Biological activities and metabolomic profiles of extracts from the marine sediment bacterium Nocardiopsis alba DP1B cultivated in different media.

The soil bacterium DP1B was isolated from a marine sediment collected off the coast of Randayan Island, Kalimantan Barat, Indonesia and identified based on 16S rDNA as Nocardiopsis alba. The bacterium was cultivated in seven different media (A1, ISP1, ISP2, ISP4, PDB, PC-1, and SCB) with three different solvents [distilled water, 5 % NaCl solution, artificial seawater (ASW)] combinations, shaken at 200 rpm, 30 °C, for 7 days. The culture broths were extracted with ethyl acetate and each extract was tested for its antimicrobial activity and brine shrimp lethality, and the chemical diversity was assessed using thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The result showed that almost all extracts showed antibacterial but not antifungal activity, whereas their brine shrimp toxicity levels vary from high to low. The best medium/solvent combinations for antibacterial activity and toxicity were PC-1 (in either distilled water, 5% NaCl solution, or ASW) and SCB in ASW. Different chemical diversity profiles were observed on TLC, GC-MS, and LC-MS/MS. Extracts from the PC-1 cultures seem to contain a significant number of cyclic dipeptides, whereas those from the SCB cultures contain sesquiterpenes, indicating that media and solvent compositions can affect the secondary metabolite profiles of DP1B. In addition, untargeted metabolomic analyses using LC-MS/MS showed many molecular ions that did not match with those in the Global Natural Products Social Molecular Networking (GNPS) database, suggesting that DP1B has great potential as a source of new natural products.

Four sulfur-containing compounds with anti-colon cancer effect from marine-derived fungus Aspergillus terreus.

Sulfur-containing natural products possess a variety of biological functions including antitumor, antibacterial, anti-inflammatory and antiviral activities. In this study, four previously undescribed sulfur-containing compounds asperteretals L and M, terreins A and B, together with 17 known compounds were obtained from a culture of marine fungus A. terreus supplemented with inorganic sulfur source Na2SO4. Their planar structures and absolute configurations were elucidated by NMR, HRESIMS, and ECD experiments. The in vitro cytotoxicities of compounds 1-21 against HCT-116 and Caco-2 were evaluated by SRB assay. Asperteretal M (2) exhibited activity against HCT-116 with the IC50 value at 30µM. The antiproliferative effect of asperteretal M was confirmed by colony formation assay and cell death staining. Furthermore, the preliminary study on the anti-colon cancer mechanism of asperteretal M was performed by RNA-seq analysis. Western blotting validated that asperteretal M significantly decreased the expression of cell-cycle regulatory proteins CDK1, CDK4, and PCNA in a concentration-dependent manner.

Synthesis of the Carba-Analogs of the α-Pyranose and ß-Pyranose Forms of Sedoheptulose 7-Phosphate and Probing the Stereospecificity of Sedoheptulose 7-Phosphate Cyclases.

Sedoheptulose 7-phosphate (SH7P) cyclases are a subset of sugar phosphate cyclases that are known to catalyze the first committed step in many biosynthetic pathways in primary and secondary metabolism. Among them are 2-epi-5-epi-valiolone synthase (EEVS) and 2-epi-valiolone synthase (EVS), two closely related SH7P cyclases that catalyze the conversion of SH7P to 2-epi-5-epi-valiolone and 2-epi-valiolone, respectively. However, how these two homologous enzymes use a common substrate to produce stereochemically different products is unknown. Two competing hypotheses have been proposed for the stereospecificity of EEVS and EVS: (1) variation in aldol acceptor geometry during enzyme catalysis, and (2) preselection of the α-pyranose or ß-pyranose forms of the substrate by the enzymes. Yet, there is no direct evidence to support or rule out either of these hypotheses. Here we report the synthesis of the carba-analogs of the α-pyranose and ß-pyranose forms of SH7P and their use in probing the stereospecificity of ValA (EEVS from Streptomyces hygroscopicus subsp. jinggangensis) and Amir_2000 (EVS from Actinosynnema mirum DSM 43827). Kinetic studies of the enzymes in the presence of the synthetic compounds as well as docking studies of the enzymes with the α- and ß-pyranose forms of SH7P suggest that the inverted configuration of the products of EEVS and EVS is not due to the preselection of the different forms of the substrate by the enzymes.

Molecular basis for the increased activity of ZMS-2 serine protease in the presence of metal ions and hydrogen peroxide.

