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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]

Abstract 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.

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]

Loeki Enggar Fitri,1,2,* Agustina Tri Endharti,1 Hafshah Yasmina Abidah,3,4,* Alif Raudhah Husnul Khotimah,3,4,* Heni Endrawati1 1Department of Parasitology, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia; 2Malaria Research Group, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia; 3Master Program in Biomedical Science, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia; 4Medical Doctor Profession Education, Faculty of Medical and Health Science, Maulana Malik Ibrahim State Islamic University, Malang, Indonesia*These authors contributed equally to this workCorrespondence: Hafshah Yasmina Abidah, Master Program in Biomedical Science, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia, Tel +62 895 397 064 350, Fax +62 341 564755, Email hafyasmina@student.ub.ac.id; hafshahyasmina@gmail.comPurpose: The study was conducted to investigate the effectivity and the cytotoxicity of fractions 14 and 36K of metabolite extract of Streptomyces hygroscopicus subsp. Hygroscopicus as an antimalarial compounds against Plasmodium berghei in vitro.Methods: Fractions 14 and 36K of metabolite extract of Streptomyces hygroscopicus subsp. Hygroscopicus produced by the fractionation process utilizing the Flash Column Chromatography (FCC) BUCHI Reveleris® PREP. Plasmodium berghei culture was used to assess the antimalarial activity of fractions 14 and 36K. Parasite densities and the ability of parasite growth were determined under microscopic. The cytotoxicity of the fractions was assessed using MTT assays on the MCF-7 cell line.Results: Streptomyces hygroscopicus subsp. Hygroscopicus fractions 14 and 36K have antimalarial activity against Plasmodium berghei, with fraction 14 having the more potent activity. The percentage of Plasmodium berghei-infected erythrocytes was decreased as well as the increase of fraction concentration. Fraction 14 has the highest inhibition of parasite growth at a concentration of 156,25 μg/mL, with an inhibition percentage of 67.73% (R2 = 0.953, p = 0.000). IC50 of fractions 14 and 36K were found at 10.63 μg/mL and 135,91 μg/mL, respectively. The fractions caused morphological damage in almost all asexual stages of the parasite. Both fractions were not toxic against MCF-7, indicating that the fractions have a safe active metabolite.Conclusion: Fractions 14 and 36K of metabolite extract Streptomyces hygroscopicus subsp. Hygroscopicus contains non-toxic compounds that could damage the morphology and inhibit the growth of Plasmodium berghei in vitro.Graphical Abstract: Keywords: antimalarial, cytotoxicity, fraction 14 and 36K, Streptomyces hygroscopicus, Plasmodium berghei

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]

Context: Malaria is still a serious global health problem due to the development of drug resistance. It is necessary to find new drugs with renewable mechanisms that are effective in killing parasites. Our previous research has analyzed more than one compound of polyether ionophore group in ethyl acetate Streptomyces hygroscopicus subsp. hygroscopicus extract. Polyether ionophore is known to have a similar mechanism of action to chloroquine which is potent in inhibiting Plasmodium falciparum glutathione S-transferase (PfGST). Aims: To evaluate the potential effect of polyether ionophore toward PfGST as a target protein through molecular docking. Methods: PfGST was obtained from Protein Data Bank. Test ligands (polyether ionophore) and control ligands (chloroquine) were obtained from PubChem. Pharmacokinetic analysis was done using SwissADME, molecular docking using PyRx 0.9, visualization using LigPlot and PyMOL, and molecular dynamics using YASARA for the best ligand activity. Results: Lenoremycin had the highest binding affinity to PfGST (-8.53 kcal/mol) among other polyether ionophores, and nigericin had the best residue bonding with hydrophobic and hydrogen with a binding affinity of -8.25 kcal/mol compared to chloroquine complex in molecular docking and molecular dynamic simulation. Conclusions: Polyether ionophore could serve as an antimalarial agent better than chloroquine, with nigericin as the best compound candidate in inhibiting PfGST compared to other polyether ionophores.

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]

Abstract Background Ascomycin is a highly valuable multifunctional drug which exhibits numerous biological properties. Being an immunosuppressant, it is known to prevent graft rejection in humans and has potential to treat varying skin ailments. Its derivatives represent a novel class of anti-inflammatory macrolactams. But the biosynthetic machinery of ascomycin is still unclear. Main body of the abstract Due to the structural complexity, there occurs difficulty in its chemical synthesis; therefore, microbial production has been preferred by using Streptomyces hygroscopicus subsp. ascomyceticus. Through several genetic manipulation and mutagenesis techniques, the yield can be increased by several folds without any difficulties. Genetic engineering has played a significant role in understanding the biosynthetic pathway of ascomycin. Short conclusion Recently, many efforts have been made to utilize the therapeutic effects of ascomycin and its derivatives. This article covers concepts related to the production kinetics of ascomycin including an update of the ongoing yield improvement techniques as well as screening method of novel strains for ascomycin production.

