Establishment of an Efficient Expression and Regulation System in Streptomyces for Economical and High-Level Production of the Natural Blue Pigment Indigoidine.

Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceuticals and is mainly synthesized from l-glutamine via a condensation reaction by indigoidine synthetases, most of which originates from Streptomyces species. However, due to the complex metabolic switches of Streptomyces, most of the researchers choose to overexpress indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine. Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, here, an updated regulation system derived from the Streptomyces self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in Streptomyces lividans TK24. The updated system was constructed via promoter mining and σhrdB expression optimization, and this system was applied to precisely and continuously regulate the expression of indigoidine synthetase IndC derived from Streptomyces albus J1704. Finally, the engineered strain was cultured with cheap industrial glycerol as a supplementary carbon source, and 14.3 and 46.27 g/L indigoidine could be achieved in a flask and a 4 L fermentor, respectively, reaching the highest level of microbial synthesis of indigoidine. This study will lay a foundation for the industrial application of Streptomyces cell factories to produce indigoidine.

Microbial Degradation, Spectral analysis and Toxicological Assessment of Malachite Green Dye by Streptomyces exfoliatus.

Malachite green (MG) dye is a common environmental pollutant that threatens human health and the integrity of the Earth's ecosystem. The aim of this study was to investigate the potential biodegradation of MG dye by actinomycetes species isolated from planted soil near an industrial water effluent in Cairo, Egypt. The Streptomyces isolate St 45 was selected according to its high efficiency for laccase production. It was identified as S. exfoliatus based on phenotype and 16S rRNA molecular analysis and was deposited in the NCBI GenBank with the gene accession number OL720220. Its growth kinetics were studied during an incubation time of 144 h, during which the growth rate was 0.4232 (µ/h), the duplication time (td) was 1.64 d, and multiplication rate (MR) was 0.61 h, with an MG decolorization value of 96% after 120 h of incubation at 25 °C. Eleven physical and nutritional factors (mannitol, frying oil waste, MgSO4, NH4NO3, NH4Cl, dye concentration, pH, agitation, temperature, inoculum size, and incubation time) were screened for significance in the biodegradation of MG by S. exfoliatus using PBD. Out of the eleven factors screened in PBD, five (dye concentration, frying oil waste, MgSO4, inoculum size, and pH) were shown to be significant in the decolorization process. Central composite design (CCD) was applied to optimize the biodegradation of MG. Maximum decolorization was attained using the following optimal conditions: food oil waste, 7.5 mL/L; MgSO4, 0.35 g/L; dye concentration, 0.04 g/L; pH, 4.0; and inoculum size, 12.5%. The products from the degradation of MG by S. exfoliatus were characterized using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The results revealed the presence of several compounds, including leuco-malachite green, di(tert-butyl)(2-phenylethoxy) silane, 1,3-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 1,4-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 1,2-benzenedicarboxylic acid, di-n-octyl phthalate, and 1,2-benzenedicarboxylic acid, dioctyl ester. Moreover, the phytotoxicity, microbial toxicity, and cytotoxicity tests confirmed that the byproducts of MG degradation were not toxic to plants, microbes, or human cells. The results of this work implicate S. exfoliatus as a novel strain for MG biodegradation in different environments.

Multi-omic characterisation of Streptomyces hygroscopicus NRRL 30439: detailed assessment of its secondary metabolic potential.

The emergence of multidrug-resistant pathogenic bacteria creates a demand for novel antibiotics with distinct mechanisms of action. Advances in next-generation genome sequencing promised a paradigm shift in the quest to find new bioactive secondary metabolites. Genome mining has proven successful for predicting putative biosynthetic elements in secondary metabolite superproducers such as Streptomycetes. However, genome mining approaches do not inform whether biosynthetic gene clusters are dormant or active under given culture conditions. Here we show that using a multi-omics approach in combination with antiSMASH, it is possible to assess the secondary metabolic potential of a Streptomyces strain capable of producing mannopeptimycin, an important cyclic peptide effective against Gram-positive infections. The genome of Streptomyces hygroscopicus NRRL 30439 was first sequenced using PacBio RSII to obtain a closed genome. A chemically defined medium was then used to elicit a nutrient stress response in S. hygroscopicus NRRL 30439. Detailed extracellular metabolomics and intracellular proteomics were used to profile and segregate primary and secondary metabolism. Our results demonstrate that the combination of genomics, proteomics and metabolomics enables rapid evaluation of a strain's performance in bioreactors for industrial production of secondary metabolites.