Serine proteases are important enzymes widely used in commercial products and industry. Recently, we identified a new serine protease from the desert bacterium Bacillus subtilis ZMS-2 that showed enhanced activity in the presence of Zn2+, Ag+, or H2O2. However, the molecular basis underlying this interesting property is unknown. Here, we report comparative studies between the ZMS-2 protease and its homolog, subtilisin E (SubE), from B. subtilis ATCC 6051. In the absence of Zn2+, Ag+, or H2O2, both enzymes showed the same level of proteolytic activity, but in the presence of Zn2+, Ag+, or H2O2, ZMS-2 displayed increased activity by 22%, 8%, and 14%, whereas SubE showed decreased activity by 16%, 12%, and 9%, respectively. In silico studies showed that both proteins have almost identical amino acid sequences and folding structures, except for two amino acids located in the protruding loops of the proteins. ZMS-2 contains Ser236 and Ser268, whereas SubE contains Thr236 and Thr268. Replacing Ser236 or Ser268 in ZMS-2 with threonine resulted in variants whose activities were not enhanced by Zn2+ or Ag+. However, this single mutation did not affect the enhancement by H2O2. This finding may be used as a basis for engineering better proteases for industrial uses.

Modifications of Protein-Bound Substrates by Trans-Acting Enzymes in Natural Products Biosynthesis.

Enzymatic modifications of small molecules are a common phenomenon in natural product biosynthesis, leading to the production of diverse bioactive compounds. In polyketide biosynthesis, modifications commonly take place after the completion of the polyketide backbone assembly by the polyketide synthases and the mature products are released from the acyl-carrier protein (ACP). However, exceptions to this rule appear to be widespread, as on-line hydroxylation, methyl transfer, and cyclization during polyketide assembly process are common, particularly in trans-AT PKS systems. Many of these modifications are catalyzed by specific domains within the modular PKS systems. However, several of the on-line modifications are catalyzed by stand-alone proteins. Those include the on-line Baeyer-Villiger oxidation, α-hydroxylation, halogenation, epoxidation, and methyl esterification during polyketide assembly, dehydrogenation of ACP-bound short fatty acids by acyl-CoA dehydrogenase-like enzymes, and glycosylation of ACP-bound intermediates by discrete glycosyltransferase enzymes. This review article highlights some of these trans-acting proteins that catalyze enzymatic modifications of ACP-bound small molecules in natural product biosynthesis.

Catalytic Mechanism of Nonglycosidic C-N Bond Formation by the Pseudoglycosyltransferase Enzyme VldE.

The pseudoglycosyltransferase (PsGT) enzyme VldE is a homologue of the retaining glycosyltransferase (GT) trehalose 6-phosphate synthase (OtsA) that catalyzes a coupling reaction between two pseudo-sugar units, GDP-valienol and validamine 7-phosphate, to give a product with α,α-N-pseudo-glycosidic linkage. Despite its biological importance and unique catalytic function, the molecular bases for its substrate specificity and reaction mechanism are still obscure. Here, we report a comparative mechanistic study of VldE and OtsA using various engineered chimeric proteins and point mutants of the enzymes, X-ray crystallography, docking studies, and kinetic isotope effects. We found that the distinct substrate specificities between VldE and OtsA are most likely due to topological differences within the hot spot amino acid regions of their N-terminal domains. We also found that the Asp158 and His182 residues, which are in the active site, play a significant role in the PsGT function of VldE. They do not seem to be directly involved in the catalysis but may be important for substrate recognition or contribute to the overall architecture of the active site pocket. Moreover, results of the kinetic isotope effect experiments suggest that VldE catalyzes a C-N bond formation between GDP-valienol and validamine 7-phosphate via an SNi-like mechanism. The study provides new insights into the substrate specificity and catalytic mechanism of a member of the growing family of PsGT enzymes, which may be used as a basis for developing new PsGTs from GTs.

Characterization and Pilot-Scale Application of the ZMS-2 Serine Protease with Novel Properties for the Eco-friendly Leather Processing.