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]

Apple fruit diseases caused by phytopathogenic species of the genus Alternaria have become current in recent years in Serbia as well as in other parts of the world. Due to the fact that pesticides have a number of side effects on the environment and human health, scientists around the world are concerned with finding alternative ways to control this important food. Usage of microorganisms, optimization of the medium and process conditions for their cultivation and production of biofungicides are the actual research direction. The effect of aeration on biofungicide production by Streptomyces hygroscopicus was studied in a 3 l stirred tank bioreactor using a previously optimized cultivation medium with glycerol as a carbon source. Aeration rates of 0.5, 1.0, and 1.5 vvm with constant agita­tion speed of 100 rpm were studied. It was found that the greatest production of antifungal metabolites effective against two Alternaria alternata isolates occurred with an aeration of 1.5 vvm and an agitation speed of 100 rpm. Statistical analysis showed that the largest inhi­bition zone diameters of A. alternata KA10 (78 mm) and T1Jg3 (78.67 mm) mycelial growth were achieved under the indicated conditions in 96 h of cultivation.

Abstract Ascomycin, a key intermediate for chemical synthesis of immunosuppressive drug pimecrolimus, is produced by Streptomyces hygroscopicus var. ascomyceticus. In order to improve the strain production, the original S. hygroscopicus ATCC 14891 strain was treated here with atmospheric and room temperature plasma to obtain a stable high-producing S. hygroscopicus SFK-36 strain which produced 495.3 mg/L ascomycin, a 32.5% increase in ascomycin compared to the ATCC 14891. Then, fermentation medium was optimized using response surface methodology to further enhance ascomycin production. In the optimized medium containing 81.0 g/L soluble starch, 57.4 g/L peanut meal, and 15.8 g/L soybean oil, the ascomycin yield reached 1466.3 mg/L in flask culture. Furthermore, the fermentation process was carried out in a 5 L fermenter, and the ascomycin yield reached 1476.9 mg/L, which is the highest ascomycin yield reported so far. Therefore, traditional mutagenesis breeding combined with medium optimization is an effective approach for the enhancement of ascomycin production.

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]

The limited availability of nutrient Fe severely impairs the health of almost all organisms. Endophytic actinobacteria can benefit the host plant in different ways. We previously inferred that the rice (Oryza) endophytic Streptomyces hygroscopicus OsiSh-2 possesses a highly efficient Fe-acquisition system. In this work, we first evaluated the effects of OsiSh-2 on the Fe-deficiency resilience of the host rice. The results demonstrated that the inoculation of OsiSh-2 considerably increased the plant biomass, Fe concentration and translocation factor, and chlorophyll content, and net leaf photosynthetic rate under Fe limiting condition. The expression of genes involved with Fe3+-reduction-related strategy in rice was up-regulated, while that involved with Fe3+-chelation-related strategy was down-regulated by OsiSh-2 treatment. Meanwhile, the OsiSh-2-rice symbiont showed enhancement of Fe3+-chelate reductase activity, total siderophore production, and acidification trend in the rhizosphere under Fe deficiency compared to plants without this endophyte. In conclusion, endophytic OsiSh-2 could protect plants against Fe-deficient stress by a sophisticated interaction with the host, including modulating Fe chelation, solubilization, reduction and translocation, ultimately leading to enhanced fitness of plant. Image 1 • Endophytic Streptomyces hygroscopicus OsiSh-2 enhanced rice tolerance to Fe deficiency. • OsiSh-2 modified root architecture, photosynthesis, Fe absorption and translocation of rice under Fe deficiency conditions. • OsiSh-2-rice interaction involving two Fe-acquisition processes enhanced Fe-deficiency resilience in rice. • OsiSh-2 has the potential to be utilized as a biofertilizer in rice cultivation. [ABSTRACT FROM AUTHOR]