Enhancement of undecylprodigiosin production from marine endophytic recombinant strain Streptomyces sp. ALAA-R20 through low-cost induction strategy.

Genetic manipulation of the undecylprodigiosin-producing strains and engineered culture medium approaches were applied as the most economical induction strategy for improving production. The hyper-producing recombinant strain ALAA-R20 was obtained after applying protoplast fusion strategy between the potent producer marine endophytic strains Streptomyces sp. ESRAA-10 (P1) and Streptomyces sp. ESRAA-31 (P2) of Dendronephthya hemprichi. Recombinant strain ALAA-R20 produced undecylprodigiosin yield higher than its parental strains ESRAA-10 and ESRAA-31 by 82.45% and 105.52% under submerged fermentation using modified R2YE medium. In order to reduce the costs of producing undecylprodigiosin, a solid-state fermentation (SSF) was applied. Scaled-up of optimized SSF parameters consisting of groundnut oil cake (GOC) sized to 3 mm, initial moisture content 80% with a mixture of dairy mill and fruit processing wastewaters (1:1), pH 7.0, inoculum size equal to 3 × 105 spores/g dry substrate (gds), incubation temperature 30 °C, and 7-day incubation period yielded the highest yield of 181.78 mg/gds of undecylprodigiosin by the recombinant strain Streptomyces sp. ALAA-R20. Extraction and purification of the pigment using the chromatographic techniques as well as mass spectral analysis exhibited maximum absorbance at 539 nm which is physiological property of the undecylprodigiosin. Undecylprodigiosin was stable over a wide temperature ranged from - 20 to 35 °C even after storage for 6 months. The maximum yield and stability of pigment was obtained at the acidic pH (acidified methanol, pH 4.0). Undecylprodigiosin obtained from the recombinant strain Streptomyces sp. ALAA-R20 demonstrated strong antimicrobial activity against all multidrug-resistant bacterial and fungal strains tested with minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations ranged between 0.5 and 4.0, 0.5 to 4.0, and 1.0 to 8.0 µg/mL, respectively. It also showed complete inhibition of cancer cells; HCT-116, HepG-2, MCF-7 and A-549 at 5, 8, 4, and 7 µM with IC50 equal to 2.0, 4.7, 1.2, and 2.8 µM, respectively.

Metabolomic Comparison and Assessment of Co-cultivation and a Heat-Killed Inducer Strategy in Activation of Cryptic Biosynthetic Pathways.

Co-cultivation has been used as a promising tool to turn on or up-regulate cryptic biosynthetic pathways for microbial natural product discovery. Recently, a modified culturing strategy similar to co-cultivation was investigated, where heat-killed inducer cultures were supplemented to the culture medium of producer fermentations to induce cryptic pathways. In the present study, the repeatability and effectiveness of both methods in turning on cryptic biosynthetic pathways were unbiasedly assessed using UHPLC-HRESIMS-based metabolomics analysis. Both induction methods had good repeatability, and they resulted in very different induced metabolites from the tested producers. Co-cultivation generated more induced mass features than the heat-killed inducer cultures, while both methods resulted in the induction of mass features not observed using the other induction method. As examples, pathways leading to two new natural products, N-carbamoyl-2-hydroxy-3-methoxybenzamide (1) and carbazoquinocin G (5), were induced and up-regulated through co-culturing a producer Streptomyces sp. RKND-216 with inducers Alteromonas sp. RKMC-009 and M. smegmatis ATCC 120515, respectively.

Aqueous two-phase partitioning and characterization of xylanase produced by Streptomyces geysiriensis from low cost lignocellulosic substrates.