Microbial alkaline proteases are dominating the global enzyme market with a share of over 65% due to their multifarious catalytic potentials. Hence, production of proteases with novel properties of commercial significance is highly desirable to meet the global enzyme demand. Here, we report the purification, characterization, and pilot-scale application of a serine protease from the desert soil bacterium Bacillus subtilis ZMS-2 with novel properties as dehairing agent in leather processing. The enzyme was purified 16.5-fold with a specific activity of 1543.5 U mg-1 and recovery percentage of 33.6% using ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The purified enzyme was characterized as a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular weight of 36.1 kDa with an optimum activity at pH 8.5 and 60 °C. The catalytic activity of the enzyme was enhanced by Zn+2 (204%), Ag+ (110%), H2O2 (123%), Triton X-100 (110%), iso-octane (109%), chloroform (110%), ethanol (105%), ethyl acetate (110%), and acetonitrile (128%). During pilot-scale applications, the optimum condition was found to be a combination of enzyme (1.5%, 460 U mL-1), sodium sulfide (2%), and calcium hydroxide (lime) (3%). Under this condition, the time required for complete dehairing was 90 min. Chemoenzymatically processed skins exhibited better physical properties than chemically processed skin, including tensile strength (16.35 ± 6.68 N/mm), ball burst (452.88 ± 6.06 N/mm), percent elongation (38.85 ± 1.06 N), tear strength (50.16 ± 4.42 N/mm), and softness (6.5 mm). Electron microscopy analysis of the treated skin showed complete removal of hairs with roots, confirming the keratin specificity of the enzyme. Moreover, the enzyme-assisted dehairing process reduced chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) by 68, 77, 34, and 39%, respectively. Thus, the alkaline serine protease from B. subtilis ZMS-2 is a potential dehairing agent for the eco-friendly processing of animal skins on industrial scales.

Genome features and secondary metabolite potential of the marine symbiont Streptomyces sp. RS2.

Streptomyces sp. RS2 was isolated from an unidentified sponge collected around Randayan Island, Indonesia. The genome of Streptomyces sp. RS2 consists of a linear chromosome of 9,391,717 base pairs with 71.9% of G + C content, 8270 protein-coding genes, as well as 18 rRNA and 85 tRNA loci. Twenty-eight putative secondary metabolites biosynthetic gene clusters (BGCs) were identified in the genome sequence. Nine of them have 100% similarity to BGCs for albaflavenone, α-lipomycin, coelibactin, coelichelin, ectoine, geosmin, germicidin, hopene, and lanthionine (SapB). The remaining 19 BGCs have low (< 50%) or moderate (50-80%) similarity to other known secondary metabolite BGCs. Biological activity assays of extracts from 21 different cultures of the RS2 strain showed that SCB ASW was the best medium for the production of antimicrobial and cytotoxic compounds. Streptomyces sp. RS2 has great potential to be a producer of novel secondary metabolites, particularly those with antimicrobial and antitumor activities.

Modifications of Acyl Carrier Protein-Bound Glycosylated Polyketides in Pactamycin Biosynthesis.

The potent antitumor antibiotic pactamycin is an aminocyclopentitol-containing natural product produced by the soil bacterium Streptomyces pactum. Recent studies showed that the aminocyclopentitol unit is derived from N-acetyl-D-glucosamine, which is attached to an acyl carrier protein (ACP)-bound polyketide by a glycosyltransferase enzyme, PtmJ. Here, we report a series of post-glycosylation modifications of the sugar moiety of the glycosylated polyketide while it is still attached to the carrier protein. In vitro reconstitution of PtmS (an AMP-ligase), PtmI (an ACP), PtmJ, PtmN (an oxidoreductase), PtmA (an aminotransferase), and PtmB (a putative carbamoyltransferase) showed that the N-acetyl-D-glucosamine moiety of the glycosylated polyketide is first oxidized by PtmN and then transaminated by PtmA to give ACP-bound 3-amino-3-deoxy-N-acetyl-D-glucosaminyl polyketide. The amino group is then coupled with carbamoyl phosphate by PtmB to give a urea functionality. We also show that PtmG is a deacetylase that hydrolyses the C-2 N-acetyl group to give a free amine.

Synthesis and Anti-Inflammatory Activity of the Natural Cyclooxygenase-2 Inhibitor Axinelline A and Its Analogues.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used medications to treat conditions such as arthritis, pain, and fever. They reduce inflammation by inhibiting cyclooxygenase (COX) enzymes that catalyze the committed step in prostaglandin (PG) biosynthesis. Despite their significant therapeutic benefits, many NSAIDS have undesirable adverse effects. The aim of this study was to discover novel COX inhibitors from natural sources. Here, we describe the synthesis and anti-inflammatory activity of the COX-2 inhibitor axinelline A (A1), which was isolated from Streptomyces axinellae SCSIO02208, and its analogues. Compared to the synthetic analogues, the natural product A1 has stronger COX inhibitory activity. Although A1 is more active against COX-2 than COX-1, its selectivity index is low; therefore, it may be classified as a nonselective COX inhibitor. Its overall activity is comparable to the clinically used drug diclofenac. In silico studies showed that A1 binds to COX-2 in a similar manner to diclofenac. Inhibition of COX enzymes by A1 in LPS-stimulated murine RAW264.7 macrophages resulted in suppression of the NF-κB signaling pathway, leading to reduced expression of pro-inflammatory factors such as iNOS, COX-2, TNF-α, IL-6, and IL-1ß and reduced production of PGE2, NO, and ROS. The potent in vitro anti-inflammatory activity of A1, together with its lack of cytotoxicity, makes it an attractive candidate for a new anti-inflammatory lead.

