A randomized, double-blind, placebo-controlled study on immune improvement effects of ethanolic extract of Echinacea purpurea (L.) Moench in Korean adults.

Echinacea purpurea (L.) Moench (EP), a medicinal plant native to North America, is now cultivated in various regions including Europe. With increasing popularity of Echinacea in Korea recently, a human clinical trial was conducted to evaluate immune-enhancing efficacy and safety of EP 60% ethanolic extract (EPE) in Koreans. Eighty volunteers were recruited for this randomized, double-blind, placebo-controlled clinical trial. They were randomly divided into two groups and given either a daily dose of 200 mg of EPE or a placebo. All participants underwent testing for Natural Killer (NK) cell cytotoxic activity, serum cytokine levels (IL-2, IL-6, IL-10, IL-12, IFN-γ, TNF-α), Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21), and Multidimensional Fatigue Scale (MFS) during this study to assess changes in outcomes. After 8 weeks of EPE consumption, a significant increase in NK cell cytotoxic activity compared to the placebo was observed. Additionally, serum cytokine levels of IL-2, IFN-γ, and TNF-α also significantly increased following EPE consumption. However, no significant changes were observed in WURSS-21 and MFS before and after EPE consumption. Throughout the 8-week study period, no adverse reactions were reported in relation to EPE consumption, and there were no clinically significant changes in diagnostic laboratory tests or vital signs in the EPE group. These results indicate that consumption of EPE could lead to immune improvement without any adverse effects. This clinical trial was the first to demonstrate beneficial effects of EPE consumption on immunity in Korean adults.

Rapid Impregnating Resins for Fiber-Reinforced Composites Used in the Automobile Industry.

As environmental regulations become stricter, weight- and cost-effective fiber-reinforced polymer composites are being considered as alternative materials in the automobile industry. Rapidly impregnating resin into the reinforcing fibers is critical during liquid composite molding, and the optimization of resin impregnation is related to the cycle time and quality of the products. In this review, various resins capable of rapid impregnation, including thermoset and thermoplastic resins, are discussed for manufacturing fiber-reinforced composites used in the automobile industry, along with their advantages and disadvantages. Finally, vital factors and perspectives for developing rapidly impregnated resin-based fiber-reinforced composites for automobile applications are discussed.

Magnolia kobus Extract Suppresses Porphyromonas gingivalis LPS-Induced Proinflammatory Cytokine and MMP Expression in HGF-1 Cells and Regulates Osteoclastogenesis in RANKL-Stimulated RAW264.7 Cells.

Clinical prevention is of utmost importance for the management of periodontal diseases. Periodontal disease starts with an inflammatory response in the gingival tissue, and results in alveolar bone destruction and subsequent tooth loss. This study aimed to confirm the anti-periodontitis effects of MKE. To confirm this, we studied its mechanism of action using qPCR and WB in LPS-treated HGF-1 cells and RANKL-induced osteoclasts. We found that MKE suppressed proinflammatory cytokine protein expression by inhibiting the TLR4/NF-κB pathway in LPS-PG-induced HGF-1 cells and blocking ECM degradation by regulating the expression of TIMPs and MMPs. We also confirmed that TRAP activity and multinucleated cell formation were reduced in RANKL-stimulated osteoclasts after exposure to MKE. These results were confirmed by inhibiting TRAF6/MAPK expression, which led to the suppression of NFATc1, CTSK, TRAP, and MMP expression at the gene and protein levels. Our results confirmed that MKE is a promising candidate for the management of periodontal disease based on its anti-inflammatory effects and inhibition of ECM degradation and osteoclastogenesis.

Scrophularia buergeriana Extract (Brainon) Attenuates Neuroinflammation in BV-2 Microglia Cells and Promotes Neuroprotection in SH-SY5Y Neuroblastoma Cells.

