A Stimulus-Responsive Ternary Heterojunction Boosting Oxidative Stress, Cuproptosis for Melanoma Therapy.

Cuproptosis, a recently discovered copper-dependent cell death, presents significant potential for the development of copper-based nanoparticles to induce cuproptosis in cancer therapy. Herein, a unique ternary heterojunction, denoted as HACT, composed of core-shell Au@Cu2O nanocubes with surface-deposited Titanium Dioxide quantum dots and modified with hyaluronic acid is introduced. Compared to core-shell AC NCs, the TiO2/Au@Cu2O exhibits improved energy structure optimization, successfully separating electron-hole pairs for redox use. This optimization results in a more rapid generation of singlet oxygen and hydroxyl radicals triggering oxidative stress under ultrasound radiation. Furthermore, the HACT NCs initiate cuproptosis by Fenton-like reaction and acidic environment, leading to the sequential release of cupric and cuprous ions. This accumulation of copper induces the aggregation of lipoylated proteins and reduces iron-sulfur proteins, ultimately initiating cuproptosis. More importantly, HACT NCs show a tendency to selectively target cancer cells, thereby granting them a degree of biosecurity. This report introduces a ternary heterojunction capable of triggering both cuproptosis and oxidative stress-related combination therapy in a stimulus-responsive manner. It can energize efforts to develop effective melanoma treatment strategies using Cu-based nanoparticles through rational design.

Ternary heterostructure-driven photoinduced electron-hole separation enhanced oxidative stress for triple-negative breast cancer therapy.

Zinc oxide nanoparticles (ZnO NPs) stand as among the most significant metal oxide nanoparticles in trigger the formation of reactive oxygen species (ROS) and induce apoptosis. Nevertheless, the utilization of ZnO NPs has been limited by the shallowness of short-wavelength light and the constrained production of ROS. To overcome these limitations, a strategy involves achieving a red shift towards the near-infrared (NIR) light spectrum, promoting the separation and restraining the recombination of electron-hole (e--h+) pairs. Herein, the hybrid plasmonic system Au@ZnO (AZ) with graphene quantum dots (GQDs) doping (AZG) nano heterostructures is rationally designed for optimal NIR-driven cancer treatment. Significantly, a multifold increase in ROS generation can be achieved through the following creative initiatives: (i) plasmonic Au nanorods expands the photocatalytic capabilities of AZG into the NIR domain, offering a foundation for NIR-induced ROS generation for clinical utilization; (ii) elaborate design of mesoporous core-shell AZ structures facilitates the redistribution of electron-hole pairs; (iii) the incorporation GQDs in mesoporous structure could efficiently restrain the recombination of the e--h+ pairs; (iv) Modification of hyaluronic acid (HA) can enhance CD44 receptor mediated targeted triple-negative breast cancer (TNBC). In addition, the introduced Au NRs present as catalysts for enhancing photothermal therapy (PTT), effectively inducing apoptosis in tumor cells. The resulting HA-modified AZG (AZGH) exhibits efficient hot electron injection and e--h+ separation, affording unparalleled convenience for ROS production and enabling NIR-induced PDT for the cancer treanment. As a result, our well-designed mesoporous core-shell AZGH hybrid as photosensitizers can exhibit excellent PDT efficacy.

A Chiral Nanocomplex for Multitarget Therapy to Alleviate Neuropathology and Rescue Alzheimer's Cognitive Deficits.

