Synthesis, Biological Evaluation, and 3D-QSAR Studies of -(Substituted pyridine-4-yl)-1-(substituted phenyl)-5-trifluoromethyl-1-pyrazole-4-carboxamide Derivatives as Potential Succinate Dehydrogenase Inhibitors.

A series of new fungicides that can inhibit the succinate dehydrogenase (SDH) was classified and named as SDH inhibitors by the Fungicide Resistance Action Committee in 2009. To develop more potential SDH inhibitors, we designed and synthesized a novel series of -(substituted pyridine-4-yl)-1-(substituted phenyl)-5-trifluoromethyl-1-pyrazole-4-carboxamide derivatives, -, namely, -, -, and -. The bioassay results demonstrated that some title compounds exhibited excellent antifungal activity against four tested phytopathogenic fungi (, , , and ).

Production of L-Theanine Using Whole-Cell Overexpressing γ-Glutamylmethylamide Synthetase with Bakers Yeast.

L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production.

Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP.

Cadaverine, also known as 1,5-pentanediamine, is an important platform chemical with a wide range of applications and can be produced either by fermentation or bioconversion. Bioconversion of cadaverine from l-lysine is the preferred method because of its many benefits, including rapid reaction time and an easy downstream process. In our previous study, we replaced pyridoxal-5-phosphate (PLP) with pyridoxal kinase (PdxY) along with pyridoxal (PL) because it could achieve 80% conversion with 0.

Polyhydroxybutyrate production in halophilic marine bacteria Vibrio proteolyticus isolated from the Korean peninsula.

Polyhydroxybutyrates (PHB) are biodegradable polymers that are produced by various microbes, including Ralstonia, Pseudomonas, and Bacillus species. In this study, a Vibrio proteolyticus strain, which produces a high level of polyhydroxyalkanoate (PHA), was isolated from the Korean marine environment. To determine optimal growth and production conditions, environments with different salinity, carbon sources, and nitrogen sources were evaluated.

The role of NdgR in glycerol metabolism in Streptomyces coelicolor.

Streptomyces, which produces many pharmaceutical antibiotics and anticancer agents, is a genus of soil-dwelling bacteria with numerous regulators that control both primary and secondary metabolism. NdgR is highly conserved in Streptomyces spp. and is known to be involved in antibiotic production, tolerance against shock and physical stress, nitrogen metabolism, leucine metabolism, and N-acetylglucosamine metabolism.

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC.

Engineering substrate specificity of succinic semialdehyde reductase (AKR7A5) for efficient conversion of levulinic acid to 4-hydroxyvaleric acid.

Engineering enzyme substrate specificity is a promising approach that can expand the applicability of enzymes for the biocatalytic production of industrial chemicals and fuels. In this study, succinic semialdehyde reductase (AKR7A5) was engineered for the conversion of levulinic acid to 4-hydroxyvaleric acid. Levulinic acid is a derivative of cellulosic biomass, and 4-hydroxyvaleric acid is a potential precursor to bio-polymers and fuels.

Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression.

The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms.

Production of rapamycin in Streptomyces hygroscopicus from glycerol-based media optimized by systemic methodology.

Rapamycin, produced by the soil bacterium Streptomyces hygroscopicus, has the ability to suppress the immune system and is used as an antifungal, anti-inflammatory, antitumor, and immunosuppressive agent. In an attempt to increase the productivity of rapamycin, mutagenesis of wild-type Streptomyces hygroscopicus was performed using ultraviolet radiation, and the medium composition was optimized using glycerol (which is one of the cheapest starting substrates) by applying Plackett-Burman design and response surface methodology. Plackett-Burman design was used to analyze 14 medium constituents: M100 (maltodextrin), glycerol, soybean meal, soytone, yeast extract, (NH4)2SO4, L-lysine, KH2PO4, K2HPO4, NaCl, FeSO4·7H2O, CaCO3, 2-(N-morpholino) ethanesulfonic acid, and the initial pH level.

Putative role of a Streptomyces coelicolor-derived α-mannosidase in deglycosylation and antibiotic production.

SCO0948 was found to be the single open reading frame annotated to encode an α-mannosidase (AM1) in Streptomyces coelicolor M145. To characterize the protein, we overexpressed SCO0948 in Escherichia coli BL21(DE3). Recombinant AM1, with a molecular weight of 110 kDa, exhibited α-mannosidase activity toward 4-nitrophenyl-α-D-mannopyranoside with a K m of 4.