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100 rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0 at 40 °C. The scale-up was done based on the results of optimized parameters using 3 L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K2HPO4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5-8.5, with thermal stability from 20 °C to 60 °C up to 180 min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4 °C and room temperature (35 °C) for 30 and 9 days respectively. The kinetic parameters Km (0.48 mg/ml) and Vmax (8.33 U/mg) were determined using birchwood xylan as substrate.

Microbial degradation, spectral analysis and toxicological assessment of malachite green by Streptomyces chrestomyceticus S20.

Malachite green (MG), a triphenylmethane dye is extensively used for coloring silk, aquaculture and textile industries, it has also has been reported toxic to life forms. This study aimed to investigate the biodegradation potential of MG by actinobacteria. The potent actinobacterial strain S20 used in this study was isolated from forest soil (Sabarimala, Kerala, India) and identified as Streptomyces chrestomyceticus based on phenotype and molecular features. Strain S20 degraded MG up to 59.65 ± 0.68% was studied in MSM medium and MG (300 mg l-1) and degradation was increased (90-99%) by additions of 1% glucose and yeast extract into the medium at pH 7. The treated metabolites from MG by S20 characterized by FT-IR and GC-MS. The results showed MG has been degraded into nontoxic compounds evaluated by (1) phytotoxic assay on Vigna radiata, (2) microbial toxicity on Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Streptococcus sp. and Escherichia coli, (3) cytotoxicity assay in a human cell line (MCF 7). The toxicity studies demonstrated that the byproducts from MG degradation by S. chrestomyceticus S20 were no toxic to plants and microbes and less toxic to human cells as compared to the parent MG. Perhaps this is the first work reported on biodegradation of MG by S. chrestomyceticus which could be a potential candidate for the removal of MG from various environments.

Production of ammonia as a low-cost and long-distance antibiotic strategy by Streptomyces species.

Soil-inhabiting streptomycetes are nature's medicine makers, producing over half of all known antibiotics and many other bioactive natural products. However, these bacteria also produce many volatiles, molecules that disperse through the soil matrix and may impact other (micro)organisms from a distance. Here, we show that soil- and surface-grown streptomycetes have the ability to kill bacteria over long distances via air-borne antibiosis. Our research shows that streptomycetes do so by producing surprisingly high amounts of the low-cost volatile ammonia, dispersing over long distances to inhibit the growth of Gram-positive and Gram-negative bacteria. Glycine is required as precursor to produce ammonia, and inactivation of the glycine cleavage system nullified ammonia biosynthesis and concomitantly air-borne antibiosis. Reduced expression of the porin master regulator OmpR and its cognate kinase EnvZ is used as a resistance strategy by E. coli cells to survive ammonia-mediated antibiosis. Finally, ammonia was shown to enhance the activity of canonical antibiotics, suggesting that streptomycetes adopt a low-cost strategy to sensitize competitors for antibiosis from a distance.

Critical Assessment of Streptomyces spp. Able to Control Toxigenic Fusaria in Cereals: A Literature and Patent Review.

Mycotoxins produced by Fusarium species on cereals represent a major concern for food safety worldwide. Fusarium toxins that are currently under regulation for their content in food include trichothecenes, fumonisins, and zearalenone. Biological control of Fusarium spp. has been widely explored with the aim of limiting disease occurrence, but few efforts have focused so far on limiting toxin accumulation in grains. The bacterial genus Streptomyces is responsible for the production of numerous drug molecules and represents a huge resource for the discovery of new molecules. Streptomyces spp. are also efficient plant colonizers and able to employ different mechanisms of control against toxigenic fungi on cereals. This review describes the outcomes of research using Streptomyces strains and/or their derived molecules to limit toxin production and/or contamination of Fusarium species in cereals. Both the scientific and patent literature were analyzed, starting from the year 2000, and we highlight promising results as well as the current pitfalls and limitations of this approach.

Assessment of the Detrimental Impact of Polyvalent Streptophages Intended to be Used as Biological Control Agents on Beneficial Soil Streptoflora.

