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To enhance the applied value of transglutaminase (TGase), various methods have been employed to improve its catalytic properties. However, most modifications have targeted the N-terminus, while the role of the C-terminus in determining TGase properties has been overlooked. In this study, we focused on enhancing the thermal stability of Streptomyces hygroscopicus TGase by engineering its C-terminal region. Modeling revealed that the C-terminal loop interacts with the N-terminal loop through hydrogen bonds between Trp331 and N-terminal residues (Asp19, Ala20, Tyr21). Removing the last C-terminal residue (Ser322) had no significant effect on TGase stability, but deleting additional residues (Trp331, Gly330, Gln299) led to inactivation. Substituting Trp331 with Ala reduced TGase's half-life at 50 °C and specific activity by 50% and 70%, respectively, highlighting the importance of C-terminal interactions in TGase stability. We also attempted to fuse three self-assembling amphipathic peptides (SAPs) (EAK16, KL15, ELK16) and a C-terminal sequence (IGCIILT) from Sulfolobus tokodaii RNase HI to TGase. The fusion of IGCIILT increased TGase's half-life by 1.5-fold without affecting specific activity, while the three SAPs had little effect on stability. Structural analysis showed that the fusion of IGCIILT raised TGase's melting temperature by 5.2 °C and altered its tertiary structure. Our results indicate that the C-terminus is important for modulating TGase properties, and fusing "stabilization tags" like IGCIILT at the C-terminus is a promising strategy to enhance thermal stability. [ABSTRACT FROM AUTHOR]

Malaria is a disease transmitted through the bite of female Anopheles mosquitoes carrying Plasmodium parasite. The resistance of the Plasmodium parasite against antimalarial drugs is a critical health concern necessitating the development of novel antimalarial treatments. Previous investigations reported that the crude extract of Streptomyces hygroscopicus Subsp. Hygroscopicus (S. hygroscopicus) possessed antimalarial properties, with in vitro assays confirming the ability to inhibit Plasmodium growth. Therefore, this research aimed to identify derivative compounds from S. hygroscopicus using Liquid Chromatography-Mass Spectrometry (LC-MS) and assess antimalarial activity by examining the binding site, pharmacokinetic profiles, and binding interactions through in silico analysis. In silico reverse molecular docking study was conducted with target proteins including Plasmodium falciparum malarial M1 aminopeptidase (PfA-M1), Plasmodium falciparum chloroquine resistance transporter (PfCRT), and falcipain-2 protease obtained from Protein Data Bank (PDB) and active compound ligands retrieved from PubChem. In addition, the analysis of pharmacokinetic profiles and bond interactions was performed using the SwissADME web tool and LigPlot software, respectively. The LC-MS analysis results showed that four compounds including dibenzyl amine, sedanolide, levalbuterol, and dibutyl phthalate with high retention times met the drug similarity criteria and had binding affinity values comparable to control ligands of the respective target proteins. The four compounds may have antimalarial activity due to the formation of hydrogen and hydrophobic bonds identical to those found between the target proteins and control ligands. Specifically, dibenzyl amine expressed the highest binding affinity across all target proteins, suggesting it as a potential antimalarial candidate. [ABSTRACT FROM AUTHOR]

Recent research has revealed that Plasmodium falciparum (P. falciparum) possesses an Artemisinin-based Combination Therapy (ACT) resistance mechanism. To address this issue, new antimalarial chemicals are required. In vitro, in vivo, and in silico tests have been conducted on metabolite extracts of Streptomyces hygroscopicus subsp. hygroscopicus (S. hygroscopicus) and showed antimalarial effects through various mechanisms. The aim of this research was to determine the effectiveness and cytotoxicity of active fractions 38K, 38T, and 56 of S. hygroscopicus secondary metabolites as antimalarial. Fractionation procedure was performed using flash column chromatography BUCHI Reveleris® PREP Purification System. Effectiveness test was conducted on Plasmodium berghei (P. berghei) culture by parasite inhibition counts. The cytotoxicity effect of S. hygroscopicus was observed utilizing the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide Assay (MTT Assay) on Michigan Cancer Foundation-7 (MCF-7) breast cancer cell culture. Inhibition Concentration 50% (IC50) and Cytotoxicity Concentration 50% (CC50) was obtained from probit analysis. S. hygroscopicus fractions 38K, 38T, and 56 showed antimalarial activity against P. berghei, with fraction 38T having the strongest activity as antimalarial from selectivity index. Fraction 38T had the IC50 with the value of 11.66 µg/mL which included in the promising activity classification of IC50. Almost all asexual stages of parasite morphology were damaged by the fractions. MCF-7, a cell line used to study antimalarial cytotoxicity, was unaffected by any of the three fractions due to its CC50 exceeding 50 μg/mL. S. hygroscopicus fractions 38K, 38T, and 56 of the metabolite extracts contain non-toxic compounds that can damage the morphology of intra-erythrocytic stage and inhibit the growth of P. berghei in vitro. [ABSTRACT FROM AUTHOR]

Background: Enhancing rapamycin production using a cost-effective medium is crucial for wider accessibility, reduced manufacturing costs, sustainable pharmaceutical practices, and advancements in therapeutic applications. It promotes global health, biotechnological innovation, research collaboration, and societal well-being through affordable and effective treatments. This study focuses on the development of a novel cost-effective production medium for the synthesis of rapamycin from Streptomyces hygroscopicus. Results: In the initial screening, more rapamycin production was observed in medium A. Initially, the organism produced 10 µg/mL rapamycin. Based on the OFT results, a novel cost-effective medium composition was designed, incorporating soyabean, sugarcane juice, and dried tomato components. Using RSM, soyabean and tomato was found to be more significant in rapamycin production than sugarcane. In the optimized medium, the production of rapamycin increased significantly to 24 µg/mL. Furthermore, a comparative analysis of the growth kinetics between the production normal medium (referred to as production medium A) and the newly optimized cost-effective production medium revealed that the optimized cost-effective production medium significantly enhanced the production of rapamycin. Conclusion: Overall, this study demonstrates the successful development of a cost-effective production medium for rapamycin synthesis from S. hygroscopicus. The findings highlight the potential of using a cost-effective medium to enhance the production of a valuable secondary metabolite, rapamycin, while reducing production costs. [ABSTRACT FROM AUTHOR]

