Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus.

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.

Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites ß- and γ-Rhodomycinone.

BACKGROUND: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. METHODS: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. RESULTS: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as ß- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. CONCLUSIONS: This is the first report about the production of ß- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography.

Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor.

BACKGROUND: Trypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate. RESULTS: The tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL-1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg-1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL-1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. CONCLUSIONS: SGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

Engineered Biosynthesis of 5/5/6 Type Polycyclic Tetramate Macrolactams in an Ikarugamycin (5/6/5 Type)-Producing Chassis.

Combinatorial biosynthesis of 5/5/6 type polycyclic tetramate macrolactams (PoTeMs) was achieved in an engineered ikarugamycin (5/6/5 type) producer by introducing a set of 5/5/6 type PoTeM biosynthetic genes from Streptomyces griseus. This study supports the idea that PoTeMs share a common polyene tetramate precursor generated by the hybrid polyketide synthase/nonribosomal peptide synthetase enzymes. The X-ray crystal structure of pactamide G (7) sets an example for resolving the absolute configuration of 5/5/6 type PoTeMs.

SrnR from Streptomyces griseus is a nickel-binding transcriptional activator.

Nickel ions are crucial components for the catalysis of biological reactions in prokaryotic organisms. As an uncontrolled nickel trafficking is toxic for living organisms, nickel-dependent bacteria have developed tightly regulated strategies to maintain the correct intracellular metal ion quota. These mechanisms require transcriptional regulator proteins that respond to nickel concentration, activating or repressing the expression of specific proteins related to Ni(II) metabolism. In Streptomyces griseus, a Gram-positive bacterium used for antibiotic production, SgSrnR and SgSrnQ regulate the nickel-dependent antagonistic expression of two superoxide dismutase (SOD) enzymes, a Ni-SOD and a FeZn-SOD. According to a previously proposed model, SgSrnR and SgSrnQ form a protein complex in which SgSrnR works as repressor, binding directly to the promoter of the gene coding for FeZn-SOD, while SgSrnQ is the Ni(II)-dependent co-repressor. The present work focuses on the determination of the biophysical and functional properties of SgSrnR. The protein was heterologously expressed and purified from Escherichia coli. The structural and metal-binding analysis, carried out by circular dichroism, light scattering, fluorescence and isothermal titration calorimetry, showed that the protein is a well-structured homodimer, able to bind nickel with moderate affinity. DNase I footprinting and ß-galactosidase gene reporter assays revealed that apo-SgSrnR is able to bind its DNA operator and activates a transcriptional response. The structural and functional properties of this protein are discussed relatively to its role as a Ni(II)-dependent sensor.

Chemical synthesis, microbial transformation and biological evaluation of tetrahydroprotoberberines as dopamine D1/D2 receptor ligands.

Dopamine D1/D2 receptors are important targets for drug discovery in the treatment of central nervous system diseases. To discover new and potential D1/D2 ligands, 17 derivatives of tetrahydroprotoberberine (THPB) with various substituents were prepared by chemical synthesis or microbial transformation using Streptomyces griseus ATCC 13273. Their functional activities on D1 and D2 receptors were determined by cAMP assay and calcium flux assay. Seven compounds showed high activity on D1/D2 receptor with low IC50 values less than 1 µM. Especially, top compound 5 showed strong antagonistic activity on both D1 and D2 receptor with an IC50 of 0.391 and 0.0757 µM, respectively. Five compounds displayed selective antagonistic activity on D1 and D2 receptor. The SAR studies revealed that (1) the hydroxyl group at C-9 position plays an important role in keeping a good activity and small or fewer substituents on ring D of THPBs may also stimulate their effects, (2) the absence of substituents at C-9 position tends to be more selective for D2 receptor, and (3) hydroxyl substitution at C-2 position and the substitution at C-9 position may facilitate the conversion of D1 receptor from antagonist to agonist. Molecular docking simulations found that Asp 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386 of D1/ D2 receptors are the key residues, which have strong interactions with the active D1/D2 compounds and may influence their functional profiles.

Dolyemycins A and B, two novel cyclopeptides isolated from Streptomyces griseus subsp. griseus HYS31.

