transcription of target genes. SAV4189 is the first reported exogenous antibiotic receptor among Streptomyces MFRs.

Butenolides from Streptomyces albus J1074 Act as External Signals To Stimulate Avermectin Production in Streptomyces avermitilis.

In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as Streptomyces hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type Streptomyces hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that Streptomyces albus strain J1074 may produce butenolide-type Streptomyces hormones. Here, we describe metabolite profiling of a disruptant of the S. albusaco gene, which encodes a key biosynthetic enzyme for butenolide-type Streptomyces hormones, and identify four butenolide compounds from S. albus J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for in vivo metabolic profiling demonstrated that the butenolide compounds of S. albus J1074 stimulate avermectin production in another Streptomyces species, Streptomyces avermitilis, illustrating the complex chemical interactions through interspecies signals in streptomycetes.IMPORTANCE Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, Streptomyces hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of Streptomyces secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type Streptomyces hormones. Moreover, the diffusible butenolide compounds from a donor Streptomyces strain were recognized by the recipient Streptomyces strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via Streptomyces hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits.

AvaR1, a Butenolide-Type Autoregulator Receptor in Streptomyces avermitilis, Directly Represses Avenolide and Avermectin Biosynthesis and Multiple Physiological Responses.

Avermectins are commercially important anthelmintic antibiotics produced by Streptomyces avermitilis. The homologous TetR-family transcriptional regulators AvaR1 and AvaR2 in this species were identified previously as receptors of avenolide, a novel butenolide-type autoregulator signal required for triggering avermectin biosynthesis. AvaR2 was found to be an important pleiotropic regulator in repression of avermectin and avenolide production and cell growth, whereas the regulatory role of AvaR1 remains unclear. Investigation of AvaR1 function in the present study showed that it had no effect on cell growth or morphological differentiation, but inhibited avenolide and avermectin production mainly through direct repression of aco (the key enzyme gene for avenolide biosynthesis) and aveR (the cluster-situated activator gene). AvaR1 also directly repressed its own gene (avaR1) and two adjacent homologous genes (avaR2 and avaR3). Binding sites of AvaR1 on these five target promoter regions completely overlapped those of AvaR2, leading to the same consensus binding motif. However, AvaR1 and AvaR2 had both common and exclusive target genes, indicating that they cross-regulate diverse physiological processes. Ten novel identified AvaR1 targets are involved in primary metabolism, stress responses, ribosomal protein synthesis, and cyclic nucleotide degration, reflecting a pleiotropic role of AvaR1. Competitive EMSAs and GST pull-down assays showed that AvaR1 and AvaR2 competed for the same binding regions, and could form a heterodimer and homodimers, suggesting that AvaR1 and AvaR2 compete and cooperate to regulate their common target genes. These findings provide a more comprehensive picture of the cellular responses mediated by AvaR1 and AvaR2 regulatory networks in S. avermitilis.

Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting.

Avermectins, produced by Streptomyces avermitilis, are important antiparasitic agents. The use of traditional microbial breeding methods for this organism has been limited by the low-throughput shake flask-based screening process. The unique growth cycle of actinomycetes makes the establishment of a reliable high-throughput screening (HTS) process difficult. To enhance the efficiency of screening strains with high yields of avermectin, a HTS process aided by fluorescence-activated cell sorting (FACS) was established. Four different spore solutions were investigated for maintaining a relatively high viability of spores. Propidium iodide (PI) and fluorescein diacetate (FDA) were used to discriminate between dead and live spores using the FACS system. Spores stained with 7-μg/mL PI and 15-μg/mL FDA at 4°C in the dark for 30min resulted in optimum sorting. Spores were treated by atmospheric and room temperature plasma (ARTP). Single live spores were sorted and sprayed into 96-well microtiter plates containing 50μL of solid agar culture medium. Solid-liquid combinatorial microculture was used for high-throughput avermectin culture. A high-titer avermectin producer (G9) was obtained from 5760 mutants after mutagenesis and HTS. Compared with the original strain, the titer was improved by 18.9% on flask culture and 20.6% on fermenter, respectively. The HTS process established in this study could easily be transferred to other similar target products produced by actinomycetes.