The advancement in pharmaceutical research has led to the discovery and development of new combinatorial life-saving drugs. Rapamycin is a macrolide compound produced from Streptomyces hygroscopicus. Rapamycin and its derivatives are one of the promising sources of drug with broad spectrum applications in the medical field. In recent times, rapamycin has gained significant attention as of its activity against cytokine storm in COVID-19 patients. Rapamycin and its derivatives have more potency when compared to other prevailing drugs. Initially, it has been used exclusively as an anti-fungal drug. Currently rapamycin has been widely used as an immunosuppressant. Rapamycin is a multifaceted drug; it has anti-cancer, anti-viral and anti-aging potentials. Rapamycin has its specific action on mTOR signaling pathway. mTOR has been identified as a key regulator of different pathways. There will be an increased demand for rapamycin, because it has lesser adverse effects when compared to steroids. Currently researchers are focused on the production of effective rapamycin derivatives to combat the growing demand of this wonder drug. The main focus of the current review is to explore the origin, development, molecular mechanistic action, and the current therapeutic aspects of rapamycin. Also, this review article revealed the potential of rapamycin and the progress of rapamycin research. This helps in understanding the exact potency of the drug and could facilitate further studies that could fill in the existing knowledge gaps. The study also gathers significant data pertaining to the gene clusters and biosynthetic pathways involved in the synthesis and production of this multi-faceted drug. In addition, an insight into the mechanism of action of the drug and important derivatives of rapamycin has been expounded. The fillings of the current review, aids in understanding the underlying molecular mechanism, strain improvement, optimization and production of rapamycin derivatives. [ABSTRACT FROM AUTHOR]

Background: Malaria has become a major global health disease among humans. Globally, an estimated 228 million cases of malaria occurred and resulted in 405,000 deaths in 2018. The treatment of Plasmodium falciparum malaria relies on derivatives of artemisinin (ART) as a core component of ART-based combination therapy (ACTs). However, some malaria-endemic areas have started showing cases of artemisinin resistance. Thus, research related to antimalarial drugs from plant and microbial sources is needed. Methods: The keywords Streptomyces hygroscopicus, Antimalarial/antiplasmodium, and secondary metabolite were used to search the important literature materials on PubMed, Google scholar and Science direct. Results: From the literature review results, it was found that 61 compounds were isolated from Streptomyces hygroscopicus. Of the isolates, seven compounds have been tested for antimalarial activity. Two compounds were tested in silico, five compounds tested in vitro on Plasmodium culture, and three compounds tested in vivo. Eponemycin and geldanamycin are the two compounds that have been tested in vitro and in vivo as antimalarials that can inhibit Plasmodium spp. growth and inhibit cerebral malaria in the experimental animals. Conclusion: In silico, in vitro and in vivo studies of S. hygroscopicus exhibited potential antimalarial activity. [ABSTRACT FROM AUTHOR]

Background: Microbiome interactions are important determinants for ecosystem functioning, stability, and health. In previous studies, it was often observed that bacteria suppress potentially pathogenic fungal species that are part of the same plant microbiota; however, the underlying microbe-microbe interplay remains mostly elusive. Here, we explored antagonistic interactions of the fungus Fusarium graminearum and bacterium Streptomyces hygroscopicus at the molecular level. Both are ubiquitous members of the healthy wheat microbiota; under dysbiosis, the fungus causes devastating diseases. Results: In co-cultures, we found that Streptomyces alters the fungal acetylome leading to substantial induction of fungal autophagy. The bacterium secrets rapamycin to inactivate the target of rapamycin (TOR), which subsequently promotes the degradation of the fungal histone acetyltransferase Gcn5 through the 26S proteasome. Gcn5 negatively regulates fungal autophagy by acetylating the autophagy-related protein Atg8 at the lysine site K13 and blocking cellular relocalization of Atg8. Thus, degradation of Gcn5 triggered by rapamycin was found to reduce Atg8 acetylation, resulting in autophagy induction in F. graminearum. Conclusions: Autophagy homeostasis plays an essential role in fungal growth and competition, as well as for virulence. Our work reveals a novel post-translational regulation of autophagy initiated by a bacterial antibiotic. Rapamycin was shown to be a powerful modulator of bacteria–fungi interactions with potential importance in explaining microbial homeostasis in healthy plant microbiomes. The autophagic process provides novel possibilities and targets to biologically control pathogens. 3Zu6EQMv78RbwrwJHF2G1T Video abstract [ABSTRACT FROM AUTHOR]

The production of different metabolites by Streptomyces spp. is directly influenced by oxygen transfer. On the other hand, intensive agitation and aeration may have negative effects on productivity due to high shear stress which causes damage to cells. Therefore, the aim of this study was multiobjective optimization of main scale‐up parameters using genetic algorithms. Between diverse solutions, an increase in aeration rate leads to the maximal production of biocontrol agents effective against storage apple pathogens of the Alternaria and Fusarium genera. Simultaneously, an increase in aeration rate enables the usage of a lower stirrer speed and significantly shorter bioprocess duration which supports the profitability of a defined production. [ABSTRACT FROM AUTHOR]

Background: Crude extract of Streptomyces hygroscopicus has antimalarial effect in vivo and its active fractions showed Plasmodium inhibition in vitro. This study aims to analyze the antimalarial activity of fraction 41 and 44 of S.hygroscopicus in vivo and predict their antimalarial mechanism in silico. Material and methods: Secondary metabolite of S.hygroscopicus was identified using LC/MS. In silico analysis was done using PfA-M1, Falcipain 2 protease, and PfCRT as protein targets from PDB and active compound ligand from PubChem. The pharmacokinetic analysis was done using SwissADME. In vivo study was conducted using Plasmodium berghei infected mice that divided into 1 positive control group, 1 Artesunate group, and 8 treatment groups that administrated with fraction 41 and 44 each in dose 8,125 µg/kgBW, 32,5 µg/kgBW, 130 µg/kgBW, and 520 µg/kgBW. Result: Tryptanthrin was identified from S.hygroscopicus. Tryptanthrin has fulfilled drug-likeness criteria and stronger binding affinity to PfA-M1, Falcipain 2 protease, and PfCRT than control ligand. Fraction 41 showed a significant difference in parasite density between positive control group and 130µg/kgBW group (p=0.046) as well as 520µg/kgBW group (p=0.004). Fraction 44 showed a significant difference in parasite density between positive control group and 130µg/kgBW group (p=0.024) as well as 520µg/kgBW group (p=0.004). Correlation test of fraction 41 showed a strong negative relationship between doses and parasite density on day 3 (p=0.001; r=-0.607) and day 4 (p=0.000; r=-0.700) as well as fraction 44 on day 3 (p=0.044; r=-0.589). Conclusion: Active fractions of Streptomyces hygroscopicus subsp Hygroscopicus that contained Tryptanthrin has a potential antimalarial agent. [ABSTRACT FROM AUTHOR]

