RESUMEN
Streptomyces peucetius produces doxorubicin and daunorubicin, which are important anticancer drugs. In this study, we activate peucemycin, a new antibacterial compound, using an OSMAC strategy. In general, bioactive compounds are produced in a higher amount at room temperature; however, in this study, we have demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius DM07. Through LC-MS/MS, IR spectroscopy, and NMR analysis, we identified the structure of this compound as a γ-pyrone macrolide. This compound was found to be novel, thus named peucemycin. It is an unusual 14-membered macrocyclic γ-pyrone ring with cyclization. Also, peucemycin exhibits potential antibacterial activity and a suppressive effect on the viability of various cancer cell lines.
RESUMEN
The members of Streptomyces have been identified as a major source of antimicrobial agents with broad spectrum. This study is mainly focused on bioactivity-guided isolation and characterization of bioactive molecule from strain Streptomyces sp. T1317-0309 and its whole-genome sequence analysis for possible isolation of novel natural products. Strain Streptomyces sp. T1317-0309 showed 100% sequence similarity with strain Streptomyces lannensis TA4-8T consisting 10, 453,255 bp of genome with 5 scaffolds and 69.9 mol% G + C content. The genome analyses revealed a total of 17 putative biosynthetic gene clusters (BGCs) responsible for various secondary metabolites including actinomycin, bacteriocin, ectoine, melanin, terpene, siderophore, betalactone, NRPS, T2PKS, and T3PKS. The BGC and bioactivity-guided purification of ethyl acetate extract of strain T1317-0309 showed the great potency of antimicrobial activities against various gram-positive multi-drug resistant human pathogens including MRSA. The BGC-predicted bioactive secondary metabolite was identified by various NMR analyses and confirmed as actinomycin D. In addition, this study reveals the first genome study of Streptomyces lannensis as a novel source for actinomycin D.
Asunto(s)
Dactinomicina/biosíntesis , Genoma Bacteriano/genética , Streptomyces/genética , Fermentación , Genes Bacterianos/genética , Pruebas de Sensibilidad Microbiana , Familia de Multigenes/genética , Filogenia , ARN Ribosómico 16S/genética , Metabolismo Secundario/genética , Streptomyces/aislamiento & purificación , Streptomyces/metabolismo , Secuenciación Completa del GenomaRESUMEN
Nargenicin A1(1) is an antibacterial macrolide with effective activity against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Due to the promising properties of this compound in inhibiting cell proliferation, immunomodulation, and the cell protective effect, there has been significant interest in this molecule. Recently, the biosynthetic gene cluster (BGC) of 1 was reported from Nocardia argentinesis and Nocardia arthritidis. In addition, two crucial enzymes involved in the formation of the core decalin moiety and postmodification of the decalin moiety by an ether bridge were characterized. This study reports on the BGC of 1 from Nocardia sp. CS682. In addition, the direct capture and heterologous expression of nar BGC from Nocardia sp. CS682 in Streptomyces venezuelae led to the production of 1. Further metabolic profiling of wild type, Nocardia sp. CS682 in optimized media (DD media) resulted in the isolation of two acetylated derivatives, 18-O-acetyl-nodusmicin and 18-O-acetyl-nargenicin. The post-PKS modification pathway in biosynthesis of 1 was also deciphered by identifying intermediates and/or in vitro enzymatic reactions of NgnP1, NgnM, and NgnO3. Different novel analogues of 1, such as compound 6, compound 7, 23-demethyl 8,13-deoxy-nodusmicin (8), 23-demethyl 8,13-deoxynargenicin (9), 8,13-deoxynodusmicin (10), and 8,13-deoxynargenicin (11), were also characterized, which extended our understanding of key post-PKS modification steps during the biosynthesis of 1. In addition, the antimicrobial and anticancer activities of selected analogues were also evaluated, whereas compound 9 was shown to exhibit potent antitumor activity by induction of G2/M cell cycle arrest, apoptosis, and autophagy.
Asunto(s)
Antibacterianos/farmacología , Antineoplásicos/farmacología , Vías Biosintéticas , Nocardia/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Genes Bacterianos , Humanos , Lactonas/química , Lactonas/metabolismo , Lactonas/farmacología , Familia de Multigenes , Neoplasias/tratamiento farmacológico , Nocardia/genética , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline-L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purification yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confirmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products.