Alzheimer's disease (AD) is a severe neurodegenerative condition characterized by inflammation, beta-amyloid (Aß) plaques, and neurodegeneration, which currently lack effective treatments. Chiral nanomaterials have emerged as a promising option for treating neurodegenerative disorders due to their high biocompatibility, strong sustained release ability, and specific enantiomer selectivity. The development of a stimulus-responsive chiral nanomaterial, UiO-66-NH2 @l-MoS2 QDs@PA-Ni (MSP-U), for the treatment of AD is reported. MSP-U is found to stimulate neural stem cell (NSCs) differentiation, promote in situ hydrogen (H2 ) production, and clear Aß plaques. l-MoS2 QDs modified with l-Cysteine (l-Cys) effectively enhance the differentiation of NSCs into neurons through circularly polarized near-infrared radiation. Doped-phytic acid nickel (PA-Ni) improves the activity of l-MoS2 QDs in scavenging reactive oxygen species at the lesion site via photocatalytic H2 production. Loading l-MoS2 QDs with UiO-66 type metal oxide suppresses electron-hole recombination effect, thereby achieving rapid charge separation and improving transport of photogenerated electrons, leading to significantly improved H2 production efficiency. The photothermal effect of MSP-U also clears the generated Aß plaques. In vivo evaluations show that MSP-U improves spatial cognition and memory, suggesting a promising potential candidate for the treatment of AD using chiral nanomaterials.

New insights into the dihydro-mureidomycin biosynthesis controlled by two unusual proteins in Streptomyces roseosporus.

BACKGROUND: Uridyl peptide compounds are renowned as a subclass of nucleoside antibiotics for their highly specific antibacterial activity against Gram-negative bacteria and the unique target of action. We previously activated the biosynthetic gene cluster of a uridyl peptide antibiotic, mureidomycin, in Streptomyces roseosporus NRRL 15998 by introducing an exogenous positive regulator gene ssaA, and the generated strain was designated as Sr-hA. This study aims to further explore mureidomycin analogs from Sr-hA as well as the collaborative roles of two wide-spread genes, SSGG-02980 and SSGG-03002 encoding putative nuclease/phosphatase and oxidoreductase respectively, in mureidomycin diversification. RESULTS: In order to understand how SSGG-02980 and SSGG-03002 contribute to mureidomycin biosynthesis, the gene disruption mutants and complementary strains were constructed. Mass spectrometry analyses revealed that two series of pairwise mureidomycin analogs were synthesized in Sr-hA with a two-dalton difference in molecular weight for each pair. By disruption of SSGG-03002, only mureidomycins with lower molecular weight (MRDs, 1-6) could be specifically accumulated in the mutant (∆03002-hA), whereas the other series of products with molecular weight plus 2 Da (rMRDs, 1'-6') became dominant in SSGG-02980 disruption mutant (∆02980-hA). Further comprehensive NMR analyses were performed to elucidate the structures, and three MRDs (3, 4, 5) with unsaturated double bond at C5-C6 of uracil group were characterized from ∆03002-hA. In contrast, the paired rMRDs analogs (3', 4', 5') from ∆SSGG-02980 corresponding to 3, 4 and 5 were shown to contain a single bond at this position. The results verified that SSGG-03002 participates in the reduction of uracil ring, whereas SSGG-02980 antagonizes the effect of SSGG-03002, which has been rarely recognized for a phosphatase. CONCLUSIONS: Overall, this study revealed the key roles of two wide-spread families of enzymes in Streptomyces. Of them, oxidoreductase, SSGG-03002, is involved in dihydro-mureidomycin biosynthesis of S. roseosporus, whereas nuclease/phosphatase, SSGG-02980, has an adverse effect on SSGG-03002. This kind of unusual regulation model between nuclease/phosphatase and oxidoreductase is unprecedented, providing new insights into the biosynthesis of mureidomycins in Streptomyces. The findings would be of significance for structural diversification of more uridyl peptide antibiotics against Gram-negative bacteria.

Transmission paths and source areas of near-surface ozone pollution in the Yangtze River delta region, China from 2015 to 2021.