Functional analysis of the gene SCO1782 encoding Streptomyces hemolysin (S-hemolysin) in Streptomyces coelicolor M145.

In the process of evaluating the growth of Streptomyces coelicolor on rich media such as blood agar, we found that S. coelicolor a non-pathogenic, well-known antibiotic producer had the ability to grow and produce a prominent hemolytic zone. By comparing the growth with an agarase gene mutant of S.

Phosphorylation of chloramphenicol by a recombinant protein Yhr2 from Streptomyces avermitilis MA4680.

Although phosphorylation of chloramphenicol has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3'-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic.

Identification and functional characterization of an α-amylase with broad temperature and pH stability from Paenibacillus sp.

Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase from the genomic DNA library of Paenibacillus sp. was identified and characterized.

Deletion of an architectural unit, leucyl aminopeptidase (SCO2179), in Streptomyces coelicolor increases actinorhodin production and sporulation.

Several reports state that three architectural units, including integration host factor, leucyl aminopeptidase (PepA), and purine regulator, are involved in transcriptional process with RNA polymerase in Escherichia coli. Similarly, Streptomyces species possess the same structural units. We previously identified a protein, Streptomyces integration host factor (sIHF), involved in antibiotic production and sporulation.

Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase.

Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV)--a monomer of bio-polyester and a precursor of bio-fuels--using 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis. Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity (kcat/Km=578.

Increased sensitivity to chloramphenicol by inactivation of manB in Streptomyces coelicolor.

Phosphomannomutase (ManB) is involved in the biosynthesis of GDP-mannose, which is vital for numerous processes such as synthesis of carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins. Here, we discovered that a deletion mutant of manB (BG101) in Streptomyces coelicolor (S. coelicolor) showed higher sensitivity to bacteriostatic chloramphenicol (CM) than the wild-type strain (M145), along with decreased production of CM metabolites.

Engineering of daidzein 3'-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450.

A cytochrome P450 (CYP) enzyme, 3'-daidzein hydroxylase, CYP105D7 (3'-DH), responsible for daidzein hydroxylation at the 3'-position, was recently reported. CYP105D7 (3'-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product.

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS).

Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process.

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor.

γ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters.

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor.

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S.

Development of inhibitors against TraR quorum-sensing system in Agrobacterium tumefaciens by molecular modeling of the ligand-receptor interaction.

The quorum sensing (QS) inhibitors that antagonize TraR, a receptor protein for N-3-oxo-octanoyl-L-homoserine lactones (3-oxo-C8-HSL), a QS signal of Agrobacterium tumefaciens were developed. The structural analogues of 3-oxo-C8-HSL were designed by in silico molecular modeling using SYBYL packages, and synthesized by the solid phase organic synthesis (SPOS) method, where the carboxamide bond of 3-oxo-C8-HSL was replaced with a nicotinamide or a sulfonamide bond to make derivatives of N-nicotinyl-L-homoserine lactones or N-sulfonyl-L-homoserine lactones. The in vivo inhibitory activities of these compounds against QS signaling were assayed using reporter systems and compared with the estimated binding energies from the modeling study.

Cell-free Escherichia coli-based system to screen for quorum-sensing molecules interacting with quorum receptor proteins of Streptomyces coelicolor.

Quorum sensing (QS) is mediated by small molecules and involved in diverse cellular functions, such as virulence, biofilm formation, secondary metabolism, and cell differentiation. In this study, we developed a rapid and effective screening tool based on a cell-free Escherichia coli-based expression system to identify QS molecules of Streptomyces. The binding of QS molecules to gamma-butyrolactone receptor ScbR was monitored by changes in the expression levels of the green fluorescent protein reporter in E.

Structural understanding of quorum-sensing inhibitors by molecular modeling study in Pseudomonas aeruginosa.

Inhibitors of 3OC12, an initial signal molecule of the quorum sensing (QS) signaling cascade in Pseudomonas aeruginosa have been developed. Eight inhibitor candidates were synthesized by substituting the head part of 3-oxododecanoyl-homoserine lactone (3OC12) with different aromatic rings, and their docking poses and scores (binding energies) were predicted by in silico modeling study. All compounds gave better docking scores than 3OC12 and good inhibition effects on LasR activity in the in vivo bioassay.

Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening.

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened.

Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa.

The biofilm formation of Pseudomonas aeruginosa, an opportunistic human pathogen, is developed by cell-to-cell signaling, so-called quorum sensing (QS). To control the biofilm formation, we designed and synthesized new QS inhibitors of P. aeruginosa based on the structure of the previously known QS inhibitor, furanone.