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A fosmid metagenomic library containing 9.7 × 104 clones was constructed. A novel esterase, XtjR8, was isolated through functional screening. XtjR8 shared the maximum amino acid identity (44%) with acetyl-hydrolase from Streptomyces hygroscopicus, and was classified into family IV esterase. XtjR8 exhibited the highest hydrolytic activity for p-nitrophenyl acetate at 40 °C and pH 8.0, and presented more than 40% activity from 20 °C to 80 °C. More importantly, XtjR8 displayed the ability to hydrolyze both phthalate monoesters and diesters, this feature is extremely rare among previously reported esterases. Site-directed mutagenesis experiments revealed that the catalytic triad residues were Ser152, Glu246, and His276. Among them, Ser152 formed a hydrogen bond with dibutyl phthalate (DBP) by molecular docking, Gly84, Gly85, and Leu248 of conserved motifs formed hydrophobic interactions with DBP, respectively, which were important for the catalytic activity. Considering its wide range of temperature and hydrolytic potential toward phthalate esters, XtjR8 will be served as an interesting candidate for biodegradation and industrial applications.

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 H2O2 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.

Microbial antagonists and their bioactive metabolites provide one of the best alternatives to chemical pesticides to control crop disease for sustainable agriculture and global food security. The rice endophyte Streptomyces hygroscopicus OsiSh-2, with remarkable antagonistic activity towards the rice blast fungus Magnaporthe oryzae, was reported in our previous study. The present study deciphered the possible direct interaction mode of OsiSh-2 against M. oryzae. An in vitro antibiotic assay for OsiSh-2 culture filtrate revealed strong suppression of mycelial growth, conidial germination and appressorial formation of M. oryzae. Meanwhile, severe morphological and internal abnormalities in M. oryzae hyphae were observed under a scanning electron microscope and transmission electron microscope. Foliar treatment of rice seedlings by OsiSh-2 culture filtrate in the greenhouse and in the field showed 23.5% and 28.3% disease reduction, respectively. Correspondingly, OsiSh-2 culture filtrate could induce disorganized chitin deposition in the cell wall and lowered ergosterol content in the cell membrane of M. oryzae. Additionally, cell wall integrity pathway activation, large cell electrolytes release, reactive oxygen species accumulation and tricarboxylic acid cycle-related enzyme activity changes were found in M. oryzae. All these results suggested that the direct antagonistic activity of OsiSh-2 against M. oryzae may be attributed to damaging the integrity of the cell wall and membrane and disrupting mitochondrial function in the pathogen.

Two new natural compounds, astolides A,B, were isolated from Streptomyces hygroscopicus collected from the alkaline soil obtained from Saratov region, Russia. The structures were elucidated by interpretation of UV spectroscopic, MS, 1D and 2D NMR data. The relative configurations were determined by analysis of NOESY/ROESY spectra and coupling constants. The isolated compounds were evaluated for their inhibitory activity against bacteria, fungi and yeasts and showed pronounced antifungal activity. Both compounds are noticeably cytotoxic, being active against doxorubicin-resistant human leukemia cell line.

Leaf spot disease causes devastating damage to oil palm seedlings and reduces both quality and quantity of the produced seedlings. This study aimed to apply the selected microorganisms Streptomyces hygroscopicus subsp. angustmyceticus NR8-2, Trichoderma harzianum TM2/1, and endophytic Trichoderma V76-12, to the control of leaf spot disease caused by Curvularia oryzae on oil palm leaves, in vitro, in vivo and in field conditions. The isolate V76-12 inhibited the growth of C. oryzae the most at 85.71% inhibition, followed by the isolates TM2/1 (75.71%) and NR8-2 (72.50%) in dual culture tests. Volatile antifungal effects were observed for V76-12 with 68% inhibition, followed by the isolates TM2/1 (45%) and NR8-2 (10%) in a bioassay. Crude metabolites of the isolates inhibited the mycelial growth of C. oryzae in agar diffusion tests. In pot experiments, the V76-12 gave disease severity index (DSI) of about 21%, similar to that with a reference chemical (20%), whereas TM2/1 and NR8-2 gave 66% and 75%, respectively. In naturally infested cases, oil palm seedlings treated with V76-12 showed 35.33% DSI, which is statistically significantly below those obtained with TM2/1, NR8-2 and the chemical (48.67%, 53.33% and 49.33%, respectively). The control treatment without antagonistic microorganisms gave the most severe DSI at 75.33%. The results also show that treatment with the selected isolates enhanced the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) enzymes in the oil palm. The V76-12 isolate was the most effective treatment tested in reducing leaf spot disease of oil palm seedlings, due to its ability to inhibit mycelial growth in vitro, to reduce disease symptoms in vivo and in natural fields, as well as to enhance PAL, POD and PPO activities in the oil palm leaves.

Zika virus (ZIKV) infection could lead to Guillain-Barre syndrome in adults and microcephaly in the newborns from infected pregnant women. To date, there is no specific drug for the treatment of ZIKV infection. In this study, we sought to screen inhibitors against ZIKV infection from a natural product library. A ZIKV replicon was used to screen a library containing 1680 natural compounds. We explored the antiviral mechanism of the compound candidate in vitro and in vivo infection models. Ascomycin, a macrolide from Streptomyces hygroscopicus, was identified with inhibitory effect against ZIKV in Vero cells (IC50 = 0.11 μM), hepatoma cell Huh7 (IC50 = 0.38 μM), and glioblastoma cell SNB-19 (IC50 = 0.06 μM), far below the cytotoxic concentrations. Mechanistic study revealed that Ascomycin suppressed ZIKV RNA replication step during the life cycle and the regulation of calcineurin-NFAT pathway maybe involved in this inhibitory effect, independent of innate immunity activation. Moreover, we found that Ascomycin also inhibited the infection of other Flaviviridae members, such as hepatitis C virus and dengue virus. Ascomycin reduced ZIKV load in blood by up to 3500-fold in A129 mice. Meanwhile, the infection in the mice brain was undetectable by immunohistochemistry staining. Together, these findings reveal a critical role of Ascomycin in the inhibition of ZIKV and related viruses, facilitating the development of novel antiviral agents.