Microglia-induced neuroinflammation is one of the causative factors in cognitive dysfunction and neurodegenerative disorders. Our previous studies have revealed several benefits of Scrophularia buergeriana extract (Brainon®) in the central nervous system, but the underlying mechanism of action has not been elucidated. This study is purposed to investigate the anti-inflammatory and neuroprotective mechanisms of Brainon in the BV-2 condition SH-SY5Y model. Lipopolysaccharide (LPS)-induced BV-2 conditioned media (CM) were used to treat SH-SY5Y cells to investigate neuroprotective effects of the extract against microglial cytotoxicity. Results demonstrated that pretreated Brainon decreased nitric oxide release, the inducible nitric oxide synthase expression level, and expression of cytokines like interleukin-6, interleukin-1ß, and tumor necrosis factor-α by blocking expression of TLR4/MyD88 and NLRP3 and suppressing nuclear factor κB/AP-1 and p38/JNK signaling pathways in LPS-induced BV-2 cells. In addition, when SH-SY5Y cells were treated with CM, pretreatment with Brainon increased neuronal viability by upregulating expression of antioxidant proteins like as SODs and Gpx-1. Increased autophagy and mitophagy-associated proteins also provide important clues for SH-SY5Y to prevent apoptosis by Brainon. Brainon also modulated mTOR/AMPK signaling to clear misfolded proteins or damaged mitochondria via auto/mitophagy to protect SH-SY5Y cells from CM. Taken together, these results indicate that Brainon could reduce inflammatory mediators secreted from BV-2 cells and prevent apoptosis by increasing antioxidant and auto/mitophagy mechanisms by regulating mTOR/AMPK signaling in SH-SY5Y cells. Therefore, Brainon has the potential to be developed as a natural product in a brain health functional food to inhibit cognitive decline and neuronal death.

Effects of Scrophularia buergeriana Extract (Brainon®) on Aging-Induced Memory Impairment in SAMP8 Mice.

Alzheimer's disease (AD) is a worldwide problem. Currently, there are no effective drugs for AD treatment. Scrophularia buergeriana Miquel (SB) is a traditional herbal medicine used in Korea to treat various diseases. Our previous studies have shown that ethanol extract of SB roots (SBE, Brainon®) exhibits potent anti-amnesic effects in Aß1-42- or scopolamine-treated memory impairment mice model and neuroprotective effects in a glutamate-induced SH-SY5Y cell model. In this study, we evaluated the therapeutic effects of Brainon® and its mechanism of action in senescence-accelerated mouse prone 8 (SAMP8) mice. Brainon® (30 or 100 mg/kg/day) was orally treated to six-month-old SAMP8 mice for 12 weeks. Results revealed that Brainon® administration effectually ameliorated cognitive deficits in Y-maze and passive avoidance tests. Following the completion of behavioral testing, western blotting was performed using the cerebral cortex. Results revealed that Brainon® suppressed Aß1-42 accumulation, Tau hyperphosphorylation, oxidative stress, and inflammation and alleviated apoptosis in SAMP8 mice. Brainon® also promoted synaptic function by downregulating the expression of AChE and upregulating the expression of p-CREB/CREB and BDNF. Furthermore, Brainon® restored SAMP8-reduced expression of ChAT and -dephosphorylated of ERK and also decreased AChE expression in the hippocampus. Furthermore, Brainon® alleviated AD progression by promoting mitophagy/autophagy to maintain normal cellular function as a novel finding of this study. Our data suggest that Brainon® can remarkably improve cognitive deficiency with the potential to be utilized in functional food for improving brain health.

Magnolia kobus Extract Inhibits Periodontitis-Inducing Mediators in Porphyromonas gingivalis Lipopolysaccharide-Activated RAW 264.7 Cells.

Periodontitis, a disease caused by inflammation of oral bacteria, contributes to the loss of alveolar bone and destruction of connective tissues. Porphyromonas gingivalis, a Gram-negative bacterium, is known to possess important pathogenic factors for periodontal disease. In this study, we investigated the anti-periodontitis effects of Magnolia kobus extract (MKE) and magnolin as a component of Magnolia kobus (MK) in murine macrophage RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Effects of MKE and magnolin on the mechanism of RAW 264.7 cellular inflammation were determined by analyzing nitric oxide (NO) production and Western blot protein expression (n = 3). MKE/magnolin inhibited NO production without affecting cell survival. MKE/magnolin treatment inhibited LPS-induced pro-inflammatory cytokines, expression levels of matrix metalloproteinases (MMPs such as MMP-1, 3, 8, 9, and 13), and protein levels of inflammatory mediators (such as TNF-α, IL-1ß, and mPGES-1). MKE/magnolin also suppressed NF-κB activation by inhibiting the TLR4 signaling pathway. These findings suggest that MKE has a therapeutic effect on inflammatory periodontal disease caused by oral bacterium P. gingivalis and that magnolin is a major functional component in the anti-inflammatory effect of MKE.

Anti-Obesity Effect of Dyglomera® Is Associated with Activation of the AMPK Signaling Pathway in 3T3-L1 Adipocytes and Mice with High-Fat Diet-Induced Obesity.