AVERMECTIN PRODUCTION BY SOLID STATE FERMENTATION-A NOVEL APPROACH

Objective: The present study was carried out to evaluate SSF process for the production of avermectin by Streptomyces avermitilis NRRL 8165 using easily available grains, millets and some agricultural by-product.Methods: Various substrates were screened for their ability to support avermectin production. Different parameters to maximize the yield of avermectin by S. avermitilis NRRL 8165 under SSF were optimized by conventional one factor at a time approach and parameters optimized earlier were adopted for the subsequent study.Results: Sorghum seeds used as solid substrate supported maximum growth and total avermectin production (4.6 mg g-1 dry substrate). The optimum values for maximum avermectin production were: moistening medium containing g l-1 KH2PO4 1; MgSO4.7H2O 0.4, inoculum size 20 % (24 h old culture in yeast extract-malt extract dextrose medium) v/w of initial dry substrate, substrate particle size 0.5 to 4 mm, incubation temperature 28 °C, initial moisture level 105%, incubation period of 15 d, 8 % w/w sucrose and 5% w/w soyameal. The avermectin yield with optimized fermentation condition was 5.8 mg g-1 dry substrate which is 1.3 fold higher as compared to non-optimized condition.Conclusion: Avermectin produced by S. avermitilis are widely used as an anthelmintic agent in the medical, veterinary and agricultural applications. In comparison with submerged fermentation, SSF can become an alternative cost-effective method for the production of avermectin. This report demonstrates the feasibility of employing agro-based substrate, that could reduce antibiotics production cost.

Activation of cryptic phthoxazolin A production in Streptomyces avermitilis by the disruption of autoregulator-receptor homologue AvaR3

The genomes of actinomycetes encode many cryptic novel/useful bioactive compounds, but access to these cryptic secondary metabolites remains limited. Streptomyces avermitilis predominantly produces three polyketide antibiotics (avermectin, filipin, and oligomycin) but has the potential to produce more secondary metabolites based on the number of cryptic biosynthetic gene clusters. Here, we extensively investigated the metabolite profiles of a gene disruptant of AvaR3 (an autoregulator receptor homologue), which is involved in the pleiotropic regulation of antibiotic production and cell morphology. Unlike the wild-type strain, the avaR3 mutant accumulated compound 3 in the culture. The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as phthoxazolin A, a cellulose synthesis inhibitor. Bioassays demonstrated that compound 3 exerts growth inhibitory activity against a broad range of plant pathogenic oomycetes. Moreover, unlike avermectin production, phthoxazolin A (3) production was negatively controlled by avenolide, a new type of autoregulator in streptomycetes, through the function of AvaR3. These results suggest that the genetic manipulation of autoregulator receptor homologues would be a valuable tool for the discovery of cryptic bioactive compounds.

An Alternative σ Factor, σ8, Controls Avermectin Production and Multiple Stress Responses in Streptomyces avermitilis.