Two novel cyclopeptides with special skeleton, namely, dolyemycins A (1) and B (2) were isolated from Streptomyces griseus subsp. griseus HYS31 by bio-guided isolation. Their structures were elucidated by detailed analysis of spectroscopic data. These two compounds were cyclopeptides containing eleven amino acids including five unusual amino acids (hydroxyglycine, 3-hydroxyleucine, 3-phenylserine, ß-hydroxy-O-methyltyrosine, 2,3-diaminobutyric acid) in both of them and an extra nonprotein amino acids (3-methylaspartic acid) in Dolyemycin B only. Dolyemycins A and B performed antiproliferative activity against human lung cancer A549 cells with IC50 values of 1.0 and 1.2 µM, respectively.

Grisemycin, a Bridged Angucyclinone with a Methylsulfinyl Moiety from a Marine-Derived Streptomyces sp.

Grisemycin (1), the first sulfur angucyclinone with an unusual ether-bridged system, was isolated from a marine-derived Streptomyces griseus strain M268. Its novel, here cage-like, structure was determined by spectroscopic analysis and single-crystal X-ray diffraction. Compound 1 exhibited modestly selective activity against the HL-60 cell line with an IC50 value of 31.54 µM. Futhermore, the absolute stereochemistry of kiamycin (2), an 1,12-epoxybenz[a]anthracene, previously obtained from the same strain, was established by X-ray diffraction analysis.

Marine-Derived Secondary Metabolite, Griseusrazin A, Suppresses Inflammation through Heme Oxygenase-1 Induction in Activated RAW264.7 Macrophages.

A new secondary metabolite, named griseusrazin A (1), was isolated from the marine-derived bacterium Streptomyces griseus subsp. griseus. The structure of the compound was determined by analysis of spectroscopic data including MS, COSY, HSQC, HMBC, and (15)N-HMBC data. Griseusrazin A (1) inhibited the production of inflammatory mediators, such as prostaglandin E2 and nitric oxide, which was mediated through the suppression of the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α, in the LPS-stimulated cells was also effectively blocked by griseusrazin A (1). Furthermore, this anti-inflammatory activity of 1 was linked to its inhibitory effects against the nuclear translocation of NF-κB p50 and p65, as wells as NF-κB binding activity. In the further study to elucidate the anti-inflammatory mechanism, 1 was shown to induce heme oxygenase-1 (HO-1) expression through the enhancement of nuclear translocation of nuclear factor E2-related factor 2. Furthermore, the anti-inflammatory activity of 1 in the LPS-stimulated cells was partially reversed by an HO inhibitor, tin protoporphyrin. These results indicate that the anti-inflammatory effect of 1 is associated with Nrf2-mediated HO-1 expression.

Biofiltration technology for the removal of toluene from polluted air using Streptomyces griseus.

Biofiltration technology has been recognized as a promising biotechnology for treating the volatile organic compounds (VOCs) present in polluted air. This study aims to investigate the performance of a biofiltration system of Streptomyces griseus sp. DSM-40759 immobilized on activated carbon (PICA S23) towards the adsorption and degradation of toluene vapour as well as to regenerate the activated carbon in situ. The batch studies were performed using nutrient agar medium and basal salt medium (BSM) for microbial growth. Initially the pre-cultures were incubated at a temperature of 28°C on a rotary shaker at 150 rpm. After two days, the strain S. griseus DSM-40759 was immobilized on a known weight of activated carbon (12 g). The results of biofilter performance showed three different stages with a quick adsorption phase with approximately 95% of toluene removal after 70 min, a slow biotransformation phase by immobilized cells. In the later, the removal efficiency decreased significantly with the extension of time and reached 60% during this stage. Moreover, a final quick removal phase by the immobilized cells had an average removal efficiency of toluene around 95% after 500 min. The toluene degradation was found to be more than 84% after the second cycle and the biofilter was still capable of removing additional toluene. Thus, the results demonstrated the feasibility and reusability of a new biofilter system for toluene removal as well as extending the activated carbon's capacity and this could be a potential solution to reuse the activated carbon in industrial application.

Dandamycin and chandrananimycin E, benzoxazines from Streptomyces griseus.

Two new benzoxazines were isolated from Streptomyces griseus (HKI 0545) and assigned as chandrananimycin E (1) and dandamycin (2). Although a number of phenoxazinone-type compounds have been reported from nature, phenoxazines are rarer, and carbon substitution at N-10 such as in 1 is unprecedented. The cyclopentene-containing ring structure of dandamycin (2) is also unique. Chandrananimycin E (1) was found to possess moderate antiproliferative activity against HUVEC cells (GI50 35.3 µM) and weak cytotoxic activity towards HeLa cells (CC50 56.9 µM). Dandamycin showed neither antiproliferative activity nor cytotoxicity towards these cell lines. Structure activity comparisons with phenoxazinones isolated from S. griseus HKI 0545 suggested that the alteration of the core ring systems in 1 and 2 diminishes their activity. Natural products 1 and 2 are interesting additions to the rich secondary metabolome of S. griseus and constitute an important addition to the body of knowledge on phenoxazinone-derived metabolites.