Introduction: Previous research has proven that ethyl acetate extract of Streptomyces hygroscopicus can inhibit Plasmodium growth in vivo and in vitro through inhibition of the Proteasome ubiquitin system. In this study, we analyzed the activity of several fractions of S. hygroscopicus toward the growth of P. falciparum culture with specific targets on the mitochondrial enzyme of P. falciparum; L-malate: quinone oxidoreductase (PfMQO) and dihydroorotate dehydrogenase (PfDHODH). Methodology: the fractionation process uses the Flash Column Chromography (FCC) BUCHI Reveleris PREP, analyzed using thin layer chromatography (TLC). The antimalarial activity of the fractions was tested on the culture of P. falciparum 3D7 using enzyme lactate dehydrogenase (PfLDH) assay and their activity toward PfMQO and PfDHODH target enzymes. The fractions analyzed using HPLC and compared to the standard dihydroeponemycin. The toxicity of the fractions, the WST-8 assay was conducted. Results: of the 30 fractions produced, 6 fractions were able to suppress the P. falciparum and two of them were able to inhibit more than 50% of P. falciparum growth. Both of these fractions were proven to inhibit the activity of the PfMQO enzyme but had no inhibition of the PfDHODH enzyme. Both fractions reduced human cell viability by less than 50% in toxicity test. HPLC analysis indicated that besides dehydroeponemycin, there were other compounds presences in the extract. Conclusions: fractions of S. hygroscopicus metabolite contains non-toxic compounds in addition to dihydroeponemycin that could suppress the growth of P. falciparum in vitro. This potential effect might be related to the inhibition of PfMQO enzyme. [ABSTRACT FROM AUTHOR]

The surplus of waste glycerol, by-product of the biodiesel production process, is available at the global market. Some species of the genera Streptomyces have the ability to assimilate glycerol and convert it into valuable metabolic products. In the present study, the ability of Streptomyces hygroscopicus to assimilate waste glycerol and convert it into metabolic compounds with antifungal activity against four phytopathogenic fungi obtained from apple fruit samples expressing rot symptoms, was investigated. Production of antifungal metabolites by S. hygroscopicus was carried out in 3 l stirred tank bioreactor through 7 days. Fermentation was carried out at 27 °C with aeration rate of 1.5 vvm and agitation rate of 100 r.p.m. The aim of this work was to analyse bioprocess parameters and to determine at which stage of bioprocess the production of antifungal metabolites occurs. Activity of the cultivation liquid on two isolates of Alternaria alternata and two isolates of Fusarium avenaceum were determined every 12 h using in vitro well diffusion method. It was found that the maximum production of antifungal metabolites occurred at 108 hour of cultivation. Formed inhibition zones have shown that the produced antifungal metabolites have high efficacy on tested phytopathogenic fungi (inhibition zone diameter higher than 35 mm for all test organisms). [ABSTRACT FROM AUTHOR]

Copyright of Matica Srpska Journal for Natural Sciences is the property of Matica Srpska, Department of Natural Studies and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Ascomycin (FK520) is a macrocyclic antibiotic that also exhibits antifungal and immunosuppressive activity. However, its relatively low titer and yield have hampered commercial application. Here, we have successfully constructed an efficient ascomycin‐producing strain of Streptomyces hygroscopicus with high titer and yield, using a novel combinatorial engineering approach based on the identification of targets involved in both metabolic and transcriptional regulation. First, we investigated the effects of different chemicals on ascomycin accumulation and found that dimethyl sulfoxide best stimulated ascomycin overproduction. We next compared intracellular metabolic and transcriptional profiles after dimethyl sulfoxide and control treatments and identified potential target genes (zwf and aroA, involved in metabolic precursor pathways; and luxR, iclR, fadR, and fkbN, involved in transcriptional regulation). These candidate genes were then engineered to produce strains with individual and combinatorial overexpression. Combined overexpression of aroA, fkbN, and luxR resulted in the highest yield of ascomycin (1258.30 ± 33.49 mg/L), 4.12‐fold higher than the control yield (305.60 ± 16.90 mg/L). This integrative multilevel approach identified novel determinants involved in both metabolic and transcriptional regulation, resulting in the diversion of carbon flux towards ascomycin accumulation. This approach could be applied to boost the production of a variety of useful bacterial metabolites. [ABSTRACT FROM AUTHOR]

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production. [ABSTRACT FROM AUTHOR]

Artificial neural networks (ANNs) have been used to dynamically model cross-flow microfiltration of Streptomyces hygroscopicus fermentation broths. The aim is to predict permeate flux as a function of temperature, feed flow, transmembrane pressure and processing time. Dynamic modeling of microfiltration performance of complex systems (such as broths) is very important for design of new processes and better understanding of the present. The results of ANN model analysis suggest that the coefficients of the determination have high values. The application of the Bayesian regularization gave better results to the performance of the neural network compared to the Levenberg-Marquet algorithm. The optimal number of neurons in the hidden layer is eight. Analysis of the absolute relative error showed excellent permeate flux estimates for 100 % of the data points, with an error less than 5 % for the data obtained during microfiltration in the presence of a turbulence promoter. Whilst in the case of microfiltration without turbulence promoter 90 % of predictions have an error less than 10 %. The results of applying the concept of neural networks in the dynamic modeling of microfiltration of Streptomyces hygroscopicus fermentative broths with and without a turbulence promoter clearly show the validity of proposed method for simulation and prediction of microfiltration experimental results. [ABSTRACT FROM AUTHOR]

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular‐biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′‐phosphotransferase‐Ia [APH(7′′)‐Ia] from Streptomyces hygroscopicus and aminoglycoside 4‐phosphotransferase‐Ia [APH(4)‐Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)‐Ia was reported as a ternary complex with HygB and 5′‐adenylyl‐β,γ‐imidodiphosphate (AMP‐PNP). To investigate the differences in the substrate‐recognition mechanism between APH(7′′)‐Ia and APH(4)‐Ia, the crystal structure of APH(7′′)‐Ia complexed with HygB is reported. The overall structure of APH(7′′)‐Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)‐Ia, and consists of an N‐terminal lobe (N‐lobe) and a C‐terminal lobe (C‐lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)‐Ia and APH(4)‐Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C‐lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)‐Ia and APH(4)‐Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes. [ABSTRACT FROM AUTHOR]