Near-ground ozone in the Yangtze River Delta (YRD) region has become one of the main air pollutants that threaten the health of residents. However, to date, the transport behavior and source areas of ozone in the YRD region have not been systematically analyzed. In this study, by combining the ozone observational record with a HYSPLIT (hybrid single-particle Lagrangian integrated trajectory) model, we tried to reveal the spatiotemporal regularity of the airflow transport trajectory of ozone. Spatially, high ozone concentrations mainly clustered in industrial cities and resource-based cities. Temporally, the center of the ozone pollution shifted westward of Nanjing from 2015 to 2021. With the passage of time, the influence of meteorological elements on the ozone concentration in the YRD region gradually weakened. Marine atmosphere had the most significant impact on the transmission path of ozone in Shanghai, of which the trajectory frequency in 2021 accounted for 64.21% of the total frequency. The transmission trajectory of ozone in summer was different from that in other seasons, and its transmission trajectory was mainly composed of four medium-distance transmission paths: North China-Bohai Sea, East China Sea-West Pacific Ocean, Philippine Sea, and South China Sea-South China. The contribution source areas mainly shifted to the southeast, and the emission of pollutants from the Shandong Peninsula, the Korean Peninsula-Japan, and the Philippine Sea-Taiwan area increased the impact of ozone pollution in the Shanghai area from 2019 to 2021. This study identified the regional transport path of ozone in the YRD region and provided a scientific reference for the joint prevention and control of ozone pollution in this area.

A butenolide signaling system synergized with biosynthetic gene modules led to effective activation and enhancement of silent oviedomycin production in Streptomyces.

Secondary metabolic gene clusters widely exist in the genomes of Streptomyces but mostly remain silent. To awaken this hidden reservoir of natural products, various strategies concerning secondary metabolic pathways are applied. Here, we describe that butenolide signaling molecule deficiency and glucose addition can interdependently activate the expression of silent oviedomycin biosynthetic gene clusters in Streptomyces ansochromogenes and Streptomyces antibioticus. Since oviedomycin is a promising anti-tumor lead compound, in order to improve its yield, we use the cluster-situated genes (ovmF, ovmG, ovmI and ovmH) encoding the enzymes for acyl carrier protein modification and precursor biosynthesis, and the discrete precursor biosynthetic genes (pyk2, gap1 and accA2) involved in glycolysis to assemble two gene modules (pFGIH and pPGA). Their co-overexpression in ΔsabA (a disruption mutant of sabA encoding SAB synthase) has superimposed effect on the yield of oviedomycin, which can be further increased to 59-fold in the presence of galactose as optimal carbon source. This is the most unambiguous evidence that butenolide signaling system can synergize with the optimization of primary metabolism to regulate the expression of secondary metabolic gene clusters, providing efficient strategies for mining natural products of Streptomyces.

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants.

Identification of a butenolide signaling system that regulates nikkomycin biosynthesis in Streptomyces.

Butenolides are an emerging family of signaling molecules in Streptomyces. They control complex physiological traits, such as morphological differentiation and antibiotic production. However, how butenolides regulate these processes is poorly investigated because of obstacles in obtaining these signaling molecules. This study reports the identification of a butenolide-type signaling system for nikkomycin biosynthesis in Streptomyces ansochromogenes with distinct features. We identified a gene cluster, sab, consisting of three genes, sabAPD, for butenolide biosynthesis and two regulator genes, sabR1 and sabR2, and characterized three butenolides (SAB1, -2, and -3) by heterologous expression of sabAPD. sabA disruption abolished nikkomycin production, which could be restored by the addition of SABs or by deletion of sabR1 in ΔsabA. Electrophoretic mobility-shift assays and transcriptional analyses indicated that SabR1 indirectly represses the transcription of nikkomycin biosynthetic genes, but directly represses sabA and sabR1 In the presence of SABs, the SabR1 transcriptional regulator dissociated from its target genes, verifying that SabR1 is the cognate receptor of SABs. Genome-wide scanning with the conserved SabR1-binding sequence revealed another SabR1 target gene, cprC, whose transcription was strongly repressed by SabR1. Intriguingly, CprC positively regulated the pleiotropic regulatory gene adpA by binding to its promoter and, in turn, activated nikkomycin biosynthesis. This is the first report that butenolide-type signaling molecules and their cognate receptor SabR1 can regulate adpA via a newly identified activator, CprC, to control nikkomycin production. These findings pave the way for further studies seeking to unravel the regulatory mechanism and functions of the butenolide signaling system in Streptomyces.

Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576.

Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with PkasO*. Compared to the yield (1282 mg/L) of Streptomyces fradiae CGMCC 4.576, the engineered strain Sf/pKCZ03 had a 36% enhancement of neomycin production. Quantitative real-time PCR analysis revealed the increased transcription of structural genes (neoE, neoB, neoL, aacC8) and regulatory genes (neoR, neoH) in Sf/pKCZ03. Additionally, under the supplementation of 1 g/L N-acetyl-D-glucosamine and 5 g/L L-glutamine, the yield of engineered strain Sf/pKCZ03 showed 62% and 107% improvements compared to that of the wild-type strain in the original medium, respectively. These findings demonstrated that engineering the antibiotic gene cluster in combination with precursors feeding was an effective approach for strain improvement, and would be potentially extended to other Streptomyces for large-scale production of commercialized antibiotics.

Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes.

Genome sequencing analysis has revealed at least 35 clusters of likely biosynthetic genes for secondary metabolites in Streptomyces ansochromogenes. Disruption of adpA encoding a global regulator (AdpA) resulted in the failure of nikkomycin production, whereas other antibacterial activities against Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis were observed with the fermentation broth of ΔadpA but not with that of the wild-type strain. Transcriptional analysis showed that a cryptic gene cluster (pks7), which shows high identity with an oviedomycin biosynthetic gene cluster (ovm), was activated in ΔadpA. The corresponding product of pks7 was characterized as oviedomycin by MS and NMR spectroscopy. To understand the molecular mechanism of ovm activation, the roles of six regulatory genes situated in the ovm cluster were investigated. Among them, proteins encoded by co-transcribed genes ovmZ and ovmW are positive regulators of ovm AdpA directly represses the transcription of ovmZ and ovmW Co-overexpression of ovmZ and ovmW can relieve the repression of AdpA on ovm transcription and effectively activate oviedomycin biosynthesis. The promoter of ovmOI-ovmH is identified as the direct target of OvmZ and OvmW. This is the first report that AdpA can simultaneously activate nikkomycin biosynthesis but repress oviedomycin biosynthesis in one strain. Our findings provide an effective strategy that is able to activate cryptic secondary metabolite gene clusters by genetic manipulation of global regulatory genes.

Coordinative Modulation of Chlorothricin Biosynthesis by Binding of the Glycosylated Intermediates and End Product to a Responsive Regulator ChlF1.

Chlorothricin, isolated from Streptomyces antibioticus, is a parent member of spirotetronate family of antibiotics that have long been appreciated for their remarkable biological activities. ChlF1 plays bifunctional roles in chlorothricin biosynthesis by binding to its target genes (chlJ, chlF1, chlG, and chlK). The dissociation constants of ChlF1 to these genes are ∼ 102-140 nm. A consensus sequence, 5'-GTAANNATTTAC-3', was found in these binding sites. ChlF1 represses the transcription of chlF1, chlG, and chlK but activates chlJ, which encodes a key enzyme acyl-CoA carboxyl transferase involved in the chlorothricin biosynthesis. We demonstrate that the end product chlorothricin and likewise its biosynthetic intermediates (demethylsalicycloyl chlorothricin and deschloro-chlorothricin) can act as signaling molecules to modulate the binding of ChlF1 to its target genes. Intriguingly, a correlation between the antibacterial activity and binding ability of signaling molecules to the regulator ChlF1 is clearly observed. These features of the signaling molecules are associated with the glycosylation of spirotetronate macrolide aglycone. The findings provide new insights into the TetR family regulators responding to special structure of signaling molecules, and we reveal the regulatory mini-network mediated by ChlF1 in chlorothricin biosynthesis for the first time.