Background: Ascomycin is a highly valuable multifunctional drug which exhibits numerous biological properties. Being an immunosuppressant, it is known to prevent graft rejection in humans and has potential to treat varying skin ailments. Its derivatives represent a novel class of anti-inflammatory macrolactams. But the biosynthetic machinery of ascomycin is still unclear. Due to the structural complexity, there occurs difficulty in its chemical synthesis; therefore, microbial production has been preferred by using Streptomyces hygroscopicus subsp. ascomyceticus. Through several genetic manipulation and mutagenesis techniques, the yield can be increased by several folds without any difficulties. Genetic engineering has played a significant role in understanding the biosynthetic pathway of ascomycin. Short conclusion: Recently, many efforts have been made to utilize the therapeutic effects of ascomycin and its derivatives. This article covers concepts related to the production kinetics of ascomycin including an update of the ongoing yield improvement techniques as well as screening method of novel strains for ascomycin production. [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]

• Positive effect of furfural on validamycin-A (VAL-A) fermentation was found. • Xylose mother liquor medium with 1 g/L furfural enhanced the VAL-A titer by 40 % compared to control. • The work is helpful to VAL-A fermentation using hemicellulose hydrolysates. Furfural is one of main inhibitors in hemicellulose hydrolysates such as xylose mother liquor, but its positive effect on the production of validamycin-A (VAL-A), a widely used agricultural antibiotic, was interestingly found in fermentation of Streptomyces hygroscopicus 5008. The furfural level in medium up to 1 g/L was effectively converted to furfuryl alcohol and furoic acid by the microorganism. Both intracellular H 2 O 2 level and ValG enzyme activity of the cells were enhanced by furfural addition. Xylose mother liquor medium with supplementation of about 1 g/L furfural could enhance the VAL-A titer by 39 %. This work is helpful to VAL-A fermentation using the hemicellulose hydrolysate. [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]

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]

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]

Abstract Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of "multiple integrases-multiple attB sites". This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts. Highlights • An innovative methodology was established for multi-locus genomic integration of genes/pathways in microbial systems. • A plug-and-play, multi-copy integration toolkit derived from five orthogonal SSR systems was developed in actinomycetes. • Multi-copy overexpression of presurcor biosynthetic genes accA2BE facilitated actinorhodin biosynthesis by 4.6-fold. • Stable amplification of 5-oxomilbemycin BGC achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L). [ABSTRACT FROM AUTHOR]

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Biotechnologically produced antifungal compounds for control of plant diseases caused by phytopathogenic fungi represent a promising alternative to synthetic pesticides. Fungi from the genera Alternaria and Fusarium are listed among important storage pathogens of apple fruits. A. alternata causes significant annual losses of apple fruit. Also, F. avenaceum is one of the most commonly encountered Fusarium species identified as the causal agent of a wet apple core rot. Species of the Streptomyces genera are soil bacteria that produce significant quantities of bioactive compounds in appropriate media. Defining the culture medium composition is the basis of bioprocesses development. Nitrogen source is critical component of cultivation medium and also the most useful tool for stimulation of the antifungal metabolites production. The aim of this study was to select the best nitrogen sources in medium for the production of antifungal compounds effective against A. alternata and F. avenaceum by S. hygroscopicus. Activity of the cultivation liquids on A. alternata and F. avenaceum isolates was tested in vitro using well diffusion method. The results in­dicate that maximum inhibition zone was reached in medium with soybean meal as nitrogen source with inhibition diameter more than 35 mm for both tested fungi. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR-31002]

The effect of isatin derivatives as a nitrogen source on antibiotic (Hexaene H-85 and Azalomycine B) production by Streptomyces hygroscopicus CH-7 was studied. Isatin-3-hydrazone, 5-chloroisatin-3-hydrazone, isatin-3-tosylhydrazone, 5-chloroisatin-3-tosylhydrazone, isatin-3-(4`-hidroxy)benzoilhydrazone and 5-chloroisatin-3-(4`-hidroxy)benzoilhydrazone were synthesized in a crude glycerol, obtained during the biodiesel production from edible sunflower oil. The highest concentration of Hexaene H-85 is achieved with 5-chloroisatin-3-hydrazone (197 mg/cm3) in medium, while isatin-3-hydrazone has a greatest impact on Azalomycine B production (72 μg/cm3). [Projekat Ministarstva nauke Republike Srbije, br. III 45001]

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]