Asunto(s)
Antineoplásicos/metabolismo , Productos Biológicos/metabolismo , Furanos/metabolismo , Streptomyces/metabolismo , Células A549 , Antibacterianos/metabolismo , Bioensayo/métodos , Línea Celular Tumoral , Genoma Bacteriano/genética , Células HCT116 , Humanos , Familia de Multigenes/genética , Filogenia , Streptomyces/genéticaRESUMEN
The application of steroids has steadily increased thanks to their therapeutic effects. However, alternatives are required due their severe side effects; thus, studies on the activities of steroid derivatives are underway. Sugar derivatives of nandrolone, which is used to treat breast cancer, as well as cortisone and prednisone, which reduce inflammation, pain, and edema, are unknown. We linked O-glucose to nandrolone and testosterone using UDP-glucosyltransferase (UGT-1) and, then, tested their bioactivities in vitro. Analysis by NMR showed that the derivatives were 17ß-nandrolone ß-D-glucose and 17ß-testosterone ß-D-glucose, respectively. The viability was higher and cytotoxicity was evident in PC12 cells incubated with rotenone and, testosterone derivatives, compared to the controls. SH-SY5Y cells incubated with H2O2 and nandrolone derivatives remained viable and cytotoxicity was attenuated. Both derivatives enhanced neuronal protective effects and increased the amounts of cellular ATP.
Asunto(s)
Bacillaceae/enzimología , Glucosiltransferasas/metabolismo , Glicósidos/metabolismo , Congéneres de la Testosterona/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Biotransformación , Línea Celular Tumoral , Metabolismo Energético/efectos de los fármacos , Glucosa/química , Glucosa/metabolismo , Glicósidos/química , Glicósidos/farmacología , Humanos , Nandrolona/química , Nandrolona/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Células PC12 , Ratas , Testosterona/química , Testosterona/metabolismo , Congéneres de la Testosterona/química , Congéneres de la Testosterona/farmacologíaRESUMEN
Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.
Asunto(s)
Antibacterianos/biosíntesis , Antineoplásicos/metabolismo , Coproporfirinas/biosíntesis , Ingeniería Metabólica/métodos , Fármacos Fotosensibilizantes/metabolismo , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismo , Antibacterianos/farmacología , Antineoplásicos/farmacología , Bacillus subtilis/efectos de los fármacos , Vías Biosintéticas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Coproporfirinas/farmacología , Medios de Cultivo/química , Humanos , Microscopía Electrónica de Rastreo , Fármacos Fotosensibilizantes/farmacología , Streptomyces/genética , Streptomyces/ultraestructuraRESUMEN
Two promiscuous Bacillus licheniformis glycosyltransferases, YdhE and YojK, exhibited prominent stereospecific but nonregiospecific glycosylation activity of 20 different classes of 59 structurally different natural and non-natural products. Both enzymes transferred various sugars at three nucleophilic groups (OH, NH2, SH) of diverse compounds to produce O-, N-, and S-glycosides. The enzymes also displayed a catalytic reversibility potential for a one-pot transglycosylation, thus bestowing a cost-effective application in biosynthesis of glycodiversified natural products in drug discovery.
Asunto(s)
Bacillus licheniformis/enzimología , Productos Biológicos/metabolismo , Glicosiltransferasas/metabolismo , Biocatálisis , Productos Biológicos/química , Glicosilación , Hidróxidos/química , Hidróxidos/metabolismo , Estructura Molecular , Fenoles/química , Fenoles/metabolismoRESUMEN
Two sustainable and cost-effective cascade enzymatic systems were developed to regenerate uridine diphosphate (UDP)-α-D-glucose and UDP-ß-L-rhamnose from sucrose. The systems were coupled with the UDP generating glycosylation reactions of UDP sugar-dependent glycosyltransferase (UGT) enzymes mediated reactions. As a result, the UDP generated as a by-product of the GT-mediated reactions was recycled. In the first system, YjiC, a UGT from Bacillus licheniformis DSM 13, was used for transferring glucose from UDP-α-D-glucose to naringenin, in which AtSUS1 from Arabidopsis thaliana was used to synthesize UDP-α-D-glucose and fructose as a by-product from sucrose. In the second system, flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from A. thaliana was used to transfer rhamnose from UDP-ß-L-rhamnose to quercetin, in which AtSUS1 along with UDP-ß-L-rhamnose synthase (AtRHM1), also from A. thaliana, were used to produce UDP-ß-L-rhamnose from the same starter sucrose. The established UDP recycling system for the production of naringenin glucosides was engineered and optimized for several reaction parameters that included temperature, metal ions, NDPs, pH, substrate ratio, and enzymes ratio, to develop a highly feasible system for large-scale production of different derivatives of naringenin and other natural products glucosides, using inexpensive starting materials. The developed system showed the conversion of about 37 mM of naringenin into three different glucosides, namely naringenin, 7-O-ß-D-glucoside, naringenin, 4'-O-ß-D-glucoside, and naringenin, 4',7-O-ß-D-diglucoside. The UDP recycling (RCmax) was 20.10 for naringenin glucosides. Similarly, the conversion of quercetin to quercetin 7-O-α-L-rhamnoside reached a RCmax value of 10.0.