[Enhancement of salinomycin production and its activity optimization - A review].

Salinomycin is a monocarboxylic acid polyether antibiotics produced by Streptomyces albus. It has strong inhibiting and killing activity against most gram-positive bacteria and various coccidiums with low adverse impact on environment. In addition, salinomycin can specifically inhibit the growth of a variety of cancer cells and cancer stem cells via targeting to multiple sites, and is a promising anti-tumor drug candidate. To obtain high yield salinomycinproducing strain, conventional mutation techniques and modern molecular genetic methods have been used. Meanwhile, bioactivity and selectivity of salinomycin could be improved by modifying the chemical structure and changing drug delivery methods. Here, we summarize the key strategies for enhancing salinomycin production and review the progresses in optimizing its drug activity and targeting properties. The future research focus is also addressed.

[Functional characterization of gouC and gouD in gougerotin biosynthesis].

OBJECTIVE: To determine the functions of gouC and gouD in gougerotin biosynthesis, disruption of these two genes was performed. As gougerotin producing strain Streptomyces graminearus lacks efficient genetic manipulation system, the gene cluster for gougerotin biosynthesis was heterologously expressed in Streptomyces coelicolor M1146 to facilitate genetic manipulations of gouC and gouD. METHODS: By using fosmid D6-4H containing the complete gougerotin biosynthetic gene cluster, gouC and gouD were disrupted by PCR-targeting method to generate pGOUe-ΔC and pGOUe-ΔD. Both pGOUe-ΔC and pGOUe-ΔD were introduced into Streptomyces coelicolor M1146 by intergeneric conjugation, thus gouC and gouD disrpution mutants (Ml146-GOUe-AC and M1146-GOUe-ΔD) were obtained. The gougerotin production of M1146-GOUe-ΔC and M1146-GOUe-ΔD were assayed by HPLC analysis. The intermediates accumulated in these mutants were purified and subjected to MS and NMR analyses for structure determinations. Bioassay of these intermediates against tumor cell line were also carried out. RESULTS: Disruption mutants of gouC and gouD failed to produce gougerotin and the mutants accumulated different gougerotin intermediates, which lost their ability to inhibit cancer cell proliferation. CONCLUSION: gouC and gouD are key structual genes in the biosynthesis of gougerotin peptidyl moieties. This study will pave the way for the elucidation of gougerotin biosynthetic pathway.

A γ-butyrolactone-sensing activator/repressor, JadR3, controls a regulatory mini-network for jadomycin biosynthesis.

Two regulatory genes, jadR2 and jadR3, in the jadomycin (jad) biosynthetic gene cluster of Streptomyces venezuelae encode homologues of γ-butyrolactone receptor. JadR2 was previously shown to be a pseudo γ-butyrolactone receptor. jadR3 is situated at the upstream of jadW123 encoding putative enzymes for γ-butyrolactone biosynthesis. Disruption of jadR3 resulted in markedly decreased production of jadomycin. Transcriptional analysis revealed that JadR3 represses jadW1, jadR2 and jadR3 but activates jadR1, the key activator gene for jadomycin biosynthesis. DNase I footprinting showed that JadR3 has four binding sites in the intergenic regions of jadR2-jadR1 and jadR3-jadW1. A JadR3 interactive molecule, SVB1, was purified from a large-scale fermentation and its structure found to be the same as SCB3, a γ-butyrolactone from Streptomyces coelicolor, and was absent from a jadW123 mutant lacking jadomycin production. Addition of SVB1 or extract from S. coelicolor to the mutant restored jadomycin production. Overall, our results revealed that the association of JadR3 and SVB1 plays an important role in controlling a regulatory mini-network governing jadomycin biosynthesis, providing new insights into the ways in which γ-butyrolactone/receptor systems modulate antibiotic biosynthesis in Streptomyces.