Streptophages are currently being investigated to control potato common scab, however, since a majority of streptophages are reported to be polyvalent, their potential to infect beneficial soil streptomycetes during the application process may have unintended consequences. To test this hypothesis, two phytopathogenic fungi, namely Fusarium solani and Rhizoctonia solani, were tested for their detrimental effect on the test crop wheat (Triticum aestivum cv. Gutha). F. solani caused a significant root weight reduction (34%) in the wheat plant and therefore was tested further in the pot trials with actinomycetes present. Sixty-seven streptomycete isolates from a Tasmanian potato farm were screened for their antifungal abilities against the two phytopathogenic fungi. Four actinomycetes found to be strongly antifungal were then tested for their disease-protective abilities against F. solani in pot trials again using wheat. Addition of the streptomycetes into the container media protected the plants against F. solani, indicating that streptomycetes have a disease-suppressive effect. A further pot trial was conducted to evaluate whether these beneficial streptomycete species would be affected by streptophage treatment and subsequently result in an increased risk of fungal infections. When streptophages were added to the pots, the shoot and root growth of wheat declined by 23.6% and 8.0%, respectively, in the pots with the pathogenic fungus compared to the control pots. These differences might suggest that removal of antifungal streptomycetes by polyvalent phages from plant rhizosphere when biocontrol of plant pathogenic streptomycetes (e.g. Streptomyces scabiei) is targeted might encourage secondary fungal infections in the farm environment. The presented data provide preliminary evidence that streptophage treatment of pathogenic streptomycetes may lead to an aggravated disease risk by soil-borne fungal pathogens when naturally present antagonists are removed. As a result, extensive farm site trials are required to determine the long-term detrimental impact of polyvalent streptophage treatments on beneficial soil streptoflora.

Biodiversity of Actinobacteria from the South Pacific and the Assessment of Streptomyces Chemical Diversity with Metabolic Profiling.

Recently, bioprospecting in underexplored habitats has gained enhanced focus, since new taxa of marine actinobacteria can be found, and thus possible new metabolites. Actinobacteria are in the foreground due to their versatile production of secondary metabolites that present various biological activities, such as antibacterials, antitumorals and antifungals. Chilean marine ecosystems remain largely unexplored and may represent an important source for the discovery of bioactive compounds. Various culture conditions to enrich the growth of this phylum were used and 232 bacterial strains were isolated. Comparative analysis of the 16S rRNA gene sequences led to identifying genetic affiliations of 32 genera, belonging to 20 families. This study shows a remarkable culturable diversity of actinobacteria, associated to marine environments along Chile. Furthermore, 30 streptomycete strains were studied to establish their antibacterial activities against five model strains, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa, demonstrating abilities to inhibit bacterial growth of Gram-positive bacteria. To gain insight into their metabolic profiles, crude extracts were submitted to liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis to assess the selection of streptomycete strains with potentials of producing novel bioactive metabolites. The combined approach allowed for the identification of three streptomycete strains to pursue further investigations. Our Chilean marine actinobacterial culture collection represents an important resource for the bioprospection of novel marine actinomycetes and its metabolites, evidencing their potential as producers of natural bioproducts.

In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04.

The chromanequinone (BIQ) compound produced by the mangrove estuary derived strain, Streptomyces sp. JRG-04 was effective even at low MIC level concentration against Methicillin resistant S. aureus and other clinical pathogens. In this study, we have investigated the antimicrobial potential of chromanequinone compound by using various microscopy and imaging techniques. The flow cytometry (FACS) analysis suggested the BIQ aromatic polyketide compound produced by the Streptomyces sp. JRG-04 has toxic effect on MRSA cell membrane by increased up take of propidium iodide dye. The bacterial imaging analysis by high content screening experiment (HCS) revealed the increased number of dead MRSA cells than the live MRSA populations with chromanequinone treatment. Furthermore, atomic force microscopic study proved the MRSA cell surface ultra-structure changes when the cells exposed to chromanequinone compound at 3 h and 6 h. Further, in-vitro lymphocytotoxicity effect of chromanequinone compound at different concentrations with the combination of complement was performed on human lymphocytes by cell lysis assay. Interestingly, we have found that the higher concentration of BIQ chromanequinone (10 mg/mL) compound without complement induced apoptosis of human lymphocytes. The present investigation reveals that the toxic potential of chromanequinone on human lymphocytes might be associated with the complement dependent. This study strongly suggests that the chromanequinone compound produced by the Streptomyces strain with bioactive property can be developed as a therapeutic leads for various pharmaceutical applications.