The textile industry is a rapidly developing industrial sector that produces – wastewater laden with various pollutants including synthetic dyes and heavy metals. Remazol Black-B (RB-B) is a group of azo dyes that are extensively used in textile industries. The objective of this study was to evaluate the potential of the Streptomyces hygroscopicus PTCC1132 to remove RB-B and to determine optimal conditions for decolorizing. The Taguchi optimization approach was used to reduce the number of experiments and the time needed to find optimum conditions. The effect of operating parameters such as temperature, pH, initial RB-B concentrations, and salt dosage were evaluated by defined factor 4 on four-level Taguchi L16 orthogonal array. Qualitek-4 software was used for data analysis. Maximum efficiency for RB-B decolorization was obtained at 33°C; wherein the solution pH was set to 9.0; and RB-B concentration and salt concentration were 5,000 mg/L and 1%, respectively. The corresponding decolorization efficiency was obtained as 95.27% at under optimized conditions confirming that the bacteria exhibits resistance against the toxic effects of RB-B, especially at high dye concentrations. Our findings indicate of the effectiveness of the evaluated microorganisms for the removal of RB-B from dye-containing effluents. [ABSTRACT FROM AUTHOR]

Milbemycins produced by several Streptomyces species are a group of 16-membered macrolides with potent insecticidal and anthelminthic activity. Milbemycin A3/A4, the main components of the milbemycins biosynthetic pathway, and 5-oxomilbemycin A3/A4, the analogs of milbemycin A3/A4 without the reduction of the C-5 keto group, have been developed as acaricides, insecticides, and anthelmintics. However, so far, little is known about the regulation of milbemycins biosynthesis, which has greatly hampered the generation of high producing strains by metabolic engineering. Herein, a TetR family regulator MilR2 (encoded by sbi_00792) was identified being involved in activation of 5-oxomilbemycin A3/A4 biosynthesis in a high 5-oxomilbemycins-producing strain Streptomyces hygroscopicus SIPI-KF. The ΔmilR2 mutant with an in-frame deletion of the MilR2 DNA-binding domain resulted in significantly reduced 5-oxomilbemycin A3/A4 production (approximately 36.9 and 39.7%) at tested two time points, and accordingly introduction of an extra copy of milR2 into SIPI-KF led to enhanced production by 12.6 and 34.4%. We further showed that MilR2 could directly repress the transcription of the gene sbi_00791 encoding a putative hydrolase, which is located divergently from milR2. The precise MilR2-binding site consisting of a 7-nt perfect inverted repeat separated by 10-nt (5′-ACCAACCAGCTGGTAAGGGTTGGT-3′) was defined. In situ mutagenesis of the MilR2-binding site resulted in 19.7 and 13.5% decreases in 5-oxomilbemycin A3/A4 production, which is much lower than the decreased rates of ΔmilR2. Collectively, the results demonstrated that MilR2 serves as an activator for 5-oxomilbemycin A3/A4 production and the function of MilR2 is only partially mediated through its repression on the transcription of sbi_00791. [ABSTRACT FROM AUTHOR]

Background: Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand ascomycin biosynthesis and improve its production. Results: The metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA-Hcd-Ccr with hcd and ccr overexpression exhibited the highest ascomycin production (up to 438.95 mg/L), 1.43-folds improvement than that of the parent strain FS35 (305.56 mg/L). Conclusions: The successful constructing and experimental validation of the metabolic model of S. hygroscopicus var. ascomyceticus showed that the general metabolic network model of Streptomyces species could be used to analyze the intracellular metabolism and predict the potential key limiting steps for target metabolites overproduction. The corresponding overexpression strains of the two identified genes (hcd and ccr) using the constructed model all displayed higher ascomycin titer. The strategy for yield improvement developed here could also be extended to the improvement of other secondary metabolites in Streptomyces species. [ABSTRACT FROM AUTHOR]

The genetics of the Streptomyces hygroscopicus strain 10-22 is of interest due to the ability of this strain to produce antifungal compounds. Strain T110 was obtained through insertional mutagenesis of strain 10-22 and was found to have undergone DNA amplification, as determined by both conventional and pulsed-field gel electrophoresis (PFGE). pIJ702, the vector used for insertional mutagenesis, was shown to have integrated into and co-amplified with the chromosomal DNA sequence of T110, as pIJ702 hybridized predominantly with two of the three amplified BamHI fragments. The amplified DNA sequence in T110 is 10.8 kb in length and consists of 5.18 kb of Streptomyces chromosomal DNA and the entire 5.62 kb pIJ702 sequence. Sequence analysis of the 5.18 kb chromosomal sequence revealed two open reading frames, one encoding a putative IS5 family transposase and the other encoding a putative dihydroxy-acid dehydratase. Real-time PCR analysis showed that expression of the putative dehydratase gene in T110 is about 50 times greater than in the wild-type strain, consistent with the high level of amplification of this DNA region, and therefore this system has the potential for producing economically or clinically important molecules. [ABSTRACT FROM AUTHOR]

Copyright of Matica Srpska Journal for Natural Sciences is the property of Matica Srpska, Department of Natural Studies and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Rapamycin is a polyketide with a 31-membered macrolide ring that possesses powerful immunosuppressant activity. In this study, we firstly obtained a mutant, shikimate-resistant Streptomyces hygroscopicus strain UV-II, which displayed about 3.20-fold higher rapamycin production (305.9 mg/L) than the wild-type S. hygroscopicus ATCC29253 (95.5 mg/L). Under optimal conditions, with the addition of 2 g/L shikimic acid, the strain's rapamycin production was further increased by approximately 34.9%, to 412.6 mg/L. To gain deeper insights into the effects of shikimic acid resistance and supplementation, the fermentation properties, metabolite concentrations, and transcriptional levels of relevant genes were analyzed and evaluated for differences between this improved mutant and its parental strain. The results showed that most of the metabolic modules involved in rapamycin biosynthesis were upregulated in the mutant strain. Analysis of metabolic pathways and gene expression levels further revealed that shikimic acid metabolism plays a crucial role in the synthesis of rapamycin, and identified the rapK gene as a potential target for genetic manipulation to obtain rapamycin-producing strains with improved product yield. Consequently, the rapK gene was overexpressed in the UV-II strain, which to our delight further improved rapamycin production to 457.3 mg/L. These findings thus provide a theoretical basis for further improvements in the production of not only rapamycin, but also of other, analogous macrolide compounds. [ABSTRACT FROM AUTHOR]