Dyglomera® is an aqueous ethanol extract of the fruit pods of Dichrostachys glomerata, a Cameroonian spice. Several studies have shown its anti-diabetic and anti-obesity effects. However, the underlying mechanisms for such effects remain unclear. Thus, the objective of this study was to investigate the anti-obesity effect of Dyglomera® and its underlying mechanisms in mice with high-fat diet-induced obesity and 3T3-L1 adipocytes. Our results revealed that Dyglomera® inhibited adipogenesis and lipogenesis by regulating AMPK phosphorylation in white adipose tissues (WATs) and 3T3-L1 adipocytes and promoted lipolysis by increasing the expression of lipolysis-related proteins. These results suggest that Dyglomera® can be used as an effective dietary supplement for treating obesity due to its modulating effect on adipogenesis/lipogenesis and lipolysis.

Anti-Obesity Effects of a Mixture of Atractylodes macrocephala and Amomum villosum Extracts on 3T3-L1 Adipocytes and High-Fat Diet-Induced Obesity in Mice.

Since the potential of (3:1) mixtures of Atractylodes macrocephala and Amomum villosum extracts has been proposed in the management of obesity, the purpose of present study was to investigate the effects of AME:AVE (3:1) mixture on weight loss, obesity-related biochemical parameters, adipogenesis and lipogenesis related proteins in 3T3-L1 cells and HFD-induced obesity in a mouse model. Treatment with AME:AVE (3:1) mixture inhibited lipid accumulation. Furthermore, the treatment with 75 and 150 mg/kg of AME:AVE (3:1) significantly decreased the body weight gain, white adipose tissue (WAT) weight, and plasma glucose level in HFD-induced obese mice. Moreover, treatment with 75 and 150 mg/kg AME:AVE (3:1) also significantly lowered the size of adipocytes in adipose tissue and reduced the lipid accumulation in liver. AME:AVE (3:1) treatment significantly decreased the expression of proteins related to adipogenesis and lipogenesis in 3T3-L1 adipocytes and WAT of HFD-induced obese mice. These results suggest that the AME:AVE herbal mixture (3:1) has anti-obesity effects, which may be elicited by regulating the expression of adipogenesis and lipogenesis-related proteins in adipocytes and WAT in HFD-induced obesity in mice.

Quality assurance for nanomaterial inhalation toxicity testing.

The recent revision of OECD inhalation toxicology test guidelines 412 and 413 presents new challenges for both the study director (SD) and quality assurance (QA) personnel when conducting GLP (good laboratory practice) studies. In the case of nanomaterial inhalation exposure studies, GLP has rarely been applied, yet the new revisions are applicable to soluble and insoluble nanomaterials, as well as conventional chemicals. For example, the new guidelines require an additional bronchoalveolar lavage (BAL) fluid assay and lung burden measurement during the post-exposure observation (PEO) period, plus nanomaterial physicochemical characterization before and after nano-aerosol generation when exposing experimental animals. Implementing these revised guidelines will prove especially challenging for QA measures related to the physicochemical characterization and aerosolization of test nanomaterials. Therefore, this review examines the key elements involved in nanomaterial inhalation GLP testing under the revised OECD guidelines, suggests an alternative to the increased animal numbers, in consideration of animal welfare and with scientific merits, and discusses the limitation of toxicokinetic estimation using the new testing guidelines.

Ameliorating effect of Citrus aurantium extracts and nobiletin on ß­amyloid (1­42)­induced memory impairment in mice.

The aim of the present study was to evaluate the neuroprotective effect of Citrus aurantium extract (CAE) and nobiletin against amyloid ß 1­42 (Aß 1­42)­induced spatial learning and memory impairment in mice. After injecting Aß 1­42 (5 µl/2.5 min, intracerebroventricular injection), amnesic mice were orally administered CAE and nobiletin for 28 days. Memory, spatial and cognitive ability were measured using passive avoidance and a Morris water maze task. Acetylcholinesterase (AchE) activity was investigated in the hippocampus and cortex using commercial kits and the analysis of Bax, Bcl­2, and cleaved caspase­3 protein expression by western blot assays was used to confirm the anti­apoptotic mechanism of CAE and nobiletin. The present study confirmed impairments in learning and memory in the Aß­induced neurodegenerative mice with increased AchE activity in the brain. However, the daily administration of CAE and nobiletin reduced the spatial learning deficits and increased the AchE activity in the cortex and hippocampus. Furthermore, CAE and nobiletin significantly downregulated the Bax and cleaved caspase­3 protein expression and upregulated the Bcl­2 and Bcl­2/Bax expression in the cortex and hippocampus of Aß­treated mice. These results suggest that CAE and nobiletin exert a neuroprotective effect by regulating anti­apoptotic mechanisms, including reduced AchE activity in the cortex and hippocampus of the cognitive deficit mouse model.