Transglutaminase 2 (TGase2), a multifunctional enzyme exhibiting both transamidase and kinase activity, is involved in a variety of cellular processes and diseases. However, details of the regulation of TGase2 have not been reported due to the lack of a suitable assay to examine both activities on the same platform under near-physiologic conditions. Thus, we developed an on-chip dual enzyme activity assay for TGase2 to simultaneously monitor the transamidase and kinase activities. Reaction mixtures specific for each enzyme activity were applied onto osteopontin arrays, and enzyme activity was monitored by sequential probing with Cy5-strepavidin and Pro-Q Diamond stain. This approach was used to determine the optimal concentrations of ATP, Mg 2+ , and Ca 2+ for dual-activity assays. The optimized assay was then used to investigate regulation of TGase2 transamidase and kinase activities by various cofactors that could potentially affect its conformation. Monothiol- and disulfide-containing compounds differentially regulated TGase2 transamidase and kinase activities. Acetylation of TGase2 activated the kinase activity but had no effect on the transamidase activity. Phosphorylation and dephosphorylation of TGase2 reciprocally regulated the transamidase and kinase activities. The new approach described here is thus useful for screening potential regulators of TGase2 transamidase and kinase and investigating the pathogenesis of TGase2-associated diseases. [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]

An approach toward (−)-enterocin, an antibiotic isolated from Streptomyces hygroscopicus, is described. Its compact, heavily oxidized protoadamantane core represents a daunting challenge for an efficient synthesis. Convergent assembly of its 2-oxabicyclo[3.3.1]nonane core with a cuprate-mediated Barbier reaction is disclosed. Its functionalization to a suitable substrate for a biomimetic aldol to close the final ring of the natural product is evaluated. [ABSTRACT FROM AUTHOR]

Validamycin A (Val-A) is produced by Streptomyces as a secondary metabolite with wide agricultural applications of controlling rice sheath blight, false smut and damping-off diseases. The effect of alkaline pH shock on enhancing Val-A production and its mechanism were investigated. A higher yield of Val-A was achieved by NaOH shock once or several times together with faster protein synthesis and sugar consumption and alkaline pH shock can increase Val-A production by 27.43%. Transcription of genes related to amino acid metabolism, carbon metabolism and electron respiratory chain was significantly up-regulated, accompanied by the substantial increase of respiratory activity and glutamate concentration. Val-A production was promoted by a series of complex mechanisms and made a response to pH stress signal, which led to the enhancement of glutamate metabolism and respiration activity. The obtained information will facilitate future studies for antibiotic yield improvement and the deep revealment of molecular mechanism. [ABSTRACT FROM AUTHOR]

Postharvest mango (Mangifera indica L.) fruit suffer tremendous loss due to anthracnose inflicted by Colletotrichum spp. Fungicides and heat treatment are often used to control the disease, while biocontrol agents are eco-friendly alternatives. Streptomyces are antagonistic organisms against plant pathogens; however, the mechanisms and active components are unclear. Here, we evaluated the antifungal actions of S. hygroscopicus metabolites tanespimycin and rapamycin against anthracnose. Within the range of 0.01–0.1 μM, tanespimycin and rapamycin reduced fungal growth by at least 60.4% and 40.4%, respectively in vitro. In mango fruit, tanespimycin upregulated MiHSP101 , MiHSP90 , MiHSP70 , and MiHSP18.2 by 5.8-, 1.9-, 4.8-, and 3.3-fold, respectively, and increased HSP101 and HSP70 protein by 2.2-fold. On the other hand, rapamycin did not affect HSP expression. Pathogen-challenge assays revealed tanespimycin and rapamycin exerted antifungal and defense-eliciting dual actions, where pre-challenge treatment of tanespimycin and rapamycin inhibited pathogen growth by 73.7% and 78.4%, respectively in mango 5 d post infection. Finally, in a 20 °C storage experiment, tanespimycin and rapamycin extended the fruit shelf life by 6 d and exerted comparable antifungal effects as hot water treatment without deteriorating fruit quality. In conclusion, we elucidated the antifungal and defense-eliciting actions of S. hygroscopicus metabolites and revealed mechanistic discrepancies regarding HSP regulation. These findings support the development of new disease management strategies and advance the knowledge regarding the roles of HSP in crop defense. • Tanespimycin and rapamycin are antibiotics derived from Streptomyces hygroscopicus. • Anti-anthracnose effect of tanespimycin and rapamycin was tested for the first time. • Tanespimycin and rapamycin (0.1–1 μM) inhibit Colletotrichum gloeosporioides. • Tanespimycin induces HSP101 , HSP90 , HSP70 , and HSP18.2 expression in mango. • Tanespimycin and rapamycin maintain quality and extend shelf-life of mango by 6 d. [ABSTRACT FROM AUTHOR]