Identification of novel tylosin analogues generated by a wblA disruption mutant of Streptomyces ansochromogenes.

BACKGROUND: Streptomyces, as the main source of antibiotics, has been intensively exploited for discovering new drug candidates to combat the evolving pathogens. Disruption of wblA, an actinobacteria-specific gene controlling major developmental transition, can cause the alteration of phenotype and morphology in many species of Streptomyces. One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool. It is interesting to identify whether novel secondary metabolites could be produced by the wblA disruption mutant as evidenced in other Streptomyces. RESULTS: The wblA disruption mutant of S. ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed to produce spores and nikkomycin, the major product of S. ansochromogenes 7100 (wild-type strain) during fermentation. Antibacterial activity against Staphylococcus aureus and Bacillus cereus was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. To identify the antibacterial compounds, the two compounds (compound 1 and compound 2) produced by ΔwblA were characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical structure of these compounds shows similarity with tylosin, and the bioassays indicated that the two compounds inhibited the growth of a number of gram-positive bacteria. It is intriguing that they displayed much higher activity than tylosin against Streptococcus pneumoniae. CONCLUSIONS: Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA. Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.

Activation and enhancement of Fredericamycin A production in deepsea-derived Streptomyces somaliensis SCSIO ZH66 by using ribosome engineering and response surface methodology.

BACKGROUND: Marine microorganisms are an important source of new drug leads. However, the discovery and sustainable production of these compounds are often hampered due to the unavailable expression of cryptic biosynthetic gene clusters or limited titer. Ribosome engineering and response surface methodology (RSM) integrated strategy was developed in this study to activate cryptic gene cluster in the deepsea-derived Streptomyces somaliensis SCSIO ZH66, and subsequently isolation, structural analysis, and the yield enhancement of the activated compound, anticancer drug lead Fredericamycin A (FDM A), were performed. RESULTS: In order to discover novel natural products from marine Streptomyces strains by genome mining strategy, the deepsea-derived S. somaliensis SCSIO ZH66 was subject to ribosome engineering to activate the expression of cryptic gene clusters. A resistant strain ZH66-RIF1 was thereby obtained with 300 µg/mL rifampicin, which accumulated a brown pigment with cytotoxicity on MS plate while absent in the wild type strain. After screening of fermentation conditions, the compound with pigment was purified and identified to be FDM A, indicating that the activation of a cryptic FDM A biosynthetic gene cluster was taken place in strain ZH66-RIF1, and then it was identified to be ascribed to the mutation of R444H in the ß subunit of RNA polymerase. To further improve the yield efficiently, nine fermentation medium components were examined for their significance on FDM A production by Plackett-Burman design and Box-Behnken design. The optimum medium composition was achieved by RSM strategy, under which the titer of FDM A reached 679.5 ± 15.8 mg/L after 7 days of fermentation, representing a 3-fold increase compared to the original medium. In terms of short fermentation time and low-cost fermentation medium, strain ZH66-RIF1 would be an ideal alternative source for FDM A production. CONCLUSIONS: Our results would hasten the efforts for further development of FDM A as a drug candidate. Moreover, this ribosome engineering and RSM integrated methodology is effective, fast and efficient; it would be applicable to genome mining for novel natural products from other strains.

Regulation of myo-inositol catabolism by a GntR-type repressor SCO6974 in Streptomyces coelicolor.