Evaluation of nystatin isolated from Streptomyces griseus SDX-4 against the ciliate, Ichthyophthirius multifiliis.

The present study was conducted to evaluate the in vitro and in vivo antiparasitic efficacy of active compounds from the bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on n-butanol extract of S. griseus yielding a pure bioactive compound, nystatin (Nys), identified by comparing spectral data (EI-MS, (1)H NMR, and (13)C NMR) with literature values. Results from in vitro antiparasitic assays revealed that Nys could be 100% effective against I. multifiliis theronts and encysted tomonts at the concentration of 6.0 mg L(-1), with the median effective concentration (EC50) values of 3.1 and 2.8 mg L(-1) for theronts and encysted tomonts (4 h), respectively. Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the gold fish treated with Nys was markedly lower than the control group at 10 days after exposed to theronts (p < 0.05). In the control group, 85.7% mortality was observed owing to heavy I. multifiliis infection at 10 days after the exposure. On the other hand, only 23.8% mortality owing to parasite infection was recorded in the groups treated with the Nys (4.0 and 6.0 mg L(-1)). In addition, our results showed that the survival and reproduction of I. multifiliis tomont exited from the fish were significantly reduced after treated with the 6.0 mg L(-1) Nys. The median lethal dose (LD50) of Nys for goldfish was 16.8 mg L(-1). This study firstly demonstrated that Nys has potent antiparasitic efficacy against I. multifiliis, and it can be a good candidate drug for chemotherapy and control of I. multifiliis infections.

Anti-parasitic activities of specific bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis.

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of bacterial extracellular products of Streptomyces griseus SDX-4 against I. multifiliis were determined. The fermentation liquor of S. griseus was extracted successively in a separating funnel with petroleum ether, ethyl acetate, and n-butanol. In vitro assays revealed that the n-butanol extracts (NBu-E) and ethyl acetate extracts (Eto-E) of S. griseus were observed to be more effective against theronts than the other extracts with median effective concentration (EC50) values of 0.86 and 12.5 mg L(-1), respectively, and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts (P<0.05). All encysted tomonts were killed when the concentration of NBu-E was 30.0 mg L(-1). Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the grass carp treated with NBu-E was markedly lower compared to the control group at 11 days after exposed to theronts (P<0.05). In the control group, 100% mortality was observed owing to heavy I. multifiliis infection at 11 days after the exposure. On the other hand, only 9.5% mortality owing to parasite infection was recorded in the groups treated with the NBu-E (30 mg L(-1)). The median lethal dose (LD50) of NBu-E for grass carp was 152.4 mg L(-1). Our results indicate that n-butanol extract of S. griseus will be useful in aquaculture for controlling I. multifiliis infections.

Improvement of catalytic efficiency and thermostability of recombinant Streptomyces griseus trypsin by introducing artificial peptide.

Streptomyces trypsin is one of the serine proteinases in Streptomyces griseus and acts as a key mediator during cell growth and differentiation. S. griseus trypsin (SGT) could be successfully expressed in Pichia pastoris by engineering the natural propeptide APNP. In this study, the recombinant Exmt with peptide YVEF and the wild-type SGT were comparatively investigated in detail. The recombinant Exmt showed significantly increased thermostability which t(½) value was 3.89-fold of that of the SGT at 40 °C. Moreover, the catalytic efficiency (referring to the specificity constant, k cat/K m) and pH tolerance of Exmt were also improved. In silico modeling analysis uncovered that introduction of the peptide YVEF resulted in a broadened substrate binding pocket and closer catalytic triad (His57, Asp¹°² and Ser¹95). The intramolecular Hydrogen bonds and the cation π-interactions were also dramatically increased. The results indicated that engineering of the N-terminus with artificial peptides might be an effective approach for optimizing the properties of the target enzymes.

Complex structure of the DNA-binding domain of AdpA, the global transcription factor in Streptomyces griseus, and a target duplex DNA reveals the structural basis of its tolerant DNA sequence specificity.