Isolation and stereospecific synthesis of indole alkaloids with lipid-lowering effects from the marine-derived fungus Colletotrichum gloeosporioides BB4.

Seven undescribed compounds, colletotrichindoles A-E, colletotrichaniline A, and colletotrichdiol A, as well as three known compounds, (-)-isoalternatine A, (+)-alternatine A and 3-hydroxybutan-2-yl 2-phenylacetate were isolated from the marine-derived fungus Colletotrichu gloeosporioides BB4. The racemic mixtures colletotrichindole A，colletotrichindole C, and colletotrichdiol A were further separated by chiral chromatography to give three pairs of enantiomers (10S,11R,13S)/(10R,11S,13R)-colletotrichindole A, (10R,11R,13S)/(10S,11S,13R)-colletotrichindole C, and (9S,10S)/(9R,10R)-colletotrichdiol A, respectively. The chemical structures of seven undescribed compounds and the known compounds, (-)-isoalternatine A, and (+)-alternatine A were determined using a combination of NMR, MS, X-ray diffraction, ECD calculations, and/or chemical synthesis. All possible enantiomers of colletotrichindoles A-E were synthesized and used to determine the absolute configurations of the natural products by comparing their spectroscopic data and HPLC retention times on a chiral column. In addition, the X-ray crystal structures of the known compounds (-)-isoalternatine A and (+)-alternatine A were also obtained to confirm their absolute configurations. (10S,11R,13S)-Colletotrichindole A, colletotrichindole B, and (+)-alternatine A significantly reduced triglyceride levels in 3T3-L1 cells with EC50 values of 5.8, 9.0, and 1.3 µM, respectively.

Acarbose May Function as a Competitive Exclusion Agent for the Producing Bacteria.

Acarbose is a well-known microbial specialized metabolite used clinically to treat type 2 diabetes. This natural pseudo-oligosaccharide (PsOS) shows potent inhibitory activity toward various glycosyl hydrolases, including α-glucosidases and α-amylases. While acarbose and other PsOSs are produced by many different bacteria, their ecological or biological role in microbial communities is still an open question. Here, we show that several PsOS-producing actinobacteria, i.e., Actinoplanes sp. SE50/110 (acarbose producer), Streptomyces glaucescens GLA.O (acarbose producer), and Streptomyces dimorphogenes ATCC 31484 (trestatin producer), can grow in the presence of acarbose, while the growth of the non-PsOS-producing organism Streptomyces coelicolor M1152 was suppressed when starch is the main source of energy. Further investigations using recombinant α-amylases from S. coelicolor M1152 and the PsOS-producing actinobacteria revealed that the S. coelicolor α-amylase was inhibited by acarbose, whereas those from the PsOS-producing bacteria were not inhibited by acarbose. Bioinformatic and protein modeling studies suggested that a point mutation in the α-amylases of the PsOS-producing actinobacteria is responsible for the resistance of those enzymes toward acarbose. Converting the acarbose-resistant α-amylase AcbE to its A304H variant diminished its acarbose-resistance property. Taken together, the results suggest that acarbose is used by the producing bacteria as a competitive exclusion agent to suppress the growth of other microorganisms in their natural environment, while the producing organisms equip themselves with α-amylase variants that are resistant to acarbose.

Biochemical Characterization of GacI, a Bifunctional Glycosyltransferase-Phosphatase Enzyme Involved in Acarbose Biosynthesis in Streptomyces glaucescens GLA.O.