Losses of apple fruit during storage are mainly caused by fungal phytopathogens. Traditionally, postharvest fungal disease is controlled by the application of synthetic fungicides. However, the harmful impact on environment as well as human health largely limits their application. To reduce these problems in agrochemicals usage, new compounds for plant protection, which are eco-friendly, should be developed. The aim of this study is optimization of medium composition in terms of glucose, soybean meal and phosphates content, by applying response surface methodology, for the production of agents with antifungal activity against Aspergillus spp. For biosynthesis was used strain of Streptomyces hygroscopicus isolated from the environment. Experiments were carried out in accordance with Box-Behnken design with three factors on three levels and three repetitions in the central point. Antifungal activity of the obtained cultivation mediums against Aspergillus oryzae and Aspergillus niger was determined, in vitro, using the diffusion - disc method. For determination optimal medium components desirability function was used. Achieved model predicts that the maximum inhibition zone diameter (40.93 mm) against test microorganisms is produced when the initial content of glucose, soybean meal and phosphates are 47.77 g/l, 24.54 g/l and 0.98 g/l, respectively. To minimize the consumption of medium components and costs of effluents processing, additional three sets of optimization were made. The chosen method for optimization of medium components was efficient, relatively simple and time and material saving. Obtained results can be used for the further techno-economic analysis of the process to select optimal medium composition for industrial application.

A media consisting of isatin-Schiff bases and its inclusion complexes with β-cyclodextrine was developed to maximize the production of antibiotics Hexaene H-85 and Azalomycine β by Streptomyces hygroscopicus CH-7. The media with β-cyclodextrine inclusion complex of isatin-3-thiosemicarbazone resulted in the maximum antibiotics concentration of 493 µg/cm3 for Hexaene H-85 and 191 µg/cm3 for Azalomycine B. The production of Hexaene H-85 and Azalomycine is higher when β-cyclodextrine complex is added as a nitrogen source, comparing to pure isatin-Schiff base. The maximum concentration of hexaene H-85 in medium with inclusion complex of isatin-Schiff base is 1.4-2.3 times higher than the basal medium. The maximum production of Azalomycine is 2.1-3.4 times higher in media with inclusion complex of Schiff base. During the fermentation process, the nutrient media with β-cyclodextrine inclusion complexes with isatin-Schiff bases affect the strain morphology, since it is in the form of compact pellets, which are formed from short and long, branched filaments [Projekat Ministarstva nauke Republike Srbije, br. III 45001]

Geldanamycin and the closely related herbimycins A, B, and C are benzoquinone-type ansamycins with antitumoral activity. They are produced by Streptomyces hygroscopicus var. geldanus, Streptomyces lydicus and Streptomyces autolyticus among other Streptomyces strains. Geldanamycins interact with the Hsp-90 chaperone, a protein that has a key role in tumorigenesis of human cells. Geldanamycin is a polyketide antibiotic and the polyketide synthase contain seven modules organized in three geldanamycin synthases genes named gdmAI, gdmAII, and gdmAIII. The loading domain of GdmI activates AHBA, and also related hydroxybenzoic acid derivatives, forming geldanamycin analogues. Three regulatory genes, gdmRI, gdmRII, and gdmRIII were found associated with the geldanamycin gene cluster in S. hygroscopicus strains. GdmRI and GdmRII are LAL-type (large ATP binding regulators of the LuxR family) transcriptional regulators, while GdmRIII belongs to the TetR-family. All three are positive regulators of geldanamycin biosynthesis and are strictly required for expression of the geldanamycin polyketide synthases. In S. autolyticus the gdmRIII regulates geldanamycin biosynthesis and also expression of genes in the elaiophylin gene cluster, an unrelated macrodiolide antibiotic. The biosynthesis of geldanamycin is very sensitive to the inorganic phosphate concentration in the medium. This regulation is exerted through the two components system PhoR-PhoP. The phoRP genes of S. hygroscopicus are linked to phoU encoding a transcriptional modulator. The phoP gene was deleted in S. hygroscopicus var geldanus and the mutant was unable to grow in SPG medium unless supplemented with 5 mM phosphate. Also, the S. hygroscopicus pstS gene involved in the high affinity phosphate transport was cloned, and PhoP binding sequences (PHO boxes), were found upstream of phoU, phoRP, and pstS; the phoRP-phoU sequences were confirmed by EMSA and nuclease footprinting protection assays. The PhoP binding sequence consists of 11 nucleotide direct repeat units that are similar to those found in S. coelicolor Streptomyces avermitilis and other Streptomyces species. The available genetic information provides interesting tools for modification of the biosynthetic and regulatory mechanisms in order to increase geldanamycin production and to obtain new geldanamycin analogues with better antitumor properties.

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]