Giant linear plasmids, which replicate independently of the chromosomes, widely exist in actinobacteria. Previous studies mostly focused on the replication and evolution of the linear plasmids or the secondary metabolite gene clusters and the resistance gene clusters therein. However, the relationships of the linear plasmids to the productivities of secondary metabolites have not been studied. In this work, we developed a method to eliminate the indigenous linear plasmid pSHJG1 in Streptomyces hygroscopicus var. jinggangensis, and validamycin A titer increased by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strain TL01 and 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strain 5008, whereas the cellular growth of the plasmid-cured mutant was reduced. Subsequently, the plasmid-cured mutant was complemented with three structure genes involved in cellular growth in pSHJG1 under the control of a strong PvalA promoter. Among them, the complementation of genes pSHJG1.069 and pSHJG1.072, encoding a putative hydrolase and putative P-loop ATPase, respectively, resulted in the restoration of cellular growth and validamycin A titer. Furthermore, the elimination of indigenous linear plasmid pHZ228 in the candicidin producer Streptomyces sp. FR008 also led to enhanced candicidin production and reduced cellular growth. Because of the wide distribution of indigenous linear plasmids in actinobacteria, the engineering strategy described here could be implemented in a variety of strains for the overproduction of various natural products. [ABSTRACT FROM AUTHOR]

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics. [ABSTRACT FROM AUTHOR]

Rapamycin, as a macrocyclic polyketide with immunosuppressive, antifungal, and anti-tumor activity produced by Streptomyces hygroscopicus, is receiving considerable attention for its significant contribution in medical field. However, the production capacity of the wild strain is very low. Hereby, a computational guided engineering approach was proposed to improve the capability of rapamycin production. First, a genome-scale metabolic model of Streptomyces hygroscopicus ATCC 29253 was constructed based on its annotated genome and biochemical information. The model consists of 1003 reactions, 711 metabolites after manual refinement. Subsequently, several potential genetic targets that likely guaranteed an improved yield of rapamycin were identified by flux balance analysis and minimization of metabolic adjustment algorithm. Furthermore, according to the results of model prediction, target gene pfk (encoding 6-phosphofructokinase) was knocked out, and target genes dahP (encoding 3-deoxy- d-arabino-heptulosonate-7-phosphate synthase) and rapK (encoding chorismatase) were overexpressed in the parent strain ATCC 29253. The yield of rapamycin increased by 30.8% by knocking out gene pfk and increased by 36.2 and 44.8% by overexpression of rapK and dahP, respectively, compared with parent strain. Finally, the combined effect of the genetic modifications was evaluated. The titer of rapamycin reached 250.8 mg/l by knockout of pfk and co-expression of genes dahP and rapK, corresponding to a 142.3% increase relative to that of the parent strain. The relationship between model prediction and experimental results demonstrates the validity and rationality of this approach for target identification and rapamycin production improvement. [ABSTRACT FROM AUTHOR]

Background: Transglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization. Results: A gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from -693 to -48. We also identified a negative element (-198 to -148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively. Conclusions: We constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces. [ABSTRACT FROM AUTHOR]

Streptomyces sp. TP-A0867 (=NBRC 109436) produces structurally complex polyketides designated alchivemycins A and B. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation, and analysis of the genome sequence. The 9.9 Mb genome of Streptomyces sp. TP-A0867 encodes 8,385 putative ORFs, of which 7,232 were assigned with COG categories. We successfully identified a hybrid polyketide synthase (PKS)/ nonribosomal peptide synthetase (NRPS) gene cluster that could be responsible for alchivemycin biosynthesis, and propose the biosynthetic pathway. The alchivemycin biosynthetic gene cluster is also present in Streptomyces rapamycinicus NRRL 5491T, Streptomyces hygroscopicus subsp. hygroscopicus NBRC 16556, and Streptomyces ascomycinicus NBRC 13981T, which are taxonomically highly close to strain TP-A0867. This study shows a representative example that distribution of secondary metabolite genes is correlated with evolution within the genus Streptomyces. [ABSTRACT FROM AUTHOR]

Ionophores such as valinomycin and nigericin are potent tools for studying the impact of ion perturbance on cellular functions. To obtain a broader picture about molecular components involved in mediating the effects of these drugs on yeast cells under respiratory growth conditions, we performed a screening of the haploid deletion mutant library covering the Saccharomyces cerevisiae nonessential genes. We identified nearly 130 genes whose absence leads either to resistance or to hypersensitivity to valinomycin and/or nigericin. The processes affected by their protein products range from mitochondrial functions through ribosome biogenesis and telomere maintenance to vacuolar biogenesis and stress response. Comparison of the results with independent screenings performed by our and other laboratories demonstrates that although mitochondria might represent the main target for both ionophores, cellular response to the drugs is very complex and involves an intricate network of proteins connecting mitochondria, vacuoles, and other membrane compartments. [ABSTRACT FROM AUTHOR]

Key message : A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker. Abstract: Sorghum ( Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7-12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40-50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement. [ABSTRACT FROM AUTHOR]

Validamycin A (Val-A) synthesized by Streptomyces hygroscopicus 5008 is widely used as a high-efficient antibiotic to protect plants from sheath blight disease. A novel fermentation strategy was introduced to stimulate Val-A production by adding oxygen carriers. About 58 % increase in Val-A production was achieved using liquid paraffin. Further, biomass, carbon source, metabolic genes, and metabolic enzymes were studied. It was also found that the supplementation of liquid paraffin increased the medium dissolved oxygen and intracellular oxidative stress level. The expression of the global regulators afsR and soxR sensitive to ROS, ugp catalyzing synthesis of Val-A precursor, and Val-A structural genes was enhanced. The change of the activities of glucose-6-phosphate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase was observed, which reflected the redirection of carbon metabolic flux. Based on these results, liquid paraffin addition as an oxygen carrier could be a useful technique in industrial production of Val-A and our study revealed a redox-based secondary metabolic regulation in S. hygroscopicus 5008, which provided a new insight into the regulation of the biosynthesis of secondary metabolites. [ABSTRACT FROM AUTHOR]

Alfalfa is the most important forage legume worldwide. However, similar to other minor forage crops, it is usually harvested along with weeds, which decrease its nutrient quality and thus reduce its high value in the market. In addition, weeds reduce alfalfa yield by about 50 %. Although weeds are the limiting factor for alfalfa production, little progress has been made in the incorporation of herbicide-tolerant traits into commercial alfalfa. This is partially due to the high times and costs needed for the production of vast numbers of transgenic alfalfa events as an empirical approach to bypass the random transgenic silencing and for the identification of an event with optimal transgene expression. In this focus article, we report the complete sequence of pPZP200BAR and the extremely high efficiency of this binary vector in alfalfa transformation, opening the way for rapid and inexpensive production of transgenic events for alfalfa improvement public programs. [ABSTRACT FROM AUTHOR]