Asunto(s)
Flavanonas/metabolismo , Glucósidos/metabolismo , Glucuronosiltransferasa/metabolismo , Hexosiltransferasas/metabolismo , Quercetina/metabolismo , Sacarosa/metabolismo , Arabidopsis/enzimología , Bacillus licheniformis/enzimología , Biocatálisis , Glucuronosiltransferasa/aislamiento & purificación , Hexosiltransferasas/aislamiento & purificaciónRESUMEN
The name of the author "Yamaguchi Tokutaro" is incorrect for the first and last name has been interchanged. The correct presentation is "Tokutaro Yamaguchi".
RESUMEN
Recently, studies on the steroidal hormone activity in the brain have attracted attention, and the influences of the varied glucosides and their artificial derivatives have been discussed; additionally, it has been suggested that glucosides are the synthetic precursors of glucuronide as a label molecule. However, glucosides are formed with 11α-hydroxyprogesterone (1), which is important as a blood pressure regulator, but anti-androgen activity remains unknown. Using UDP-glucosyltransferase, glucoside synthesis was successful in linking ß-d-glucopyranose and ß-d-laminaribiose to 11α oxygen of 1 at a high conversion ratio, and full assignment structure was analyzed for the two glucosides by high-resolution quadrupole-time flight electrospray ionization-mass spectrometry, 1D (1H and 13C) NMR and 2D (COSY, ROESY, HSQC-DEPT and HMQC) NMR. Furthermore, the bioactivity of 1 and two 11α-hydroxyprogesterone glucosides [11α-(ß-d-glucopyranosyl)oxyprogesterone, 2, and 11α-(ß-d-laminaribiosyl)oxyprogesterone, 3] was tested in vitro. On rotenone-induced PC12 cells, the two 11α-hydroxyprogesterone glucosides (2 and 3) showed superior neuroprotective effects and increased cellular ATP levels compared with those of 1.
Asunto(s)
Glucósidos/química , Glucosiltransferasas/metabolismo , Hidroxiprogesteronas/química , Hidroxiprogesteronas/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Animales , Biotransformación , Hidroxiprogesteronas/farmacología , Fármacos Neuroprotectores/farmacología , Células PC12 , RatasRESUMEN
Nargenicin A1 is major secondary metabolite produced by Nocardia sp. CS682, with an effective antibacterial activity against various Gram-positive bacteria. Most Nocardia spp. have metabolic ability to produce compounds of diverse nature, so one-strain-many-compounds (OSMAC) approach can be applied for obtaining versatile compounds from these strains. In this study, we characterized a novel 1, 3, 6, 8-tetrahydroxynaphthalene (THN) derivative by metabolic engineering approach leading to the inactivation of nargenicin A1 biosynthesis. By using genome mining, metabolite profiling, and bioinformatics, the biosynthetic gene cluster and biosynthetic mechanism were elucidated. Further, the antibacterial, anticancer, melanin formation, and UV protective properties for isolated THN compound were performed. The compound did not exhibit significant antibacterial and cytotoxic activities, but it exhibited promising UV protection effects. Thus, metabolic engineering is an effective strategy for discovering novel bioactive molecules.