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.

Magnaporthe oryzae is a major hazard to the growth and yield of rice and other graminaceous crops. The rice endophyte Streptomyces hygroscopicus OsiSh-2 has been reported to have a strong antagonistic effect on M. oryzae. However, the defence mechanism of OsiSh-2 against pathogens remains to be established. In this study, a combination of physiological, biochemical and label-free proteomics analyses were used to investigate the global response of OsiSh-2 to the toxins of M. oryzae (Mo-toxins). When exposed to Mo-toxins, OsiSh-2 regulated its growth rate to get rapid transition to the stationary growth phase where stronger antibiotic activity was achieved. Additionally, OsiSh-2 triggered reactive oxygen species (ROS) generation, hydrolase activity and biofilm formation in response to Mo-toxin stress. Finally, proteomic profiling confirmed that OsiSh-2 possesses the following sophisticated coping strategies against Mo-toxins: (1) sensing and transmittance of Mo-toxin signals by signal-transmitting proteins; (2) damaging the toxicity of M. oryzae by hydrolytic enzymes and other secondary products; and (3) guaranteeing the secretion and function of proteins by proteases and protein isomerases. Our research may promote the understanding of the interaction between antagonistic microbes and further broaden the scope of utilizing endophytic Streptomyces for biological control.

Compounds capable of inhibiting the efflux pump mechanism are a promising alternative against bacterial resistance because, when combined with antibiotics, they can increase the effectiveness of these drugs by inhibiting active efflux. Elaiophylin, derived from Streptomyces hygroscopicus, is a natural antibiotic that exhibits a variety of biological activities, including antibacterial activity. However, its potential as an inhibitor of the bacterial efflux mechanism has not been investigated. This study evaluated the ability of Elaiophylin to inhibit the NorA efflux pump in Staphylococcus aureus strains. Therefore, tests were performed to obtain the Minimum Inhibitory Concentration (MIC) and to verify the ability of Elaiophylin to potentiate the MIC of the antibiotic Norfloxacin and Ethidium Bromide (EtBr), known substrates of NorA efflux. Real-time PCR and molecular docking assays were also performed to assess the potential of Elaiophylin against NorA. The strains SA-1199 (wild type) and SA-1199B (NorA over-expressed) of S. aureus were used for this study. The results showed that Elaiophylin significantly decreased the MIC of Norfloxacin and EtBr, increasing the activity of these substrates against S. aureus, which carries the efflux protein NorA. However, Elaiophylin provided a non-significant reduction in norA gene expression, however, molecular docking demonstrated a high binding affinity between Elaiophylin and NorA efflux protein, indicating that Elaiophylin can act as a potential NorA in S. aureus.

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhy-drazine (DNPH) in 10 mmol·L -1 H 3 PO 4 at 37℃ for 45 min and the derivatives were separated on Eclipse XDB-C 18 (5 μm, 4.6 mm×150 mm) column at 30℃ eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by compu-tational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 μg·mL -1 . The intra-and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engi-neered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.

A multiplex PCR system was developed to trace GM maize events in food and feed. Genetic elements of 38 single GM maize events were investigated, and 10 screening targets were selected as follows: promoters [Cauliflower mosaic virus (CaMV) 35S, rice actin 1 gene (rAct1)], terminators [CaMV 35S, Agrobacterium nopaline synthase (nos), proteinase inhibitor II of potato (PinII), heat shock protein 17 of wheat (hsp17)] and structural genes [cry1A gene derived from Bacillus thuringiensis, phosphinothricin acetyltransferase gene derived from Streptomyces hygroscopicus (bar), and Streptomyces Viridochromogenes (pat), 5-enolpyruvulshikimate-3-phosphate synthase enzyme derived from Agrobacterium tumefaciens strain CP4 (cp4-epsps)]. These screening targets were combined in three individual multiplex PCR assays. The method specificity was confirmed by detecting the specific amplification of DNA derived from 16 different GM maize events. The limit of detection of the multiplex PCR system was tested using the certified reference materials of GM maize events with different GM % (w/w) and approximately 0.1%. To test the applicability of the developed screening method, 22 food samples containing maize as ingredients, and 36 maize kernels were analyzed. Various types of single and/or stacked GM events were found in 11 food and 20 maize kernel samples. These results demonstrate that the screening method developed may be useful for tracking GM maize events in food and feed.