Biological control agents offer one of the best alternatives to reduce the use of pesticides . Fungi from the genera Alternaria, Colletotrichum and Fusarium are listed among the most important storage pathogens of apple fruits. During storage, transport and marketing, pathogenic fungi can cause significant losses of apple fruits. This investigation studied the potential of Streptomyces hygroscopicus as a biocontrol agent against pathogenic fungi obtained from apple fruit samples expressing rot symptoms. Production of antifungal metabolites by S. hygroscopicus was carried out in 3-l bench-scale bioreactor (Biostat® Aplus, Sartorius AG, Germany) during 7 days. Fermentation was carried out at 27°C with aeration rate of 0.5 vvm and agitation rate of 200 rpm. The aim was to analyse bioprocess parameters of batch biofungicide production in medium containing glucose as a carbon source and to examine at which stage of bioprocess production of antifungal metabolite(s) against six phytopathogenic fungi occurs. In vitro antifungal activity of the produced metabolites against six fungi of the genera Colletotrichum, Fusarium and Alternaria grown on potato dextrose agar were determined every 24 h using wells technique. Antifungal activity of cell-free culture filtrate and filtrate treated with high temperature were tested. The filtrate treated with high temperature did not show any antifungal activity suggesting that active components are thermo unstable. Stationary phase of growth occurred between the third and fourth day of cultivation when production of secondary metabolites begins. Obtained results showed that maximal antifungal activity is achieved on fifth and sixth day of S. hygroscopicus cultivation under defined conditions (inhibition zone diameter higher than 30 mm for all test fungi). [ABSTRACT FROM AUTHOR]

Sirolimus (SRL) and everolimus (EVR) are mammalian targets of rapamycin inhibitors (mTOR-I) largely employed in renal transplantation and oncology as immunosuppressive/antiproliferative agents. SRL was the first mTOR-I produced by the bacterium Streptomyces hygroscopicus and approved for several medical purposes. EVR, derived from SRL, contains a 2-hydroxy-ethyl chain in the 40th position that makes the drug more hydrophilic than SRL and increases oral bioavailability. Their main mechanism of action is the inhibition of the mTOR complex 1, the regulation of factors involved in a several crucial cellular functions including: protein synthesis, regulation of angiogenesis, lipid biosynthesis, mitochondrial biogenesis and function, cell cycle, and autophagy. Most of the proteins/enzymes belonging to the aforementioned biological processes are encoded by numerous and tightly regulated genes. However, at the moment, the polygenic influence on SRL/EVR cellular effects is still not completely defined, and its comprehension represents a key challenge for researchers. Therefore, to obtain a complete picture of the cellular network connected to SRL/EVR, we decided to review major evidences available in the literature regarding the genetic influence on mTOR-I biology/pharmacology and to build, for the first time, a useful and specific "SRL/EVR genes-focused pathway", possibly employable as a starting point for future in-depth research projects. [ABSTRACT FROM AUTHOR]

Background: Transglutaminases (TGase), synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in food industry. Due to the important applications of TGase in food industry, obtaining robust and food-safe TGase-producing strains has attracted much attention during the past decade. In this study, Streptomyces hygroscopicus pro-TGase was efficiently expressed and secreted by a food-grade host, Yarrowia lipolytica, without antibiotic markers. Results: The pro-TGase gene was cloned into integrative vectors pINA1296 (monocopy) and pINA1297 (multicopy), and was used to transform the Y. lipolytica Po1g or Po1h strain, respectively. Expression was driven by a recombinant hp4d promoter and secretion obtained using a XPR2 pre-sequence as a signal peptide. The highest yield of extracellular pro-TGase produced by the recombinant Po1h strain corresponded to 5.3 U/mL of TGase, a level 8.8 fold higher than that obtained using the recombinant Po1g strain. Asparagines in two potential Asn-linked glycosylation sites (Asn160 and Asn355) from pro-TGase were mutated to glutamine individually or simultaneously, yielding the deglycosylated variants N160Q, N355Q, and N160Q/N355Q. The activities of N160Q, N355Q and N160Q/N355Q constructs were respectively 5.3 U/mL, 7.8 U/mL, and 3.0 U/mL, equivalent to 100 %, 147 %, and 57 % of that from wild-type pro-TGase. The TGase yield of N355Q variant was raised to 35.3 U/mL of by using a glycerol feeding strategy in a 3 L fermenter. The optimal pH and temperature of the activated pro-TGase, and of its deglycosylated variants, were in the range of 5.0-6.0 pH and 40-45 °C, respectively. The half-life of the recombinant wild-type pro-TGase at 37 °C reached 34.0 min, and those of the variants were from 24.2 min to 11.5 min. In contrast to the wild-type pro-TGase, all of the variants had decreased specific activities, and both the Km and kcat values of the variants decreased accordingly. Conclusions: This study constitutes the first report of the heterologous expression of a pro-TGase in Y. lipolytica, and provides new possibilities for the efficient production of TGases used in food processing. [ABSTRACT FROM AUTHOR]

Validamycin A (VAL-A) is a CN aminocyclitol antibiotic produced by Streptomyces hygroscopicus var. jinggangensis 5008, which has been widely used as antifungal agent against rice sheath blight disease. VAL-A biosynthesis has been proven to be affected by γ-butyrolactone and temperature. Herein, we showed that GlnR, a global regulator in nitrogen metabolism, is specifically associated with valK-valA intergenic promoter region by DNA-affinity chromatography and MS-based protein identification. Subsequent EMSA and DNase I footprinting assays revealed two GlnR binding sites in this promoter region. Targeted disruption of glnR in S. hygroscopicus 5008 led to a significant increase in the transcription of VAL-A structural genes, albeit the VAL-A production was reduced by 80 % and the sporulation of the mutant was impaired. Compared with the wild-type 5008, site-specific mutagenesis of GlnR binding site I enhanced VAL-A production by 2.5-fold, whereas the mutation of GlnR binding site II resulted in a 50 % reduction of VAL-A yield. Moreover, tandem mutation of site I in the site II mutant led to a 66 % increase of VAL-A production. The result suggested that GlnR not only serves as an inhibitor by binding site I but also as an activator by binding site II for VAL-A biosynthesis. Furthermore, overexpression of glnR in the site I mutant JG45 improved VAL-A production for 41 % compared with the control strain containing the vector. Therefore, the obtained data illustrate a novel regulatory feature of the global regulator GlnR. GlnR is firstly proved to act simultaneously as an activator and a repressor in validamycin biosynthesis by binding to different loci within a promoter region of the gene cluster. [ABSTRACT FROM AUTHOR]