Asunto(s)
Ingeniería Metabólica/métodos , Naftoles/química , Nocardia/crecimiento & desarrollo , Protectores contra Radiación/química , Proteínas Bacterianas/genética , Vías Biosintéticas/efectos de los fármacos , Lactonas/metabolismo , Metabolómica , Estructura Molecular , Naftoles/farmacología , Nocardia/química , Nocardia/genética , Protectores contra Radiación/farmacología , Metabolismo Secundario , Eliminación de SecuenciaRESUMEN
Quercetin and its derivatives are important flavonols that show diverse biological activity, such as antioxidant, anticarcinogenic, anti-inflammatory, and antiviral activities. Adding different substituents to quercetin may change the biochemical activity and bioavailability of molecules, when compared to the aglycone. Here, we have synthesised two novel derivatives of quercetin, quercetin-3-O-ß-d-glucopyranosyl, 4''-O-d-galactopyranosyl 3'''-O-α-N-acetyl neuraminic acid i.e. 3'-sialyllactosyl quercetin (3'SL-Q) and quercetin-3-O-ß-d-glucopyranosyl, 4''-O-ß-d-galactopyranosyl 6'''-O-α-N-acetyl neuraminic acid i.e. 6'-sialyllactosyl quercetin (6'SL-Q) with the use of glycosyltransferases and sialyltransferases enzymes. These derivatives of quercetin were characterised by high-resolution quadrupole-time-of-flight electrospray ionisation mass spectrometry (HR-QTOF-ESI/MS) and 1H and 13C nuclear magnetic resonance (NMR) analyses.
Asunto(s)
Antineoplásicos Fitogénicos/química , Lactosa/análogos & derivados , Quercetina/análogos & derivados , Quercetina/química , Ácidos Siálicos/química , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/farmacología , Técnicas de Química Sintética , Ensayos de Selección de Medicamentos Antitumorales , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Células Hep G2 , Humanos , Lactosa/síntesis química , Lactosa/química , Lactosa/farmacología , Espectroscopía de Resonancia Magnética , Estructura Molecular , Quercetina/síntesis química , Quercetina/farmacología , Ácidos Siálicos/síntesis química , Ácidos Siálicos/farmacología , Sialiltransferasas/química , Sialiltransferasas/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
A simultaneous method for analyzing sodium iron chlorophyllin (SIC) and sodium copper chlorophyllin (SCC) using high-performance liquid chromatography was developed. This method employed an Inertsil ODS-2 column and diode array detection at 395â¯nm, using methanol-water (97:3 and 80:20, v/v) containing 1% acetic acid as the mobile phase. Liquid chromatography-tandem mass spectrometry was used to identify the main components of SIC and SCC as Fe-isochlorine e4 and Cu-isochlorine e4, respectively. The limits of detection and quantitation of SIC were 1.2 and 4.1â¯mg/kg, respectively, while those of SCC were 1.4 and 4.8â¯mg/kg, respectively. For intraday and interday tests, the SIC recoveries from candy ranged from 81% to 101%, while SCC recoveries ranged from 100% to 109%. The developed method can be applied to the rapid determination of SIC and SCC in candy.
Asunto(s)
Clorofilidas/análisis , Cromatografía Líquida de Alta Presión/métodos , Análisis de los Alimentos , Espectrometría de Masas en Tándem/métodos , Cobre/análisis , Hierro/análisisRESUMEN
Flavonoids are one of the predominant groups of plant polyphenols, and these compounds have significant effects on human health and nutrition. Sulfated flavonoids have more favorable attributes compared to their parent compounds such as increased solubility, stability, and bioavailability. In this research, we developed a microbial system to produce sulfated naringenin using Escherichia coli expressing a sulfotransferase (ST) from Arabidopsis thaliana (At2g03770). This wild-type strain was used as a model system for testing clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) metabolic engineering strategies. Using synthetic sgRNA to mediate transcriptional repression of cysH, a gene encoding 3'-phosphoadenosine-5'-phosphosulfate (PAPS) ST, which is involved in sulfur metabolism, resulted in an increase in intracellular PAPS accumulation by over 3.28-fold without impairing cell growth. Moreover, naringenin 7-sulfate production by engineering E. coli with its cysH gene repressed in the open reading frame through CRISPRi was enhanced by 2.83-fold in compared with the wild-type control. To improve the efficiency of biotransformation, the concentration of SO42- , glucose, and substrate were optimized. The bioproductivity of naringenin 7-sulfate was 135.49 µM [â¼143.1 mg (47.7 mg L-1)] in a 3-L fermenter at 36 h. These results demonstrated that the CRISPRi system was successfully applied for the first time in E. coli to develop an efficient microbial strain for production of a sulfated flavonoid. In addition, antibacterial and anticancer activities of naringenin 7-sulfate were investigated and found to be higher than the parent compound.