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Miah, Farzana. John Innes Institute; Reino Unido Fil: Stevenson, Clare E. M.. John Innes Institute; Reino Unido Fil: Lawson, David M.. John Innes Institute; Reino Unido Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Bornemann, Stephen. John Innes Institute; Reino Unido

Root rot is one of the major constraints on strawberry production worldwide, and no effective control measure is available yet. Therefore, the objective of this study was to develop a new bio-agent for controlling this disease. The antagonistic strain B04 was isolated from the rhizosphere soil of a healthy strawberry plant, and it strongly inhibited the growth of phytopathogenic fungi, especially that of Fusarium oxysporum. Strain B04 was identified as Streptomyces hygroscopicus by morphological and cultural traits and by 16S rRNA gene sequence analysis. Strain B04 could utilize numerous carbon sources and produce extracellular fungal cell wall-degrading enzymes including chitinase, β-1,3-glucanase, cellulase and protease. Agro-industrial waste materials, specifically wheat bran and vermicompost, were used for solid-state fermentation with strain B04 to produce bioorganic fertilizer. On the seventh day of fermentation, the spore number peaked at 5.3 × 1010 CFU g−1. Pot and field experiments were performed to evaluate the effects of bioorganic fertilizer B04 (BOF-B04) on the infested soils. Each experiment included three groups: (1) control treatment, (2) organic fertilizer, and (3) bioorganic fertilizer B04. BOF-B04 significantly increased the fresh weight of strawberry plants compared to the control and also decreased the incidence of root rot. The biocontrol efficiencies of BOF-B04 were 63.35% and 56.77% in pot and field experiments, respectively. Moreover, application of BOF-B04 also altered the microbial community structure of the rhizosphere soil. The number of fungi in rhizosphere soil decreased markedly, by 82.43% compared to the control. In contrast, bacterial and actinomycete densities were dramatically higher than in the control. Application of BOF-B04 decreased the levels of stress-related enzyme activity and of malondialdehyde compared to the control. Consequently, this study showed that bioorganic fertilizer B04 is a potential biocontrol agent for controlling strawberry root rot.

Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that can be attached to substrate proteins in Actinobacteria. The modification process is defined as pupylation and is associated with proteasome-mediated protein degradation in mycobacteria and streptomycetes. Here, we report the pupylation of Streptomyces hygroscopicus 5008 in vitro. Each component of the Pup system was expressed in Escherichia coli and poly-Pup chains were observed by western blot analysis. Though only one potential Pup substrate (SHJG_3659) was identified using MS/MS, we verified this candidate and other predicted substrates by a reconstituted Pup system in E. coli. In addition, we discuss the depupylation activity of Dop (a Pup activation enzyme). The results presented here show that pupylation exists in S. hygroscopicus and that a reconstituted Pup system can function in vitro in a heterologous host.

Streptomyces hygroscopicus UFPEDA 3370 was fermented in submerged cultivation and the biomass extract was partitioned, obtaining a fraction purified named EB1. After purification of EB1 fraction, nigericin free acid was obtained and identified. Nigericin presented cytotoxic activity against several cancer cell lines, being most active against HL-60 (human leukemia) and HCT-116 (human colon carcinoma) cell lines, presenting IC50 and (IS) values: 0.0014 μM, (30.0) and 0.0138 μM (3.0), respectively. On HCT-116, nigericin caused apoptosis and autophagy. In this study, nigericin was also screened both in vitro and in silico against a panel of cancer-related kinases. Nigericin was able to inhibit both JAK3 and GSK-3β kinases in vitro and its binding affinities were mapped through the intermolecular interactions with each target in silico.

2-Phosphinomethylmalate synthase (PMMS) from Streptomyces hygroscopicus catalyzes the first step in the biosynthesis of the herbicide bialophos using 3-phosphinopyruvic acid and acetyl coenzyme A as substrates to form 2-phosphinomethylmalic acid and coenzyme A. PMMS belongs to the Claisen condensation-like (CC-like) subgroup of the DRE-TIM metallolyase superfamily, which uses conserved active site architecture to catalyze a functionally-diverse set of reactions. Analysis of a sequence similarity network for the CC-like subgroup identified PMMS and the related R-citrate synthase in an early-diverging cluster suggesting that this group of sequences are more distinct in relation to other Claisen-condensation subgroup members. To better understand the structure/function landscape of the CC-like subgroup PMMS was recombinantly expressed in Escherichia coli, purified, and characterized with respect to its enzymatic properties. Using oxaloacetate as a substrate analog, the recombinantly-produced enzyme exhibited improved Michaelis constants relative to the previously reported natively-produced enzyme. Results from pH rate profiles and kinetic isotope effects were consistent with results from other members of the CC-like subgroup supporting acid-base chemistry and hydrolysis of the direct Claisen-condensation product as the rate-determining step. Results of site-directed mutagenesis experiments indicate that PMMS uses an active-site architecture similar to homocitrate synthase to select for a dicarboxylic acid substrate.

Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin (FK520) in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5 L bioreactor. To find the relation between FK520 production and foaming, effects of 10 fermentation parameters including organic acids and membrane permeability were investigated. The results suggest that acetate accumulation caused by short period oxygen deficiency and fast consumption of glucose is the reason for increased foaming and declined FK520 production. Therefore, a fed-batch fermentation strategy was developed to reduce the accumulation of acetate. After optimization, the maximum acetate concentration dropped from 320 mg·L − 1 to 157 mg·L − 1 , decreased by 50.8%, and the maximum foam height reduced from 5.32 cm to 3.74 cm, decreased by 29.7%, while the maximum FK520 production increased from 375 mg·L − 1 to 421 mg·L − 1 , improved by 12%.

Validamycin A (VAL-A) is an important agricultural antibiotic produced by Streptomyces hygroscopicus 5008, which uses starch as carbon source occupying about 20% of total production cost. To reduce the medium cost, corncob hydrolysate - a hemicellulose hydrolysate was applied as a low-cost substrate to VAL-A fermentation. It was found that three major sugars in corncob hydrolysate including d-glucose, d-xylose and l-arabinose could all be utilized by S. hygroscopicus 5008 to produce VAL-A while d-xylose was the main contributor. A higher VAL-A production titer from d-xylose was achieved by using a genetically engineered strain TC03 derived from S. hygroscopicus 5008, which resulted in 1.27-fold improvement of VAL-A production from the medium containing 13% (v/v) corncob hydrolysate compared to that by its original strain. A medium cost analysis was done and compared with previous reports. This work indicates a great potential of the hemicellulose hydrolysate as substrate for antibiotic fermentation.