Preventive Effect of Citrus aurantium Peel Extract on High-Fat Diet-Induced Non-alcoholic Fatty Liver in Mice.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation, which is the most common form of chronic liver disease. Multiple clinical studies using natural compounds such as flavonoids have been conducted to treat NAFLD. In the present study, the pharmacological effect of Citrus aurantium L. (Rutaceae) peel extract (CAE), which contains over 27% of polymethoxyflavone nobiletin, on NAFLD was evaluated using a high-fat diet (HFD) animal model susceptible to developing NAFLD. C57BL/6 mice were fed an HFD (60% kcal of energy derived from fat) for 8 weeks to induce obesity. Obese mice were randomly allocated to four groups of eight mice each (HFD alone, HFD with silymarin, HFD with 50 mg/kg CAE, and HFD with 100 mg/kg CAE). After 8 weeks of treatment, all mice were euthanized, and plasma and liver tissues were analyzed biochemically and histopathologically. The results indicate that CAE treatment significantly reduced HFD-induced NAFLD, as shown by decreased serum lipid index and prevented liver histopathology. The expression of genes involved in lipid synthesis including free fatty acid (FFA), peroxisome-proliferator-activated receptor γ (PPAR-γ), sterol receptor element binding protein 1c (SREBP-1c), and fatty acid synthesis enzyme was suppressed by CAE treatment. Moreover, compared to untreated mice, CAE-treated HFD mice showed decreased pro-inflammatory cytokine expression. These results demonstrated that CAE prevented HFD-induced NAFLD by reducing plasma levels of triglyceride and cholesterol and de novo lipid synthesis.

Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) of Corynebacterium glutamicum Enhances l-Threonine Biosynthesis.

l-Threonine is an important supplement in the food industry. It is currently produced through fermentation of Escherichia coli but requires additional purification steps to remove E. coli endotoxin. To avoid these steps, it is desirable to use Corynebacterium glutamicum, a microorganism generally regarded as safe. Engineering of C. glutamicum to increase production of l-threonine has mainly focused on gene regulation as well as l-threonine export or carbon flux depletion. In this study, we focus on the negative feedback inhibition produced by l-threonine on the enzyme homoserine kinase (ThrB). Although l-threonine binds to allosteric sites of aspartate kinase (LysC) and homoserine dehydrogenase (Hom), serving as a noncompetitive inhibitor, it acts as a competitive inhibitor on ThrB. This is problematic when attempting to engineer enzymes that are nonresponsive to increasing cellular concentrations of l-threonine. Using primary structure alignment as well as analysis of the Methanocaldococcus jannaschii ThrB (MjaThrB) active site in complex with l-threonine (inhibitor of ThrB) and l-homoserine (substrate of ThrB), a conserved active-site alanine residue (A20) in C. glutamicum ThrB (CglThrB) was predicted to be important for differential interactions with l-threonine and l-homoserine. Through site-directed mutagenesis, we show that one variant of C. glutamicum ThrB, CglThrB-A20G, retains wild-type enzymatic activity, with dramatically decreased feedback inhibition by l-threonine. Additionally, by solving the first Corynebacterium X-ray crystal structure of homoserine kinase, we can confirm that the changes in l-threonine affinity to the CglThrB-A20G active site derive from loss of van der Waals interactions.

Improvement of daptomycin production via increased resistance to decanoic acid in Streptomyces roseosporus.

Daptomycin, a cyclic anionic lipopeptide compound produced by Streptomyces roseosporus, is used to treat skin infections caused by multi-drug resistant gram-positive pathogens. The biosynthesis of daptomycin is initiated by the condensation of decanoic acid (DA, a 10-carbon unit fatty acid) and the N-terminal l-tryptophan. So, the addition of DA to the fermentation medium is essential for increasing daptomycin production. However, increasing of DA concentration in the fermentation medium was not possible due to the high toxicity of DA. The previous studies reported that the cell growth of S. roseosporus was halted from 1 mM DA. In order to improve daptomycin production with increasing DA concentration in the medium, the DA-resistant S. roseosporus was developed via a sequential-adaptation method. The DA-resistant strain (DAR) showed complete resistance to 1 mM DA, and the daptomycin production was increased 1.4-fold (40.5 ± 0.7 mg/L) compared with the wild-type (28.5 ± 0.8 mg/L) at 1 mM DA. Additionally, the initial step of the daptomycin biosynthesis was enhanced by the overexpression of dptE and dptF in DAR. The dptEF overexpression DAR showed 3.9-fold (156.3 ± 8.2 mg/L) increase in the daptomycin production compared with DAR (40.1 ± 2.6 mg/L) at 1 mM DA.