Rapamycin is an important natural macrolide antibiotic with antifungal, immunosuppressive and anticancer activity produced by Streptomyces hygroscopicus. In this study, a mutant strain obtained by ultraviolet mutagenesis displayed higher rapamycin production capacity compared to the wild-type S. hygroscopicus ATCC 29253. To gain insights into the mechanism of rapamycin overproduction, comparative metabolic profiling between the wild-type and mutant strain was performed. A total of 86 metabolites were identified by gas chromatography-mass spectrometry. Pattern recognition methods, including principal component analysis, partial least squares and partial least squares discriminant analysis, were employed to determine the key biomarkers. The results showed that 22 potential biomarkers were closely associated with the increase of rapamycin production and the tremendous metabolic difference was observed between the two strains. Furthermore, metabolic pathway analysis revealed that amino acids metabolism played an important role in the synthesis of rapamycin, especially lysine, valine, tryptophan, isoleucine, glutamate, arginine and ornithine. The inadequate supply of amino acids, or namely 'nitrogen starvation' occurred in the mutant strain. Subsequently, the exogenous addition of amino acids into the fermentation medium of the mutant strain confirmed the above conclusion, and rapamycin production of the mutant strain increased to 426.7 mg/L after adding lysine, approximately 5.8-fold of that in the wild-type strain. Finally, the results of real-time PCR and enzyme activity assays demonstrated that dihydrodipicolinate synthase involved with lysine metabolism played vital role in the biosynthesis of rapamycin. These findings will provide a theoretical basis for further improving production of rapamycin. [ABSTRACT FROM AUTHOR]

ϵ-Poly- L-lysine (ϵ-PL) is an L-lysine homopolymer with strong antimicrobial activity, which is generally produced by Streptomyces strains. ϵ-PL is only produced under acidic conditions in liquid culture, and to improve the current understanding of ϵ-PL biosynthesis, the present study was undertaken to investigate the effects of ϵ-PL on its producer Streptomyces ahygroscopicus GIM8, under acidic and neutral conditions. The results indicated that a neutral pH favored ϵ-PL adsorption onto the cells, whereas minimal adsorption occurred at pH 4.0, the maximum pH for ϵ-PL production. At pH 7.0, small amounts of ϵ-PL caused considerable ATP leakage from the cells, which showed increased membrane permeability. Conversely, ATP leakage was inhibited by ϵ-PL at pH 4.0. Transmission electron microscopy investigation indicated that the cytoplasmic membrane was the primary site of ϵ-PL activity at pH 7.0, and that cell shape was maintained. Metabolic activity profiles revealed that ϵ-PL decreased cellular metabolic activity at a relatively low rate at pH 7.0. However, the toxic effect was significantly enhanced at pH 4.0. Based on these data, a mechanism for the effect of ϵ-PL on ϵ-PL-producing cells under neutral and acidic conditions is proposed. Additionally, acidic conditions may potentially be required for ϵ-PL biosynthesis in liquid culture because low pH can increase membrane permeability and prevent binding of ϵ-PL onto cells, both of which favor the secretion of the ϵ-PL produced by the cells into the broth. This research contributes to the current understanding of ϵ-PL biosynthesis. [ABSTRACT FROM AUTHOR]

Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE), to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold-specific quantitative PCR analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home ( p = 0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included Streptomyces coelicolor and Streptomyces sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included Streptomyces hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings. [ABSTRACT FROM AUTHOR]

Research work was carried out to describe the kinetics of cell growth, substrate consumption and product formation in batch fermentation of rapamycin using shake flask as well as laboratory-scale fermentor. Fructose was used as the sole carbon source in the fermentation media. Optimization of fermentation parameters and reliable mathematical models were used for the maximum production of rapamycin from Streptomyces hygroscopicus MTCC 4003. The experimental data for microbial production of rapamycin fitted well with the proposed mathematical models. Kinetic parameters were evaluated using best fit unstructured models, viz. Andrew's model, Monod model, Yano model, Aiba model. Andrew's model showed a comparatively better R value (0.9849) among all tested models. The values of maximum specific growth rate ( μ), saturation constant ( K), inhibition constant ( K), and growth yield coefficient ( Y) were found to be 0.008 (h), 2.835 (g/L), 0.0738 (g/L), and 0.1708 (g g, respectively. The optimum production of rapamycin was obtained at 300 rpm agitation and 1 vvm aeration rate in the fermentor. The final production of rapamycin in shake flask was 539 mg/L. Rapamycin titer found in bioreactor was 1,316 mg/L which is 52 % higher than the latest maximum value reported in the literature. [ABSTRACT FROM AUTHOR]

The nutritional requirements for antimicrobial agent production using Streptomyces hygroscopicus were analyzed in shake flask experiments. Antimicrobial activity was tested against Staphylococcus aureus and Bacillus cereus. The mathematical models have been generated with relative high complexity in order to give an adequate fit to the data. All the results suggest a high dependence of produced antimicrobial agent quantities on the amount of carbon, nitrogen, and phosphorus in cultivation medium. The statistical results of the generated models reflect the high predictive ability. The derived models were validated using leave-one-out cross-validation technique, and from statistical point of view, they have significantly high values of the cross-validation parameters. [ABSTRACT FROM AUTHOR]

Validamycin A (VAL-A) is a widely used antifungal antibiotic for the treatment of sheath blight disease of rice and other plants. It can be produced from agro-industrial by-products by Streptomyces hygroscopicus 5008. To enhance its production titer, in this work, the entire val gene cluster was amplified in tandem in S. hygroscopicus 5008 by integrating the zouA-mediated DNA amplification system into between the two boundaries of val gene cluster, resulting in multiple copies (mainly three to five) of the val gene cluster. The genetic stability of the amplified copies was confirmed by Southern blot and fermentation experiments. In shake flask fermentation, the recombinant strain (TC03) led to a 34 % enhancement of VAL-A production titer compared to that of the wild-type strain, while the accumulation of intermediate validoxylamine A was decreased in TC03. Additionally, both the structural gene transcription levels and the ValG enzyme activity were significantly increased in TC03. This work demonstrated that the amplification of the val gene cluster was an efficient strategy to enhance VAL-A production by S. hygroscopicus 5008, and the information obtained would be helpful for engineering other interesting antibiotic biosynthesis by gene cluster amplification. [ABSTRACT FROM AUTHOR]

Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5 % m/v HP20 added at 24 h, ascomycin production was increased to 380 from 300 mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2 % v/v n-hexadecane was added at 24 h; 1.0 g/L valine was supplemented at 48 h; 1.0 g/L lysine was added at 72 h. The ascomycin production was ultimately improved to 460 mg/L, a 53.3 % enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds. [ABSTRACT FROM AUTHOR]