Antifungal potential of Streptomyces hygroscopicus metabolites was tested against the isolates of Colletotrichum spp., obtained from rotten apple fruits. Experimental factorial design and response surface methodology were employed to optimize medium composition and enhance antifungal components productivity by S. hygroscopicus. Results of optimization suggest that the optimal medium composition is (g/L): glycerol (13.92), yeast extract (5.00), phosphates (0.63), CaCO3, (3.0), NaCl, (3.0), MgSO4, (0.5). Further research was performed to validate the obtained results and confirm their applicability in planta on artificially inoculated apple fruits. The obtained efficacy after 14 days of incubation was 99.3% against Colletotrichum acutatum and 99.8% against Colletotrichum gloeosporioides. Application of supernatant of S. hygroscopicus cultivation medium, produced under defined conditions, in biological control of apple rot could offer safe and healthy apple fruits on the market, while simultaneously helping to solve the problem of waste glycerol disposal in the biodiesel industry.

The combination of cell morphology investigation and metabolomics analysis were carried out to improve rapamycin production rationally in Streptomyces hygroscopicus. First, the correlation between morphology and rapamycin production by S. hygroscopicus was investigated with statistical experiments, and the longer filaments and higher branching frequency in hyphal growth in the periphery of the pellets were conducive to high production of rapamycin. Then, to explore the potential mechanism influencing morphology of S. hygroscopicus, metabolomics-based approach was employed to reveal intracellular metabolic change under different morphology characteristics conditions and identify the potential key metabolites in response to different morphology characteristics in submerged fermentation process. Furthermore, 19 potential key metabolites related pellet morphology and rapamycin production were revealed. Based on the analysis of key metabolites change combined with related metabolic pathway, as well as the change in pellet morphology characteristics, the dissolved oxygen supplied strategies were proposed, and rapamycin production was ultimately increased by 63.9%, reaching 536 mg/L under optimal oxygen supplied strategy condition. The strategy developed here could be extended to titer improvement and process optimization of other products.

The production of antibiotics by filamentous organisms is often dependent on the morphology and pellet size within the culture. An increase in viscosity by adding carboxymethylcellulose (CMC) could reduce pellet size and increase rapamycin production in the cultivation of Streptomyces hygroscopicus. A maximum yield of 410 mg/L of rapamycin was obtained when 3 g/L of CMC was added to a 5 L agitation bioreactor, as compared to 260 mg/L in the control batch (without the addition of CMC). Using an airlift bioreactor instead of an agitation tank can avoid the break-down of pellets due to the high shear force with the latter. The results indicate that an aeration rate of 1.5 vvm could achieve the maximum rapamycin production of 543 mg/L in an airlift bioreactor with 3 g/L of CMC adding. The application of both viscosity control and an airlift bioreactor has great potential with regard to commercialized rapamycin production.

Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant activity, produced by Streptomyces hygroscopicus var. ascomyceticus. However, the specific regulatory mechanism of the ascomycin biosynthesis pathway remains poorly understood. Here, we show that FkbN is a pathway-specific positive regulator for the ascomycin biosynthesis. Inactivation of fkbN led to a complete loss of ascomycin production ability. Compared with the parental strain, the ascomycin yield of a constructed fkbN overexpression strain increased by 400%–1800 mg/L. Transcriptional levels of the genes of encoding precursor, PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetases) were blocked in the fkbN in-frame deletion strain and greatly upregulated in the fkbN overexpression strain. Electrophoretic mobility shift assays (EMSAs) revealed that FkbN-DBD protein specifically bound to the intergenic regions of fkbR1-fkbE and fkbB-fkbO. To explore the high conservation among regulators homologous to FkbN, EMSAs showed that the FkbN-DBD protein directly bound to the putative promoters of different macrolide biosynthetic gene clusters. We did further study in the fkbN deletion strain by heterologous genetic complementation with single copy of the homologous LAL family regulators. Results showed that all the heterologous complementation strains restored the production of ascomycin. Introduction of a single copy of fkbN into the tacrolimus producing strain S. tsukubaensis or rapamycin producing strain S. hygroscopicus, respectively, both enhanced the yields of the corresponding antibiotics.

The objective of this study undertaken was to acquire maximum information by the shake flask studies for Ascomycin production with Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891. The morphological observation of culture on plate media was carried out visually and under stereo microscope. The colonies obtained were granular and attained grayish black color on maturity. The microscopic examination of mycelia was performed at 20× and 100× (oil immersion) magnification. To improve the Ascomycin production in shake flask studies, two different approaches were evaluated. In the first set of experiments, “Two stage Fermentation” approach was adopted wherein; matured lab inoculum was transferred directly in different production media. The second approach consisted of set of experiments with “Three stage Fermentation” which involved transfer of grown lab inoculum into seed media and grown seed culture into production media. The age of seed culture was investigated for the betterment of the yield. The maximum concentration of ascomycin obtained was 0.5 mg/gm in the second approach on selected P8 media. Media selection was done on the basis of carbon source, nitrogen source, trace salts, buffer to maintain pH throughout the process and precursor as supplement. Simultaneously, microscopic observation was captured in the present study. Thus it could be concluded that “Three stage Fermentation” on selected media was the best option for scale up of ascomycin.