Alternative σ factors in bacteria redirect RNA polymerase to recognize alternative promoters, thereby facilitating coordinated gene expression necessary for adaptive responses. The gene sig8 (sav_741) in Streptomyces avermitilis encodes an alternative σ factor, σ8, highly homologous to σB in Streptomyces coelicolor. Studies reported here demonstrate that σ8 is an important regulator of both avermectin production and stress responses in S. avermitilis. σ8 inhibited avermectin production by indirectly repressing expression of cluster-situated activator gene aveR, and by directly initiating transcription of its downstream gene sav_742, which encodes a direct repressor of ave structural genes. σ8 had no effect on cell growth or morphological differentiation under normal growth conditions. Growth of a sig8-deletion mutant was less than that of wild-type strain on YMS plates following treatment with heat, H2O2, diamide, NaCl, or KCl. sig8 transcription was strongly induced by these environmental stresses, indicating response by σ8 itself. A series of σ8-dependent genes responsive to heat, oxidative and osmotic stress were identified by EMSAs, qRT-PCR and in vitro transcription experiments. These findings indicate that σ8 plays an important role in mediating protective responses to various stress conditions by activating transcription of its target genes. Six σ8-binding promoter sequences were determined and consensus binding sequence BGVNVH-N15-GSNNHH (B: C, T or G, V: A, C or G, S: C or G, H: A, C or T, N: any nucleotide) was identified, leading to prediction of the σ8 regulon. The list consists of 940 putative σ8 target genes, assignable to 17 functional groups, suggesting the wide range of cellular functions controlled by σ8 in S. avermitilis.

Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis

The regulatory role of redox-sensing regulator Rex was investigated in Streptomyces avermitilis. Eleven genes/operons were demonstrated to be directly regulated by Rex; these genes/operons are involved in aerobic metabolism, morphological differentiation, and secondary metabolism. Rex represses transcription of target genes/operons by binding to Rex operator (ROP) sequences in the promoter regions. NADH reduces DNA-binding activity of Rex to target promoters, while NAD+ competitively binds to Rex and modulates its DNA-binding activity. Rex plays an essential regulatory role in aerobic metabolism by controlling expression of the respiratory genes atpIBEFHAGDC, cydA1B1CD, nuoA1-N1, rex-hemAC1DB, hppA, and ndh2. Rex also regulates morphological differentiation by repressing expression of wblE, which encodes a putative WhiB-family transcriptional regulator. A rex-deletion mutant (Drex) showed higher avermectin production than the wild-type strain ATCC31267, and was more tolerant of oxygen limitation conditions in regard to avermectin production.

SAV742, a Novel AraC-Family Regulator from Streptomyces avermitilis, Controls Avermectin Biosynthesis, Cell Growth and Development.

Avermectins are useful anthelmintic antibiotics produced by Streptomyces avermitilis. We demonstrated that a novel AraC-family transcriptional regulator in this species, SAV742, is a global regulator that negatively controls avermectin biosynthesis and cell growth, but positively controls morphological differentiation. Deletion of its gene, sav_742, increased avermectin production and dry cell weight, but caused delayed formation of aerial hyphae and spores. SAV742 directly inhibited avermectin production by repressing transcription of ave structural genes, and also directly regulated its own gene (sav_742) and adjacent gene sig8 (sav_741). The precise SAV742-binding site on its own promoter region was determined by DNase I footprinting assay coupled with site-directed DNA mutagenesis, and 5-nt inverted repeats (GCCGA-n10/n12-TCGGC) were found to be essential for SAV742 binding. Similar 5-nt inverted repeats separated by 3, 10 or 15 nt were found in the promoter regions of target ave genes and sig8. The SAV742 regulon was predicted based on bioinformatic analysis. Twenty-six new SAV742 targets were identified and experimentally confirmed, including genes involved in primary metabolism, secondary metabolism and development. Our findings indicate that SAV742 plays crucial roles in not only avermectin biosynthesis but also coordination of complex physiological processes in S. avermitilis.

Enhanced production of avermectin by deletion of type III polyketide synthases biosynthetic cluster rpp in Streptomyces avermitilis.

Because of the wide use of avermectins, many efforts have been made to improve the productivity via conventional genetic engineering technique. However, due to the lack of the molecular and genetic basis underlying such a yield enhancement after iterative rounds of mutagenesis and selection, it is often difficult to improve the titre in overproducers. Here, we report knocking out rpp biosynthetic gene cluster improved the production of an industrial overproducer by 24%. This work enriched the strategy to improve the production of antibiotics in industrial strains and may help further understanding of the interaction between type III polyketide synthases and other types.