Improvement of FK506 Production in the High-Yielding Strain Streptomyces sp. RM7011 by Engineering the Supply of Allylmalonyl-CoA Through a Combination of Genetic and Chemical Approach.

FK506, a widely used immunosuppressant, is a 23-membered polyketide macrolide that is produced by several Streptomyces species. FK506 high-yielding strain Streptomyces sp. RM7011 was developed from the discovered Streptomyces sp. KCCM 11116P by random mutagenesis in our previous study. The results of transcript expression analysis showed that the transcription levels of tcsA, B, C, and D were increased in Streptomyces sp. RM7011 by 2.1-, 3.1-, 3.3-, and 4.1- fold, respectively, compared with Streptomyces sp. KCCM 11116P. The overexpression of tcsABCD genes in Streptomyces sp. RM7011 gave rise to approximately 2.5-fold (238.1 µg/ml) increase in the level of FK506 production compared with that of Streptomyces sp. RM7011. When vinyl pentanoate was added into the culture broth of Streptomyces sp. RM7011, the level of FK506 production was approximately 2.2-fold (207.7 µg/ml) higher than that of the unsupplemented fermentation. Furthermore, supplementing the culture broth of Streptomyces sp. RM7011 expressing tcsABCD genes with vinyl pentanoate resulted in an additional 1.7-fold improvement in the FK506 titer (498.1 µg/ml) compared with that observed under nonsupplemented condition. Overall, the level of FK506 production was increased approximately 5.2-fold by engineering the supply of allylmalonyl-CoA in the high-yielding strain Streptomyces sp. RM7011, using a combination of overexpressing tcsABCD genes and adding vinyl pentanoate, as compared with Streptomyces sp. RM7011 (95.3 µg/ml). Moreover, among the three precursors analyzed, pentanoate was the most effective precursor, supporting the highest titer of FK506 in the FK506 high-yielding strain Streptomyces sp. RM7011.

Glutathione S-transferase pi (GST-pi) inhibition and anti-inflammation activity of the ethyl acetate extract of Streptomyces sp. strain MJM 8637.

To investigate the anti-cancer properties of soil-borne actinobacteria, MJM 8637, the glutathione S-transferase pi (GST-pi) assay, anti-tumor necrosis factor (TNF)-α assay, the level of antioxidant potential by DPPH radical scavenging activity, NO scavenging activity, and ABTS radical scavenging activity in ethyl acetate extract were determined. The 16S rDNA sequencing analysis revealed that Streptomyces sp. strain MJM 8637, which was isolated from Hambak Mountain, Korea, has 99.5% similarity to Streptomyces atratus strain NBRC 3897. The physiological and the morphological characteristics of the strain MJM 8637 were also identified. The ethyl acetate extract of MJM 8637 inhibited TNF-α production approximately 61.8% at concentration 100 µg/ml. The IC50 value of the strain MJM 8637 extract on GST-pi was identified to be 120.2 ± 1.6 µg/ml. In DPPH, NO, and ABTS radical scavenging assays, the IC50 values of the strain MJM 8637 extract were found to be 977.2 µg/ml, 1143.7 µg/ml, and 454.4 µg/ml, respectively. The ethyl acetate extract of the strain MJM 8637 showed 97.2 ± 1.3% of cell viability at 100 µg/ml in RAW 264.7 cell viability assay. The results obtained from this study suggest that the ethyl acetate extract of Streptomyces sp. strain MJM 8637 could be considered as a potential source of drug for the cancers that have multidrug resistance with its GST-pi inhibition and anti-inflammation activities, and low cytotoxicity.

Improvement in Oil Production by Increasing Malonyl-CoA and Glycerol-3-Phosphate Pools in Scenedesmus quadricauda.