Shikimic acid resistance and addition to enhance immunosuppressant ascomycin production were investigated in this study. A shikimic acid resistant strain SA68 was obtained with ascomycin production of 330 mg/L, displaying approximately 22% increment compared with the parental strain FS35 (270 mg/L). Under the optimal condition of 3 g/L shikimic acid added at 24 h, ascomycin production was further increased by 36%, reaching 450 mg/L. To get deep insights into the effects of shikimic acid resistance and addition on ascomycin biosynthesis, fermentation properties, enzymes activities, metabolites and genes transcriptional levels were analyzed and evaluated. The potential targets were revealed for higher ascomycin production, which were the amplification of carbon flux toward shikimic acid by enhancing 3-deoxy- d -arabino-heptulosonate-7-phosphate synthase (DAHPS) activities and eliminating the feedback inhibition of aromatic amino acids on DAHPS activities, and the elevation of FkbO activities by amplifying fkbO gene to increase the flux of shikimic acid to chorismic acid further to 4,5-dihydroxycyclohex-1-enecarboxylic acid in Streptomyces hygroscopicus var. ascomyceticus. The results indicated that the developed methods would be helpful for ascomycin large scale fermentation. Moreover, the potential targets revealed in this study are valuable to engineer the higher-yield ascomycin-producing strain, as well as other analogous macrolide compounds-producing strains.

Rapamycin, a ring macrolide compound, is recognized as a potent immunosuppressive drug which works by inhibiting T-cell activation and proliferation and is also characterized as having cancer-fighting and anti-aging functions. The pellet morphology of Streptomyces hygroscopius was suggested to have the high rapamycin production titer. The change of culturing pH could alter the morphology of actinomycetes. Therefore, the influence of pH level on rapamycin production was examined. No significant effects of initial pH on rapamycin production were observed in the pH range of 6–8 in the flask trials. Nevertheless, a two-stage pH control strategy was proposed in a bench-scale fermenter, with no pH control in the 1st stage and with the pH controlled at 5.5 in the 2nd stage after the pH rebounded from the bottom. A recorded high rapamycin level of 537 ± 23 mg/L was obtained in a simple batch operation, far higher than the 45 ± 5 and 98 ± 5 mg/L levels in the batches with the pH maintained at a fixed level of 7.0 and without the pH controlled, respectively. The strategy of a two-stage pH control was simple to operate and potentially applied in the scaled-up production of rapamycin.

Streptomyces transglutaminase (TGase) is naturally synthesized as a zymogen (pro-TGase), which is then processed to produce the active enzyme through removal of its pro-peptide. In this study, we investigated the effect of the pro-peptide on the secretion of Streptomyces hygroscopicus TGase in Escherichia coli by modifying its pro-peptide. Four N-terminal amino acid residues (Tyr12, Asn27, Asn30, and Arg32) in the pro-peptide predicted to interact with TGase region through hydrogen bonds. When the four amino acid residues were mutated into Ala, the secretion of TGase was partially or completely inhibited. Furthermore, deletion of the C-terminal α-helix 37–42 in the pro-peptide concomitantly decreased the secretion of TGase. However, deletion of the C-terminal loop 43–52 of the pro-peptide, resulted in increased secretion of TGase by approximately 70% as compared with the control native pro-peptide. These findings indicate that modification of the pro-peptide has a significant impact on the secretory efficiency of TGase in E. coli .

Validamycin A is widely used to control Basidiomycetes, which causes sheath blight disease in rice, potatoes, vegetables, and other crops as well as dumping-off disease in vegetable seedlings, cotton, sugar beets, and other plants. In order to improve the content of validamycin A in the commercial products, valG from Streptomyces hygroscopicus was successfully cloned into Escherichia coli BL21(DE3) and was directly employed as the biocatalyst in the biotransformation from validoxylamine A to validamycin A with the existence of d-cellobiose using the free resting cells in the present study. The fermentation medium was optimized through single factor experiment and response surface method. With the optimized medium, which contained lactose 4.7g/L, yeast extract 49.5g/L, ammonium chloride 2.7g/L, potassium phosphate buffer solution 110mL/L, Ca(2+) 0.0352g/L, the biomass yield and enzyme activity reached 5.5g/L and 1.49U/mL, respectively, which were nearly twice higher than those with initial medium.

Transglutaminase (TGase) is an important industrial enzyme that catalyzes the cross-linking of proteins. In this study, the N-terminal residues were deleted and substituted to improve the activity and thermostability of Streptomyces hygroscopicus TGase. Seven N-terminal residues of TGase were chosen to be deleted individually. The mutated TGase missing the first four residues showed an increase in specific activity of 32.92%. The fifth residue (E5) in the N-terminus was then selected for substitution with the 19 other amino acids. The mutant replacing the fifth residue with an aspartic acid exhibited a 1.85-fold higher specific activity and a 2.7-fold longer half-life at 50 °C when compared with the wild-type enzyme. The melting temperature of the mutated TGase increased from 68.9 to 79.1 °C by circular dichroism spectroscopy analysis. This study showed that substitution combined with deletion of the N-terminal amino acids could enhance the activity and thermostability of TGase.

Currently, the detection methods with lower cost and higher throughput are the major trend in screening genetically modified (GM) food or feed before specific identification. In this study, we developed a quadruplex degenerate PCR screening approach for more than 90 approved GMO events. This assay is consisted of four PCR systems targeting on nine DNA sequences from eight trait genes widely introduced into GMOs, such as CP4-EPSPS derived from Acetobacterium tumefaciens sp. strain CP4, phosphinothricin acetyltransferase gene derived from Streptomyces hygroscopicus ( bar ) and Streptomyces viridochromogenes ( pat ), and Cry1Ab , Cry1Ac , Cry1A ( b / c ), mCry3A , and Cry3Bb1 derived from Bacillus thuringiensis . The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute limit of detection (LOD) of approximate 80 target copies. Furthermore, the applicability of the quadruplex PCR assay was confirmed by screening either several artificially prepared samples or samples of Grain Inspection, Packers and Stockyards Administration (GIPSA) proficiency program.