Feather hydrolysate from Streptomyces sampsonii GS 1322: A potential low cost soil amendment.

Process parameters for obtaining hydrolysate from hen feathers, i.e., initial pH (5.0-9.0) and incubation period (1-6 day), were set and studied, using Streptomyces sampsonii GS 1322 in submerged and solid state conditions. Under submerged conditions, complete hydrolysis of feathers was observed on fifth day [initial pH 8.0, 28 ± 2°C, shaking (150 rpm)] with release of soluble protein (2985 µg ml(-1)) and amino acids (2407 µg ml(-1)). Cell free hydrolysate showed hydrolysis of casein (18 mm), gelatin (26 mm), collagen (31 mm), hemoglobin (23 mm) and Tween 80 (35 mm) while 445 U keratinase activity. Total soluble protein reached 5 mg ml(-1) in solid state conditions. During Pot experimentation using barren agriculture soil the effect of feather hydrolysate on wheat crop were recorded. Significant increase (p<0.01) in wheat seed germination was observed in treated soils as compared to untreated. Treatment significantly increased plant height from 30 to 60 days and 30-90 days (p<0.001). Treated soil showed an increase in total microbial count, proteolytic activity, phosphate solubilizing bacteria and ammonifying bacteria, whereas pathogenic fungi load was reduced. S. sampsonii GS 1322 can be used for bio-processing of poultry wastes yielding feather hydrolysate rich in proteins and amino acids for development of low-cost organic amendment to accelerate wheat crop growth in barren agricultural land.

The influence of effective microorganisms (EM) and yeast on the degradation of strobilurins and carboxamides in leafy vegetables monitored by LC-MS/MS and health risk assessment.

The aim of this study was to determine the behaviour of strobilurin and carbocyamides commonly used in chemical protection of lettuce depending on carefully selected effective microorganisms (EM) and yeast (Y). Additionally, the assessment of the chronic health risk during a 2-week experiment was performed. The statistical method for correlation of physico-chemical parameters and time of degradation for pesticides was applied. In this study, the concentration of azoxystrobin, boscalid, pyraclostrobin and iprodione using liquid chromatography-mass spectrometry (LC-MS/MS) in the matrix of lettuce plants was performed, and there was no case of concentration above maximum residues levels. Before harvest, four fungicides and their mixture with EM (1 % and 10 %) and/or yeast 5 % were applied. In our work, the mixtures of 1%EM + Y and 10%EM + Y both stimulated and inhibited the degradation of the tested active substances. Adding 10%EM to the test substances strongly inhibited the degradation of iprodione, and its concentration decreased by 30 %, and in the case of other test substances, the degradation was approximately 60 %. Moreover, the addition of yeast stimulated the distribution of pyraclostrobin and boscalid in lettuce leaves. The risk assessment for the pesticides ranged from 0.4 to 64.8 % on day 1, but after 14 days, it ranged from 0.0 to 20.9 % for children and adults, respectively. It indicated no risk of adverse effects following exposure to individual pesticides and their mixtures with EM and yeast.

Economic process to co-produce poly(ε-l-lysine) and poly(l-diaminopropionic acid) by a pH and dissolved oxygen control strategy.

This study tended to apply biorefinery of indigenous microbes to the fermentation of target-product generation through a novel control strategy. A novel strategy for co-producing two valuable homopoly(amino acid)s, poly(ε-l-lysine) (ε-PL) and poly(l-diaminopropionic acid) (PDAP), was developed by controlling pH and dissolved oxygen concentrations in Streptomyces albulus PD-1 fermentation. The production of ε-PL and PDAP got 29.4 and 9.6gL(-1), respectively, via fed-batch cultivation in a 5L bioreactor. What is more, the highest production yield (21.8%) of similar production systems was achieved by using this novel strategy. To consider the economic-feasibility, large-scale production in a 1t fermentor was also implemented, which would increase the gross profit of 54,243.5USD from one fed-batch bioprocess. This type of fermentation, which produces multiple commercial products from a unified process is attractive, because it will improve the utilization rate of raw materials, enhance production value and enrich product variety.