Rapamycin is a high-value product finding immense use as a drug, in organ transplantation, and as a potential immunosuppressant. Optimization of fermentation parameters of rapamycin production by Streptomyces hygroscopicus NRRL 5491 has been carried out. The low titer value of rapamycin in the original producer strain limits its applicability at industrial level. This study aims at improving the production of rapamycin by optimizing the nutrient requirements. Addition of l-lysine increased the production of rapamycin up to a significant level which supports the fact that it acts as precursor for rapamycin production, as found in previous studies. Effect of optimized medium on the Streptomyces growth rate as well as rapamycin production has been studied. The optimization study incorporates one at a time parameter optimization studies followed by tool-based hybrid methodology. This methodology includes the Plackett-Burman design (PBD) method, artificial neural networks (ANN), and genetic algorithms (GA). PBD screened mannose, soyabean meal, and l-lysine concentrations as significant factors for rapamycin production. ANN was used to construct rapamycin production model. This strategy has led to a significant increase of rapamycin production up to 320.89 mg/L at GA optimized concentrations of 25.47, 15.39, and 17.48 g/L for mannose, soyabean meal, and l-lysine, respectively. The present study must find its application in scale-up study for industrial level production of rapamycin. [ABSTRACT FROM AUTHOR]

Total syntheses of (+)-valiolamine ( 1) and (-)-1- epi-valiolamine ( 2) from the naturally abundant (-)-shikimic acid are described. Ethyl 3- epi-5- O-methylsulfonyl-shikimate ( 3), as the key common intermediate, was first synthesized in five steps in 74 % overall yield, and then converted into the targets 1 and 2 in seven steps in 48 and 41 % overall yield, respectively. [ABSTRACT FROM AUTHOR]

The article discusses thermostabilization of a Hygromycin B Phosphotransferase gene (HYG) from bacterium species Streptomyces hygroscopicus. Topics discussed include thermophilic bacterium thermus thermophilus is an vivo-directed evolutionary strategy for thermostabilization of HYG, biochemical and biophysical analysis of recombinant HYG and duplication HYGIO, and nucleotide sequencing of cloned hyg gene in pT8S-P31-hyg. It also mentions enzymatic characterization of HYG 10.

Resistance to the non-selective herbicide dl-phosphinothricin ( PPT) was introduced into commercial Lotus corniculatus cv. Bokor by co-cultivation of cotyledons with Agrobacterium tumefaciens AGL1 harbouring the binary vector pDM805 which contains the bialaphos resistance gene ( bar) from Streptomyces hygroscopicus encoding phosphinothricin acetyltransferase ( PAT) and the uidA gene encoding β-glucuronidase. The half-cotyledon explants were precultured on regeneration Murashige and Skoog's (MS) medium supplemented with 6-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA) at 0.5 mg L−1 each, 3 days prior to infection. Upon co-cultivation, the explants were cultured on PPT-free regeneration medium for 10 days, and then subcultured on regeneration/selection media with increasing PPT concentrations (5-7 mg L−1) for about 18 weeks. Out of 480 initially co-cultivated explants, 272 regenerated shoots survived the entire PPT selection procedure. Resistant shoots were grown further, multiplied by tillering that was additionally promoted by PPT and rooted on hormone-free MS medium containing 5 mg L−1 PPT. Established shoot cultures, continuously maintained on the same medium, have preserved PPT resistance up to now (more than 2 years). Transformed plants assessed in vitro and in a greenhouse were tolerant to the herbicide PPT at 300 mg L−1 equivalent to more than twofold the recommended field dosage for weed eradication. Applied PPT treatment did not affect the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) in transformed plants. However, PPT did increase the mobility of glutamine synthetase isoforms GS1 and GS2 as well as the inhibition of an additional high mobility GS (hmGS) activity. In untransformed plants, PPT treatment reduced total GS activity by 4.4-fold while contrary the activity of NADH-GDH was increased by ninefold. All transformed herbicide-resistant plants were phenotypically normal and exhibited genomic stability, as were the untransformed plants analysed by flow cytometry. Under greenhouse conditions, they grew to maturity, flowered and set seeds. Stable integration and expression of the bar gene in T0 and T1 plants were confirmed by Southern and Western blot analysis, while integration of the reporter uidA gene did not occur. The bar gene was inherited in a Mendelian fashion by the progeny, as detected by PPT resistance. The production of PPT-resistant plants may have significant practical applications in weed control in fields of L. corniculatus. [ABSTRACT FROM AUTHOR]

Streptomyces transglutaminase (TGase) is an important industrial enzyme that catalyzes cross-linking of proteins. It is secreted as a zymogene and then is activated by proteases under physiological conditions. Although the activation process of TGase has been well investigated, the physiological function of TGase in Streptomyces has not been revealed. In this study, physiological function of TGase from Streptomyces hygroscopicus was found to be involved in differentiation by construction of a TGase gene interruption mutation strain (Δtg). The mutant Δtg showed an absence of differentiation compared with the parent strain. Furthermore, the production of TGase was found to be increased with the extending growth arrest phase of mycelium in submerged cultures. Thus, to enhance yield of TGase, the mycelium differentiation of Streptomyces was regulated via low temperature stress in a 3-L stirred-tank fermenter. The production of TGase increased by 39 % through extending the growth arrest phase for 4 h. This study found that TGase is involved in Streptomyces differentiation and proposed an approach to improve TGase production by regulation of mycelium differentiation in submerged cultures. [ABSTRACT FROM AUTHOR]

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) andN-methyl-N′-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain. [ABSTRACT FROM PUBLISHER]

An antifungal Actinomyces BS-112 strain, with Aspergillus flavus as the target pathogen, was isolated from soil in the forest land of Mountain Tai. This strain showed a strong antagonistic activity against various mold fungi in food and feed. Strain BS-112 was identified as Streptomyces hygroscopicus based on its morphologic, cultural, physiological, biochemical characteristics, cell wall components and 16S rDNA sequence. Four active components were separated and purified from strain BS-112. These four antifungal components were identified as tetrins A and B and tetramycins A and B using spectroscopic analysis including mass spectrometry and nuclear magnetic resonance spectroscopy. Tetrins A and B and tetramycins A and B strongly inhibited the growth of A. flavus, A. alutaceus, A. niger, and A. fumigatus in vitro. [ABSTRACT FROM AUTHOR]

Rapamycin is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces hygroscopicus. To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7 fermentation in medium M1 and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and 16 key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, and shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as follows: 1.0 g/L methyl oleate was added at 0 h; 1.0 g/L lysine was added at 12 h; 0.5 g/L shikimic acid was added at 24 h; 0.5 g/L sodium succinate, 0.1 g/L phenylalanine, 0.1 g/L tryptophan, and 0.1 g/L tyrosine were added at 36 h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3 was increased by 56.6 % compared with it in M2, reaching 307 mg/L at the end of fermentation (120 h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds. [ABSTRACT FROM AUTHOR]