The effect of ethanol on the production of the important agro-antibiotic validamycin A (Val-A) in medium containing agricultural by-products was investigated. Under the optimal condition of ethanol addition, the maximal Val-A production titer reached 18 g/L, which increased by 60% compared to the control. To provide an insight into cell response to ethanol, the intracellular reactive oxygen species (ROS), gene transcription and enzyme activity were determined. Intracellular ROS as the molecular signal was increased in the ethanol condition. Global regulators afsR and glnR were involved in regulation of Val-A biosynthesis, and the transcription of eight Val-A structural genes was enhanced. The activity of glucose-6-phosphate dehydrogenase (G6PD) was enhanced while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was inhibited. A signal transduction cascade from cell signal response to activated transcription of Val-A biosynthetic genes and enhanced antibiotic production is proposed. The information can be helpful for the improvement of large-scale fermentation.

During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.

BackgroundMesenteric ischemia and reperfusion (I/R) syndrome (MIRS) has been considered a clinicopathologic entity associated with a variety of clinically severe conditions with decreased intestinal blood flow and has been known to induce I/R damage in various organs. Sirolimus (SRL), a macrolide antibiotic isolated from a strain of Streptomyces hygroscopicus, is a potent and nonnephrotoxic immunosuppressant.ObjectiveThis study was designed to investigate the potential impact of sirolimus on MIRS-induced I/R damage in renal, intestinal, pulmonary, and hepatic tissues in an experimental rat model.MethodsTwenty-four male Sprague-Dawley rats, aged 6 to 8 weeks and weighing 280 (±20 g), were studied. Using computer-generated random numbers, rats were assigned to 1 of the following 3 groups: group 1 (I/R group, n = 8), group 2 (I/R + sirolimus group, n = 8), and group 3 (control group, n = 8). Sirolimus, in a 1 mg/mL (60 mL) solution, was administered intraperitoneally in a dose of 1.5 mg/kg/d to the rats assigned to group 2 starting from 3 days before the surgical procedure. In surgery, a laparotomy was performed to clamp the superior mesenteric artery and, thus, induce bowel ischemia in groups 1 and 2. After 60 minutes of ischemia, the microvascular clamp on the superior mesenteric artery was removed for 3 hours of reperfusion. Soon after experimental induction of MIRS, bowel, lung, kidney, and liver specimens from each animal were harvested for both biochemical and histopathologic analysis.ResultsThere were statistically significant differences between groups 1 and 3 with regard to degrees of intestinal (P < 0.001), hepatic (P = 0.001), renal (P < 0.001), and pulmonary (P = 0.01) I/R damage. The lung specimens from group 2 had less inflammation and perivascular edema formation compared with specimens from group 1, but no statistical significance was observed between the groups (P < 0.33). There were statistically significant differences between groups 1 and 2 with regard to degrees of intestinal, hepatic, and renal I/R damage (P = 0.001 for all).ConclusionThe findings of the present study demonstrate the attenuating effects of sirolimus on I/R damage in the intestine and remote organs, including the liver and kidney in the setting of MIRS in an experimental rat model. As a therapeutic implication, the utility of sirolimus may be of clinical value in procedures associated with a high likelihood of I/R damage, including major abdominal operations and renal transplantation. However, whether these results apply to humans is unclear. Additional experimental and clinical studies are warranted to confirm the clinical utility of sirolimus in conditions potentially associated with I/R damage.

A novel fermentation strategy to enhance antibiotics production was demonstrated by inducing reactive oxygen species (ROS), and validamycin A (VAL-A) production by Streptomyces hygroscopicus 5008 in agro-industrial residues containing medium was taken as an example. By optimizing H2O2 amount and addition time, the intracellular ROS level was increased, and VAL-A production titer was enhanced by 40% on day 4 when 25 μM H2O2 was added at 8th h of fermentation. Addition of diphenyleneiodonium chloride (ROS inhibitor) reduced the H2O2 induction effect. The transcription level of eight VAL-A structure genes was enhanced by ROS, and activities of glucose-6-phosphate dehydrogenase and ValG enzyme were increased while glyceraldehyde 3-phosphate dehydrogenase activity was inhibited. This work demonstrated that ROS induction was a useful strategy for VAL-A fermentation, and the information on gene transcription and enzyme activities may be helpful to further understanding the mechanism of ROS effect on the antibiotic biosynthesis.

Summary The antibacterial activity of hygromycin A (HA) arises from protein synthesis inhibition and is dependent upon a methylenedioxy bridged-aminocyclitol moiety. Selective gene deletions and chemical complementation in Streptomyces hygroscopicus NRRL 2388 showed that the hyg18 and hyg25 gene products, proposed to generate a myo -inositol intermediate, are dispensable for HA biosynthesis but contribute to antibiotic yields. Hyg8 and Hyg17, proposed to introduce the amine functionality, are essential for HA biosynthesis. Hyg6 is a methyltransferase acting on the aminocyclitol, and a Δ hyg6 mutant produces desmethylenehygromycin A. Deletion of hyg7 , a metallo-dependant hydrolase homolog gene, resulted in methoxyhygromycin A production, demonstrating that the corresponding gene product is responsible for the proposed oxidative cyclization step of methylenedioxy bridge formation. The methyl/methylene group is not required for in vitro protein synthesis inhibition but is essential for activity against Escherichia coli .