RESUMEN
Glucosylation of the 21-hydroxyl group of glucocorticoid changes its solubility into hydrophilicity from hydrophobicity and, as with glucocorticoid glucuronides as a moving object in vivo, it is conceivable that it exhibits the same behavior. Therefore, glucosylation to the 21-hydroxyl group while maintaining the 11ß-hydroxyl group is particularly important, and glucosylation of corticosterone was confirmed by high-resolution mass spectrometry and 1D (¹H and 13C) and 2D (COSY, ROESY, HSQC-DEPT and HMBC) NMR. Moreover, the difference in bioactivity between corticosterone and corticosterone 21-glucoside was investigated in vitro. Corticosterone 21-glucoside showed greater neuroprotective effects against H2O2-induced cell death and reactive oxygen species (ROS) compared with corticosterone. These results for the first time demonstrate that bioconversion of corticosterone through the region-selective glucosylation of a novel compound can present structural potential for developing new neuroprotective agents.
Asunto(s)
Corticosterona/química , Corticosterona/metabolismo , Glucósidos/química , Glucósidos/metabolismo , Glucosiltransferasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Glucocorticoides/química , Glucocorticoides/metabolismo , Glicosilación , Humanos , Espectroscopía de Resonancia Magnética , Estructura Molecular , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Streptomyces peucetius ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of S. peucetius ATCC 27952, which consists of 8,023,114â¯bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed â¼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, in silico analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes.
Asunto(s)
Antraciclinas/metabolismo , Genoma Bacteriano/genética , Streptomyces/genética , Secuenciación Completa del Genoma , Antraciclinas/uso terapéutico , Daunorrubicina/biosíntesis , Daunorrubicina/química , Doxorrubicina/biosíntesis , Doxorrubicina/química , Humanos , Anotación de Secuencia Molecular , Sintasas Poliquetidas/biosíntesis , Sintasas Poliquetidas/química , Metabolismo Secundario/genética , Streptomyces/química , Streptomyces/metabolismoRESUMEN
Nargenicin A1, an antibacterial produced by Nocardia sp. CS682 (KCTC 11297BP), demonstrates effective activity against various Gram-positive bacteria. Hence, we attempted to enhance nargenicin A1 production by utilizing the cumulative effect of synthetic biology, metabolic engineering and statistical media optimization strategies. To facilitate the modular assembly of multiple genes for genetic engineering in Nocardia sp. CS682, we constructed a set of multi-monocistronic vectors, pNV18L1 and pNV18L2 containing hybrid promoter (derived from ermE* and promoter region of neo r ), ribosome binding sites (RBS), and restriction sites for cloning, so that each cloned gene was under its own promoter and RBS. The multi-monocistronic vector, pNV18L2 containing transcriptional terminator showed better efficiency in reporter gene assay. Thus, multiple genes involved in the biogenesis of pyrrole moiety (ngnN2, ngnN3, ngnN4, and ngnN5 from Nocardia sp. CS682), glucose utilization (glf and glk from Zymomonas mobilis), and malonyl-CoA synthesis (accA2 and accBE from Streptomyces coelicolor A3 (2)), were cloned in pNV18L2. Further statistical optimization of specific precursors (proline and glucose) and their feeding time led to ~84.9 mg/L nargenicin from Nocardia sp. GAP, which is ~24-fold higher than Nocardia sp. CS682 (without feeding). Furthermore, pikC from Streptomyces venezuelae was expressed to generate Nocardia sp. PikC. Nargenicin A1 acid was characterized as novel derivative of nargenicin A1 produced from Nocardia sp. PikC by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. We also performed comparative analysis of the anticancer and antibacterial activities of nargenicin A1 and nargenicin A1 acid, which showed a reduction in antibacterial potential for nargenicin A1 acid. Thus, the development of an efficient synthetic biological platform provided new avenues for enhancing or structurally diversifying nargenicin A1 by means of pathway designing and engineering.