Automated identification of depsipeptide natural products by an informatic search algorithm.

Nonribosomal depsipeptides are a class of potent microbial natural products, which include several clinically approved pharmaceutical agents. Genome sequencing has revealed a large number of uninvestigated natural-product biosynthetic gene clusters. However, while novel informatic search methods to access these gene clusters have been developed to identify peptide natural products, depsipeptide detection has proven challenging. Herein, we present an improved version of our informatic search algorithm for natural products (iSNAP), which facilitates the detection of known and genetically predicted depsipeptides in complex microbial culture extracts. We validated this technology by identifying several depsipeptides from novel producers, and located a large number of novel depsipeptide gene clusters for future study. This approach highlights the value of chemoinformatic search methods for the discovery of genetically encoded metabolites by targeting specific areas of chemical space.

Microbial degradation of chitin waste for production of chitosanase and food related bioactive compounds.

Ecological samples rich in microbial diversity like cow dung, legume rhizosphere, fish waste and garden soil were used for isolation of chitosan-degrading microorganisms. Selected isolates were used for production of chitosanase and food related bioactive compounds by conversion of biowaste. Production of glucosamine (Gln), N-acetylglucosamine (NAG), chitooligosaccharides (COS), antioxidants, antibacterial compounds and prebiotics was carried out by microbial fermentation of biowaste. The highest chitosanase activity (8 U/mL) was observed in Aspergillus sp. isolated from fish market waste and it could produce Gln and NAG while Streptomyces sp. isolated from garden soil was able to produce COS along with Gln and NAG. Radical scavenging activity was observed in culture supernatants of 35% of studied isolates, and 20% isolates secreted compounds which showed positive effect on growth of Bifidobacterium. Antibacterial compounds were produced by 40% of selected isolates and culture supernatants of two microbial isolates, Streptomyces zaomyceticus C6 and one of garden soil isolates, were effective against both gram positive and negative bacteria.

Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1.

Poly(ε-L-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP) co-production by Streptomyces albulus PD-1 from cane molasses and hydrolysate of strepyomyces cells (HSC) was investigated for the first time in this study. The optimal initial total sugar concentration of the cane molasses pretreated with sulfuric acid was determined to be 20 g L(-1), and HSC could substitute for yeast extract for ε-PL and PDAP co-production. When fed-batch fermentation was performed in 1t fermentor with pretreated cane molasses and HSC, 20.6 ± 0.5 g L(-1) of ε-PL and 5.2 ± 0.6 g L(-1) of PDAP were obtained. The amount of strepyomyces cells obtained in one fed-batch fermentation is sufficient to prepare the HSC to satisfy the demand of subsequent fermentations, thus the self-cycling of organic nitrogen source becomes available. These results suggest that the low-cost cane molasses and HSC can be used for the economical production of ε-PL and PDAP by S. albulus PD-1.

The natural product domain seeker NaPDoS: a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity.

New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry.

Assessment of resistomycin, as an anticancer compound isolated and characterized from Streptomyces aurantiacus AAA5.

A new actinomycete strain, isolated from humus soils in the Western Ghats, was found to be an efficient pigment producer. The strain, designated AAA5, was identified as a putative Streptomyces aurantiacus strain based on cultural properties, morphology, carbon source utilization, and analysis of the 16S rRNA gene. The strain produced a reddish-brown pigmented compound during the secondary metabolites phase. A yellow compound was derived from the extracted pigment and was identified as the quinone-related antibiotic resistomycin based on ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, liquid chromatography and mass spectroscopy, and nuclear magnetic resonance analyses. The AAA5 strain was found to produce large quantities of resistomycin (52.5 mg/L). It showed potent cytotoxic activity against cell lines viz. HepG2 (hepatic carcinoma) and HeLa (cervical carcinoma) in vitro, with growth inhibition (GI(50)) of 0.006 and 0.005 µg/ml, respectively. The strain also exhibited broad antimicrobial activities against both Gram-positive and Gram-negative bacteria. Therefore, AAA5 may have great potential as an industrial resistomycin-producing strain.