Validamycin A (VAL-A), produced by Streptomyces hygroscopicus, is an important anti-fungal agro-antibiotic. In this work, the effect of fermentation temperature on VAL-A biosynthesis by S. hygroscopicus 5008 was investigated between 28 degrees C and 42 degrees C, and an interesting threshold of temperature for VAL-A biosynthesis was found between 35 degrees C and 37 degrees C. At a relatively higher temperature, a much higher VAL-A productivity was obtained together with faster protein synthesis and sugar consumption. Transcriptional analysis of samples from early, middle and late stages of fermentation at various temperatures demonstrated that three operons, valABC, valKLMN and valG, for all eight necessary structure genes, were dramatically promoted when temperature reached the threshold. Activities of both glucose-6-phosphate dehydrogenase (G6PDH) of pentose-phosphate pathway and ValG of VAL-A biosynthesis were also enhanced at a higher cultivation temperature. The interesting temperature effect with a 2 degrees C threshold shift from 35 degrees C to 37 degrees C on the antibiotic biosynthesis was understood to be related to the gene transcriptional levels and key enzyme activities.

Cultivation shift from 30° to 37° significantly enhanced validamycin (VAL) production. Analyzed by reverse-transcription PCR, the transcription of three val genes, valA, valK and valG, representing the three operons of the cluster was simultaneously increased at elevated temperature. Furthermore, the transcription of valP and valQ, a pair of two-component regulators in validamycin biosynthetic gene cluster, was also increased at 37°. Inactivation of valP and valQ reduced validamycin production at 37° to the yield level of wild type strain at 30°, and the val genes showed reduced expression in the mutant LL-8 at 37°. These results revealed that the two-component regulator valP and valQ contribute to the elevated validamycin production.

Nutritional assessment of transgenic crops used for human food and animal feed is an important aspect of safety evaluations. An herbicide-tolerant rice (Bar68-1) was generated by the stable insertion of bar gene, isolated from Streptomyces hygroscopicus strain HP632 into the genome of a conventional variety of Chinese rice (D68). Nutrients including proximates, amino acids, fatty acids, minerals and vitamins and anti-nutritive components such as phytic acid, lectin, and trypsin inhibitors of the brown and milled rice grain from the resulted Bar68-1 rice were compared with those of the parental line D68. The data demonstrated that the nutritional quality of both the brown and milled rice grains from the transgenic line was substantial equivalent to those of the non-transgenic counterpart, and most results of nutritional components fell within the range of values reported for other commercial lines.

Effect of ethanol on the stability and conformational changes of transglutaminase from Streptomyces hygroscopicus (MTGase) was studied. The presence of 10% ethanol increased 20% activity and about 85% activity was lost in 50% ethanol–aqueous solution after incubation for 60 min at 25 °C. The K m and V max of the enzyme were 63.13 mmol/L and 14.02 μmol/(mg protein min) in the presence of 10% ethanol, and 34.20 mmol/L and 0.68 μmol/(mg protein min) in the presence of 30% ethanol, respectively. The results of spectral determination indicated that MTGase did not undergo significant structural changes in 10% ethanol–aqueous solution. At ethanol concentration of 40%, the α-helix structure content of the enzyme decreased from 20.5 ± 0.5% to 14.3 ± 0.7% and the unordered structure content increased from 46.4 ± 0.5% to 72.6 ± 1.7%. Some polyhydroxy compounds could improve the stability of the enzyme at high ethanol concentration.

A Streptomyces fed-batch fermentation was studied with on-line measurement of dissolved carbon dioxide (DCO 2 ). A selected cell strain R060107 of Streptomyces hygroscopicus was used in the production of rapamycin. pH was controlled only in the production phase at the low limit of 5.1 after its value dropped from an initial 6.5 to 5.1. DCO 2 was used to control the feed in the fed-batch fermentation of S. hygroscopicus R060107. The experiment investigated the relationship between rapamycin production and the evolution of DCO 2 . A high limit of DCO 2 (hDCO 2 ) was used to control medium feed. In order to meet the needs for the dynamic growth and metabolism of cells in the process, the hDCO 2 was defined as a dynamic parameter, and was therefore calculated constantly in the process. A fermentation condition of limited nutrient supply was realized. It was observed that rapamycin was secreted when the glycerol concentration was well controlled in the fermentation condition. Experimental results showed that 500 mg/l of rapamycin was produced in 120 h of fermentation.

To enhance Streptomyces hygroscopicus 17997, a geldanamycin(Gdm)-producing strain, in fermentation yield, the naphthalenic 3-amino-5-hydroxybenzoic acid (AHBA) biosynthetic genes (shnSOP), encoding the products that share the common biosynthetic substrates with Gdm, were disrupted by homologous recombination technology. The resulting mutant strain (Δ SOP) was cultivated on a solid medium, and the number of spores collected from the plates was calculated from 5 to 14 days, and the yield of Gdm was measured by high-performance liquid chromatography. The Gdm fermentation yield of Δ SOP strain was 185% more than that of the parent strain. On solid medium, the spore growth of Δ SOP strain experienced two cycles; the second generation of spores produced a higher yield of Gdm.

In this study, a novel microbial transglutaminase (MTG) from Streptomyces hygroscopicus WSH03-13 was applied in the processing of wool fabrics. The results indicated that MTG treatment could improve felting properties and decrease tensile strength loss of wool fabrics. For the wool fabrics used in this study, MTG treatment following chemical and protease pretreatment led to a 2.32% of area shrinkage and about 16% recovery in tensile strength based on the samples without MTG treatment. Moreover, a traditional resin treatment was compared with the role of MTG. Although the tensile strength of wool fabrics treated by MTG was lower than that treated by resin treatment, the fabrics had similar anti-felting properties, and the chemical oxygen demand of wastewater was only half of the latter.