Asunto(s)
Antibacterianos/metabolismo , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Nocardia/genética , Nocardia/metabolismo , Biología Sintética , Medios de Cultivo/química , Expresión Génica , Vectores Genéticos , Lactonas/metabolismo , Nocardia/crecimiento & desarrollo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Herboxidiene is a natural product produced by Streptomyces chromofuscus exhibiting herbicidal activity as well as antitumor properties. Using different substrate-flexible cytochrome P450s and glycosyltransferase, different novel derivatives of herboxidiene were generated with structural modifications by hydroxylation or epoxidation or conjugation with a glucose moiety. Moreover, two isomers of herboxidiene containing extra tetrahydrofuran or tetrahydropyran moiety in addition to the existing tetrahydropyran moiety were characterized. The hydroxylated products for both of these compounds were also isolated and characterized from S. chromofuscus PikC harboring pikC from the pikromycin gene cluster of Streptomyces venezuelae and S. chromofuscus EryF harboring eryF from the erythromycin gene cluster of Saccharopolyspora erythraea. The compounds generated were characterized by high-resolution quadrupole-time-of-flight electrospray ionization mass spectrometry (HR-QTOF-ESI/MS) and (1)H- and (13)C-nuclear magnetic resonance (NMR) analyses. The evaluation of antibacterial activity against three Gram-positive bacteria, Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus, indicated that modification resulted in a transition from anticancer to antibacterial potency.
Asunto(s)
Antibacterianos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Alcoholes Grasos/metabolismo , Glicosiltransferasas/metabolismo , Ingeniería Metabólica , Piranos/metabolismo , Streptomyces/enzimología , Streptomyces/metabolismo , Antibacterianos/química , Bacillus/efectos de los fármacos , Bacillus/crecimiento & desarrollo , Biotransformación , Alcoholes Grasos/química , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrollo , Piranos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharopolyspora/enzimología , Saccharopolyspora/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Streptomyces/genéticaRESUMEN
Chromobacterium sp. strain C61 displays antifungal activities in vitro and has been used successfully for the biocontrol of plant diseases under field conditions. In this study, the roles of extracellular chitinase and an antifungal compound produced by strain C61 were investigated to elucidate their contributions to biological control activity. The bacterium possessed a locus chi54 encoding an extracellular chitinase, and mutation of chi54 eliminated chitinase production. Production of the extracellular enzyme and expression of the chi54 transcript were increased in the wild-type strain when chitin was added to the culture medium. In vitro assays showed that purified chitinase inhibited spore germination of multiple pathogens. However, the in planta biocontrol activity of filtrates of cultures grown in the presence of chitin was lower than that of filtrates grown without chitin, indicating that correlation between chitinase and biocontrol activity was lacking. The analysis of C61 culture filtrates revealed an antifungal cyclic lipopeptide, chromobactomycin, whose structure contained a unique nonameric peptide ring. The purified chromobactomycin inhibited the growth of several phytopathogenic fungi in vitro, and plant application significantly reduced disease severity for several pathogens. Furthermore, the production of chromobactomycin was reduced in cultures amended with chitin. These data suggest that the production of both the extracellular chitinase Chi54 and the newly identified antibiotic chromobactomycin can contribute, in an interconnected way, to the suppression of plant disease by Chromobacterium sp. strain C61.
Asunto(s)
Antifúngicos/farmacología , Quitinasas/farmacología , Chromobacterium/metabolismo , Lipopéptidos/farmacología , Péptidos Cíclicos/farmacología , Control Biológico de Vectores/métodos , Secuencia de Bases , Quitina/farmacología , Quitinasas/metabolismo , Chromobacterium/genética , Medios de Cultivo , Cartilla de ADN , Hongos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la PolimerasaRESUMEN
A sterol glucosyltransferase-encoded gene was isolated from Salinispora tropica CNB-440, a marine, sediment-dwelling, Gram positive bacterium that produces the potent anticancer compound, salinosporamide A. The full-length gene consists of 1284 nucleotides and encodes 427 amino acids with a calculated mass of 45.65kDa. The gene was then cloned and heterologously expressed in Escherichia coli BL21(DE3). The amino acid sequence shares 39% similarity with the glycosyltransferase from Withania somnifera, which belongs to glycosyltransferase family 1. Enzyme reactions were carried out with the various free sterols (acceptor) and NDP-sugars (donor). The purified protein only showed activity for glucosylation of ß-sitosterol with UDP-D-glucose and TDP-D-glucose donors, and optimal activity at pH 7.5 and 37°C. Among these two donors, UDP-D-glucose was preferred.