Acarbose, a pseudotetrasaccharide produced by several strains of Actinoplanes and Streptomyces, is an α-glucosidase inhibitor clinically used to control type II diabetes. Bioinformatic analysis of the biosynthetic gene clusters of acarbose in Actinoplanes sp. SE50/110 (the acb cluster) and Streptomyces glaucescens GLA.O (the gac cluster) revealed their distinct genetic organizations and presumably biosynthetic pathways. However, to date, only the acarbose pathway in the SE50/110 strain has been extensively studied. Here, we report that GacI, one of the proteins that appear to be different between the two pathways, is a bifunctional glycosyltransferase family 5 (GT5)-phosphatase (PP) enzyme that functions at two different steps in acarbose biosynthesis in S. glaucescens GLA.O. In the acb pathway, the GT and the PP reactions are performed by two different enzymes. Truncated GacI proteins having only the GT or the PP domain showed comparable catalytic activity with the full-length GacI, indicating that domain separation does not significantly affect their respective catalytic activity. GacI, which is widely distributed in many Streptomyces, represents the first example of naturally occurring GT5-PP bifunctional enzymes biochemically characterized.

ß-Carboline Alkaloids From the Deep-Sea Fungus Trichoderma sp. MCCC 3A01244 as a New Type of Anti-pulmonary Fibrosis Agent That Inhibits TGF-ß/Smad Signaling Pathway.

Pulmonary fibrosis is a scarring disease of lung tissue, which seriously threatens human health. Treatment options are currently limited, and effective strategies are still lacking. In the present study, 25 compounds were isolated from the deep-sea fungus Trichoderma sp. MCCC 3A01244. Among them, two ß-carboline alkaloids, trichocarbolines A (1) and C (4) are new compounds. The chemical structures of these compounds were elucidated based on their HRESIMS, 1D and 2D NMR spectra, optical rotation calculation, and comparisons with data reported in the literature. Trichocarboline B [(+)- and (-)-enantiomers] had previously been synthesized, and this is its first report as a natural product. Their anti-pulmonary fibrosis (PF) activity and cytotoxicity were investigated. Compounds 1, 11, and 13 strongly inhibited TGF-ß1-induced total collagen accumulation and showed low cytotoxicity against the HFL1 cell line. Further studies revealed compound 1 inhibited extracellular matrix (ECM) deposition by downregulating the expression of protein fibronectin (FN), proliferating cell nuclear antigen (PCNA), and α-smooth muscle actin (α-SMA). Mechanistic study revealed that compound 1 decreased pulmonary fibrosis by inhibiting the TGF-ß/Smad signaling pathway. As a newly identified ß-carboline alkaloid, compound 1 may be used as a lead compound for developing more efficient anti-pulmonary fibrosis agents.

Exophilone, a Tetrahydrocarbazol-1-one Analogue with Anti-Pulmonary Fibrosis Activity from the Deep-Sea Fungus Exophiala oligosperma MCCC 3A01264.

A new compound, exophilone (1), together with nine known compounds (2-10), were isolated from a deep-sea-derived fungus, Exophiala oligosperma. Their chemical structures, including the absolute configuration of 1, were elucidated using nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and electronic circular dichroism (ECD) calculation. Compounds were preliminarily screened for their ability to inhibit collagen accumulation. Compounds 1, 4, and 7 showed weaker inhibition of TGF-ß1-induced total collagen accumulation in compared with pirfenidone (73.14% inhibition rate). However, pirfenidone exhibited cytotoxicity (77.57% survival rate), while compounds 1, 4, and 7 showed low cytotoxicity against the HFL1 cell line. Particularly, exophilone (1) showed moderate collagen deposition inhibition effect (60.44% inhibition rate) and low toxicity in HFL1 cells (98.14% survival rate) at a concentration of 10 µM. A molecular docking study suggests that exophilone (1) binds to both TGF-ß1 and its receptor through hydrogen bonding interactions. Thus, exophilone (1) was identified as a promising anti-pulmonary fibrosis agent. It has the potential to be developed as a drug candidate for pulmonary fibrosis.

Functional Studies and Revision of the NFAT-133/TM-123 Biosynthetic Pathway in Streptomyces pactum.

The biosynthetic gene cluster of NFAT-133, an inhibitor of the nuclear factor of activated T cells, was recently identified in Streptomyces pactum ATCC 27456. This cluster is conspicuous by its highly disordered noncollinear type I modular polyketide synthase (PKS) genes that encode PKSs with one module more than those expected for the heptaketide NFAT-133 biosynthesis. Thus, the major metabolite NFAT-133 was proposed to derive from an octaketide analogue, TM-123. Here, we report that further bioinformatic analysis and gene inactivation studies suggest that NFAT-133 is not derived from TM-123 but rather a product of programmed KS7 extension skipping of a nascent heptaketide from the PKS assembly line that produces TM-123. Furthermore, identification of NFAT-133/TM-123 analogues from mutants of the ATCC 27456 strain suggests that NftN (a putative dehydrogenase), NftE (a cytochrome P450), and NftG (a putative hydrolase/decarboxylase) function "in trans" during the polyketide chain assembly processes.