Geldanamycin is a benzoquinone ansamycin, produced by the Actinomycete Streptomyces hygroscopicus var. geldanus, which possesses a broad spectrum antibiotic and anticancer activity. Adsorbent resins are gaining significant application in areas of product recovery, and in particular antibiotic recovery. It was decided to establish how effective such resins could be, for recovery of geldanamycin from fermentation broths, in a solid phase extraction. The extractions were batch adsorptions between the liquid phase fermentation broth, containing the geldanamycin and the solid phase adsorbent resins. The resins assessed were Amberlite XAD-4, 7, 16, 1180 and 1600, and Sepabeads SP-850 and Diaion HP-20. All resins assessed were capable of recovery of in excess of 90% of geldanamycin at a resin concentration of 15 g/l, although resins XAD-16, XAD-1600, XAD-1180 and Diaion HP-20 showed a higher capacity at lower resin concentrations than the others resins. All resins displayed a level of positive selectivity for adsorption of geldanamycin over that of contaminants. It was found that the rate of adsorption were dependant on temperature, where longer adsorption times were required at lower temperatures. The adsorbent resins displayed a wide pH range over which they were effective. The level of contaminant adsorption could be partially regulated through pH control. Inclusion of acetonitrile in the fermentation broth resulted in lower levels of geldanamycin adsorption, however for Diaion HP-20, at a mix of 80% fermentation broth, 20% acetonitrile, approximately 90% of the geldanamycin was adsorbed, but less than 10% of the contaminants present.

Transglutaminase (TGase, EC 2.3.2.13) from a Streptomyces hygroscopicus strain isolated from soil was purified from culture broth by ethanol precipitation, followed by successive chromatographies on CM-cellulose and Sephadex G-75 columns with a yield and purification-fold of 21.1% and 30%, respectively. The enzyme’s molecular weight was estimated as 38,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified microbial transglutaminase (MTG) exhibited optimum activity at 37–45 °C and in a range of pH 6.0–7.0 for hydroxamate formation from N-carboxybenzoyl- l -glutaminyl-glycine and hydroxylamine. The enzyme was not stable above 50 °C and was stable within a pH range of 5.0–8.0 at lower temperature. The MTG was not inhibited by Ca2+ and ethylenediaminetetraacetic acid, suggesting it was calcium-independent. Purified MTG was strongly inactivated by 5,5′-dithiobis (2-nitrobenzoic acid), Cu2+, Zn2+, Pb2+, and Hg2+, suggesting that this enzyme could possess a thiol group at the active site. The MTG stability was strongly affected by ethanol concentration. The enzyme activity was slightly elevated at a lower concentration of ethanol at 25 °C.

A geldanamycin (GDM) producing strain, Streptomyces hygroscopicus 17997, was isolated from Yunnan China soil by our institute researchers. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (Heat Shock Protein 90) and alter its function. Hsp90 is a chaperone protein involved in the regulation of the cell cycle, cell growth, cell survival, apoptosis, and oncogenesis. So it plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumor and antivirus bioactivity, but the hypato-toxicity and poor solubility in water limits its clinical use. Two GDM derivatives, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG), both showing lesser hepato-toxicity, are now in Phase II and Phase I clinic trials. In order to accomplish the structure modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene (gdmN) in GDM biosynthesis. The 732 bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT gene containing fragments were verified and localized by Southern blot. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. Based on BLAST analysis, this sequence contains 13 possible ORFs and their deduced functions are believed to be involved in GDM production. The ORF1 encoding products show homology (87%) with incomplete sixth module and complete seventh module of PKS, gdmA3, in S. hygroscopicus NRRL 3602. The ORF2-13 gene products are similar to gdmF(9 5%), gdmM(8 8%), gdmN (92%), gdmH (92%), I (93%), J (90%), K (93%), G (96%), gdmO (91%), gdmP (93%) and two transcription regulation genes gdmRI (83%) and gdmRII (90%). The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and to modify the structure of GDM through combinatorial biosynthesis.

The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of α-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s− 1; Km, 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.

Effect of some additives on the thermal stability of a new microbial transglugaminase from Streptomyces hygroscopicus WSH03-13, and the stabilization of MTG by spray drying were studied and compared with freeze-drying method. It was observed that MTG was not stable at 50 °C (loss of over 50% after 30 min of incubation) and became more sensitive to heat in the presence of ethanol. The addition of carbohydrates such as: maltodextrin, sucrose, mannose, trehalose, and reduced glutathione (GSH), evidently enhanced enzyme thermal stability. The highest activity yield of spray drying reached about 90% with the combined addition of maltodextrin and GSH at the recommended concentrations and at a lower air outlet temperature (60 ± 3 °C). The MTG activity retention in dried preparations during storage at 4 °C for 60 days was mainly related to the moisture content. Lower moisture content and the addition of proper stabilizers could improve the storage stabilization and thermal stability of enzyme preparations.

SummaryA 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat (Triticum turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium whereas the non-transgenic cells are actively killed by the selective agent. We compared the ‘selection efficiency’ of a commonly used negative selection method that employs the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using a biolistic system. The integration and expression of the two genes in the T0 generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pmi was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide

SummaryGeldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the “inert” groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.

A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples. The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis. The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound. Using this dose-response curve, the method facilitates definition of three distinct Minimum Inhibitory Concentration (MIC) values for use in the characterisation of the bioactive attributes of a sample. The assay uses established standard procedures to facilitate adaptation of the assay for use with a wider range of test microorganisms. Errors due to the assumption of a linear relationship between turbidity and biomass concentration are also reduced, due to incorporation of a step to convert turbidity to biomass concentration, for use in the calculation of bioactivity.