Myo-inositol is important for Streptomyces growth and morphological differentiation. Genomic sequence analysis revealed a myo-inositol catabolic gene cluster in Streptomyces coelicolor. Disruption of the corresponding genes in this cluster abolished the bacterial growth on myo-inositol as a single carbon source. The transcriptions of these genes were remarkably enhanced by addition of myo-inositol in minimal medium. A putative regulatory gene SCO6974, encoding a GntR family protein, is situated in the cluster. Disruption of SCO6974 significantly enhanced the transcription of myo-inositol catabolic genes. SCO6974 was shown to interact with the promoter regions of myo-inositol catabolic genes using electrophoretic mobility shift assays. DNase I footprinting assays demonstrated that SCO6974 recognized a conserved palindromic sequence (A/T)TGT(A/C)N(G/T)(G/T)ACA(A/T). Base substitution of the conserved sequence completely abolished the binding of SCO6974 to the targets demonstrating that SCO6974 directly represses the transcriptions of myo-inositol catabolic genes. Furthermore, the disruption of SCO6974 was correlated with a reduced sporulation of S. coelicolor in mannitol soya flour medium and with the overproduction of actinorhodin and calcium-dependent antibiotic. The addition of myo-inositol suppressed the sporulation deficiency of the mutant, indicating that the effect could be related to a shortage in myo-inositol due to its enhanced catabolism in this strain. This enhanced myo-inositol catabolism likely yields dihydroxyacetone phosphate and acetyl-CoA that are indirect or direct precursors of the overproduced antibiotics.

[Engineering and heterologous expression of a nikkomycin biosynthetic gene cluster].

OBJECTIVE: We expressed a nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146. METHODS: By using PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains nikkomycin biosynthetic gene cluster, with the hrdB promoter to generate pNIKm. We transferred pNIK and pNIKm into S. coelicolor M1146 by intergeneric conjugation and obtained M1146-NIK and M1146-NIKm, respectively. We then evaluated expression of the gene cluster in the heterologous host by RT-PCR. Furthermore, we also compared the antifugal activity and nikkomycin production of M1146-NIK and M1146-NIKm by bioassay against Alternaria longipes and HPLC analysis. RESULTS: M1146-NIK and M1146-NIKm exhibited antifungal activity, and they can produce a trace amount of nikkomycin X, nikkomycin Z and pseudo-Z. There was a substantial accumulation of uridine in M1146-NIK, whereas substantial accumulations of uridine, ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm. CONCLUSION: We successfully expressed the nikkomycin biosynthetic gene cluster in the heterologous host and identified nikkomycins and some of its key biosynthetic intermediates. This study will provide the basis for enzymatic reaction of the condensation between the two nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.

JadR*-mediated feed-forward regulation of cofactor supply in jadomycin biosynthesis.

Jadomycin production is under complex regulation in Streptomyces venezuelae. Here, another cluster-situated regulator, JadR*, was shown to negatively regulate jadomycin biosynthesis by binding to four upstream regions of jadY, jadR1, jadI and jadE in jad gene cluster respectively. The transcriptional levels of four target genes of JadR* increased significantly in ΔjadR*, confirming that these genes were directly repressed by JadR*. Jadomycin B (JdB) and its biosynthetic intermediates 2,3-dehydro-UWM6 (DHU), dehydrorabelomycin (DHR) and jadomycin A (JdA) modulated the DNA-binding activities of JadR* on the jadY promoter, with DHR giving the strongest dissociation effects. Direct interactions between JadR* and these ligands were further demonstrated by surface plasmon resonance, which showed that DHR has the highest affinity for JadR*. However, only DHU and DHR could induce the expression of jadY and jadR* in vivo. JadY is the FMN/FAD reductase supplying cofactors FMNH2/FADH2 for JadG, an oxygenase, that catalyses the conversion of DHR to JdA. Therefore, our results revealed that JadR* and early pathway intermediates, particularly DHR, regulate cofactor supply by a convincing case of a feed-forward mechanism. Such delicate regulation of expression of jadY could ensure a timely supply of cofactors FMNH2/FADH2 for jadomycin biosynthesis, and avoid unnecessary consumption of NAD(P)H.

GouR, a TetR family transcriptional regulator, coordinates the biosynthesis and export of gougerotin in Streptomyces graminearus.

Gougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities. gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed that gouR plays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of the gouL-to-gouB operon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions of gouL, gouM, and gouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export in Streptomyces graminearus.