In recent years, microalgae have attracted considerable interest as a biofuel resource owing to their rapid growth, tolerance to harsh conditions, and ability to accumulate a large amount of triacylglycerols (TAGs). However, the economic effectiveness of algal biofuel is still low. In this study, we attempted to increase oil production of the microalga Scenedesmus quadricauda by elevating intracellular malonyl-CoA and glycerol-3-phosphate (G3P) pools. To increase intracellular oil content, yeast-derived genes encoding acetyl-CoA carboxylase (ACC1), glycerol kinase (GPD1), and glycerol-3-phosphate dehydrogenase (GUT1) were overexpressed under the control of CaMV 35S and NOS promoters with SV40 large T antigen components. Fatty acid profiling, G3P content, and the number of cells with high oil content were analyzed by gas chromatography-mass spectrometry, G3P assay kit, and flow cytometry, respectively. Overexpression of ACC1 increased the total fatty acid content by 1.6-fold. Overexpression of GPD1 and GUT1 increased intracellular G3P content by 1.6- and 1.9-fold, respectively. Multi-gene expression of ACC1, GPD1, and GUT1 increased the number of cells with high oil content by 1.45-fold compared with that observed with the wild-type. This study is the first to report increased oil production by overexpression of the key genes (ACC1, GPD1, and GUT1) for TAG biosynthesis in microalgae.

Antioxidant activity and free radical scavenging activities of Streptomyces sp. strain MJM 10778.

OBJECTIVE: To investigate the antioxidant activity of soil-borne actinobacteria. METHODS: The total phenolic contents, the level of antioxidant potential by DPPH radical scavenging activity, NO scavenging activity, and ABTS radical scavenging activity in ethyl acetate extract were determined. RESULTS: The 16S rDNA sequencing analysis revealed that Streptomyces sp. strain MJM 10778, which was isolated from Hambak Mountain, Korea, has 99.9% similarity to Streptomyces misionensis (S. misionensis) NBRC 13063. The physiological and the morphological test revealed that the strain MJM 10778 has different characteristics from the strain NBRC 13063. The entire antioxidant assay with the ethyl acetate extract displayed good radical scavenging activity. The IC50 values of the strain MJM 10778 extract on DPPH, NO, and ABTS radicals were identified to be 92.8 µg/mL, 0.02 µg/mL, and 134.9 µg/mL, respectively. The ethyl acetate extract of the strain MJM 10778 showed an 81.50% of cell viability at 100 µg/mL in Raw264.7 cell viability assay. CONCLUSIONS: The results obtained suggest that the ethyl acetate extract of Streptomyces sp. strain MJM 10778 could be considered as a potential source of drug for the diseases that is caused by free radicals with its anti-oxidant activities and low cytotoxicity.

Overexpression of the putative extracytoplasmic function sigma (σ) factor FujE enhances FK506 production in Streptomyces sp. strain KCCM 11116P.

The role of the putative extracytoplasmic function sigma (σ) factor FujE, which has not been characterized as a member of the FK506 biosynthetic gene cluster, on FK506 production was identified by gene deletion, overexpression, and transcription analysis experiments in Streptomyces sp. strain KCCM 11116P. Inactivation of fujE had no effect on FK506 production, growth, or morphological differentiation. Overexpression of fujE with integrative vectors increased FK506 production by 2.87-fold (24.5 ± 1.4 mg·L(-1)) compared with the wild type (8.5 ± 0.5 mg·L(-1)). Semiquantitative reverse transcription-polymerase chain reaction analysis indicated that the overexpression of fujE stimulates the transcription of the FK506 biosynthetic genes. These results demonstrated that fujE is a new member of the FK506 biosynthetic gene cluster.

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011.

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

An ABC transporter complex containing S-adenosylmethionine (SAM)-induced ATP-binding protein is involved in antibiotics production and SAM signaling in Streptomyces coelicolor M145.

A sco3956-deletion mutant (ΔSCO3956) of Streptomyces coelicolor was generated to characterize the S-adenosylmethionine (SAM)-induced, ATP-binding cassette transporter (ABC transporter) ATP-binding protein, SCO3956. It produced actinorhodin (ACT) and undecylprodigiosin (RED) decreased by approx. 82 and 64 %, respectively. In addition, the effect of exogenous SAM was lost in the ΔSCO3956. Plasmid-based complementation of sco3956 in ΔSCO3956 restored ACT and RED levels of ΔSCO3956 to wild-type levels (ACT: 20 ± 1.4 mg g(-1) DCW and RED: 5.3 ± 0.6 mg g(-1) DCW) and the exogenous effect significantly increased ACT and RED by approx. 129 and 135 %, respectively, when compared to the exogenous SAM non-treated sco3956 complementation strain. Thus, the ABC transporter ATP-binding protein, SCO3956, plays a critical role in ACT and RED production serving as a transducer of SAM signaling.