AdpA serves as the global transcription factor in the A-factor regulatory cascade, controlling the secondary metabolism and morphological differentiation of the filamentous bacterium Streptomyces griseus. AdpA binds to over 500 operator regions with the consensus sequence 5'-TGGCSNGWWY-3' (where S is G or C, W is A or T, Y is T or C, and N is any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To elucidate the structural basis of this tolerant DNA recognition by AdpA, we focused on the interaction between the DNA-binding domain of AdpA (AdpA-DBD), which consists of two helix-turn-helix motifs, and a target duplex DNA containing the consensus sequence 5'-TGGCGGGTTC-3'. The crystal structure of the AdpA-DBD-DNA complex and the mutant analysis of AdpA-DBD revealed its unique manner of DNA recognition, whereby only two arginine residues directly recognize the consensus sequence, explaining the strict recognition of G and C at positions 2 and 4, respectively, and the tolerant recognition of other positions of the consensus sequence. AdpA-DBD confers tolerant DNA sequence specificity to AdpA, allowing it to control hundreds of genes as a global transcription factor.

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB).

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T(13)E(14)Y(15)-OMTKY3 and T(13)E(14)Y(15)G(18)M(21)P(32)V(36)-OMTKY3, and for SGPB, S(13)D(14)Y(15)-OMTKY3 and S(13)D(14)Y(15)G(18)I(19)K(21)-OMTKY3. The free energies of association (ΔG(0)) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.

Rational design of a novel propeptide for improving active production of Streptomyces griseus trypsin in Pichia pastoris.

Applying in silico simulations and in vitro experiments, the amino acid proline was proved to have a profound influence on Streptomyces griseus trypsinogen, and the hydrogen bond between H(57) and D(102) was found to be crucial for trypsin activity. By introducing an artificial propeptide, IVEF, the titer of trypsin was increased 6.71-fold.

Resin acid conversion with CYP105A1: an enzyme with potential for the production of pharmaceutically relevant diterpenoids.

Cytochrome P450s are very versatile enzymes with great potential for biotechnological applications because of their ability to oxidize unactivated CH bonds. CYP105A1 from Streptomyces griseolus was first described as a herbicide-inducible sulfonylurea hydroxylase, but it is also able to convert other substrates such as vitamin D(3) . To extend the substrate pool of this interesting enzyme further, we screened a small diterpenoid compound library and were able to show the conversion of several resin acids. Binding of abietic acid, dehydroabietic acid, and isopimaric acid to the active site was assayed, and V(max) and K(m) values were calculated. The products were analyzed by NMR spectroscopy and identified as 15-hydroxyabietic acid, 15-hydroxydehydroabietic acid, and 15,16-epoxyisopimaric acid. As the observed products are difficult to obtain by chemical synthesis, CYP105A1 has proved to be a promising candidate for biotechnological applications that combine bioconversion and chemical synthesis to obtain functionalized resin acids.

A convenient chemical-microbial method for developing fluorinated pharmaceuticals.

A significant proportion of pharmaceuticals are fluorinated and selecting the site of fluorine incorporation can be an important beneficial part a drug development process. Here we describe initial experiments aimed at the development of a general method of selecting optimum sites on pro-drug molecules for fluorination, so that metabolic stability may be improved. Several model biphenyl derivatives were transformed by the fungus Cunninghamella elegans and the bacterium Streptomyces griseus, both of which contain cytochromes P450 that mimic oxidation processes in vivo, so that the site of oxidation could be determined. Subsequently, fluorinated biphenyl derivatives were synthesised using appropriate Suzuki-Miyaura coupling reactions, positioning the fluorine atom at the pre-determined site of microbial oxidation; the fluorinated biphenyl derivatives were incubated with the microorganisms and the degree of oxidation assessed. Biphenyl-4-carboxylic acid was transformed completely to 4'-hydroxybiphenyl-4-carboxylic acid by C. elegans but, in contrast, the 4'-fluoro-analogue remained untransformed exemplifying the microbial oxidation - chemical fluorination concept. 2'-Fluoro- and 3'-fluoro-biphenyl-4-carboxylic acid were also transformed, but more slowly than the non-fluorinated biphenyl carboxylic acid derivative. Thus, it is possible to design compounds in an iterative fashion with a longer metabolic half-life by identifying the sites that are most easily oxidised by in vitro methods and subsequent fluorination without recourse to extensive animal studies.