The chemistry and biology of natural ribomimetics and related compounds.

Natural ribomimetics represent an important group of specialized metabolites with significant biological activities. Many of the activities, e.g., inhibition of seryl-tRNA synthetases, glycosidases, or ribosomes, are manifestations of their structural resemblance to ribose or related sugars, which play roles in the structural, physiological, and/or reproductive functions of living organisms. Recent studies on the biosynthesis and biological activities of some natural ribomimetics have expanded our understanding on how they are made in nature and why they have great potential as pharmaceutically relevant products. This review article highlights the discovery, biological activities, biosynthesis, and development of this intriguing class of natural products.

Complete biosynthetic pathway to the antidiabetic drug acarbose.

Acarbose is a bacterial-derived α-glucosidase inhibitor clinically used to treat patients with type 2 diabetes. As type 2 diabetes is on the rise worldwide, the market demand for acarbose has also increased. Despite its significant therapeutic importance, how it is made in nature is not completely understood. Here, we report the complete biosynthetic pathway to acarbose and its structural components, GDP-valienol and O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose. GDP-valienol is derived from valienol 7-phosphate, catalyzed by three cyclitol modifying enzymes, whereas O-4-amino-(4,6-dideoxy-α-D-glucopyranosyl)-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose is produced from dTDP-4-amino-4,6-dideoxy-D-glucose and maltose by the glycosyltransferase AcbI. The final assembly process is catalyzed by a pseudoglycosyltransferase enzyme, AcbS, which is a homologue of AcbI but catalyzes the formation of a non-glycosidic C-N bond. This study clarifies all previously unknown steps in acarbose biosynthesis and establishes a complete pathway to this high value pharmaceutical.

EDB Gene Cluster-Dependent Indole Production Is Responsible for the Ability of Pseudomonas fluorescens NZI7 to Repel Grazing by Caenorhabditis elegans.

The "EDB" (from "edible") gene cluster, a variant of the ebo cluster of genes found in many bacteria and algae, allows Pseudomonas fluorescens NZI7 (referred to here as "NZI7") to repel grazing by the nematode Caenorhabditis elegans. The mechanism underlying this phenotype is unknown. Here we report that the EDB cluster is involved in the conversion of tryptophan to (1H-indol-3-yl)-oxoacetamide, indole 3-aldehyde, and other indole-derived compounds. Inactivation of the EDB genes in NZI7 resulted in mutants that lack the ability to excrete indole-derived compounds as well as the ability to repel C. elegans. Heterologous expression of the NZI7 EDB cluster in E. coli cultivated in minimal M9 medium containing 2 mM l-tryptophan also released indole derivatives including tryptophol, 3-(hydroxyacetyl)indole, colletotryptin E, and two new dimeric indoles. Expression of the NZI7 EDB cluster in E. coli, cultured in minimal M9 medium and lacking tryptophan, did not produce detectable levels of indole derivatives. Both (1H-indol-3-yl)-oxoacetamide and indole 3-aldehyde showed repellent activity against C. elegans, revealing the mechanism underlying the ability of P. fluorescens NZI7 to repel grazing by C. elegans.

Characterization, Antioxidant and Antitumor Activities of Oligosaccharides Isolated from Evodia lepta (Spreng) Merr. by Different Extraction Methods.

Evodia lepta (E. lepta) is a traditional Chinese herbal medicine with various biological activities. One of the active components of this widely used medicinal plant is believed to be an oligosaccharide. The extraction yields, structural characteristics, antioxidant, and antitumor activities of four oligosaccharide extracts obtained by hot water extraction (HEO), ultrasound-assisted extraction (UEO), enzyme-assisted (EEO), and microwave-assisted extraction (MEO) were investigated. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), X-ray diffraction (XRD), and Scanning electron microscopy (SEM) results indicated that the extraction methods had a difference on the molecular mass distribution, structure, and morphology of the EOs. In addition, HEO and MEO showed strong antioxidant activities, which might be related to their uronic acid and protein contents. More interestingly, MEO was more active toward MDA-MB-231 cells compared to other cells, and cell growth inhibition was proposed to occur through apoptosis. Overall, microwave-assisted extraction is a promising technique for the extraction of high quality EO.