RESUMO
Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: ⢠SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. ⢠SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. ⢠SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.
Assuntos
Streptomyces coelicolor , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Galactose/química , Ágar/metabolismo , Glicosídeo Hidrolases/metabolismo , Galactosidases/metabolismo , beta-GalactosidaseRESUMO
A new secondary metabolite, ulleungdolin (1), was isolated from the co-culture of an actinomycete, Streptomyces sp. 13F051, and a fungus, Leohumicola minima 15S071. Based on the NMR, UV, and MS data, it was deduced that the planar structure of 1 comprised an isoindolinone (IsoID) with an octanoic acid, a tripeptide, and a sugar. The tripeptide has the unprecedented amino acids norcoronamic acid, 3-hydroxy-glutamine, and 4-hydroxy-phenylglycine and is linked by a C-N bond with IsoID. The absolute configurations were determined by chemical derivatization, extensive spectroscopic methods, and electronic circular dichroism calculations and supported by bioinformatic analyses. Bioactivity evaluation studies indicated that 1 had an antimigration effect on MDA-MB-231 breast cancer cells.
Assuntos
Ascomicetos , Policetídeos , Streptomyces , Streptomyces/química , Policetídeos/farmacologia , Policetídeos/química , Técnicas de Cocultura , Estrutura Molecular , PeptídeosRESUMO
Two angucyclines, pseudonocardones D (1) and E (2), were isolated from Streptomyces sp. KCB15JA151. The planar structure was elucidated by comprehensive spectroscopic analysis. The absolute configuration of the sugar unit was determined based on the basis of coupling constants, ROESY, chemical derivatization and HPLC analysis. The biological activities of compounds 1 and 2 were examined by performing a computational target prediction, which led to tests of the antiestrogenic activity. The result suggested that compound 1 might be an ERα antagonist.
Assuntos
Receptor alfa de Estrogênio/antagonistas & inibidores , Ácido Glucurônico/farmacologia , Streptomyces/química , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/metabolismo , Ácido Glucurônico/química , Ácido Glucurônico/isolamento & purificação , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Three new trichostatin analogues, ulleunganilines A-C (1-3), and seven known trichostatins (4-10) were isolated from cultures of Streptomyces sp. 13F051. NMR, UV, and MS data indicated that the planar structures of 1-3 consisted of modified side chains in the trichostatic acid moiety. The absolute configuration of the 2,4-dimethyl-branched carbon chains in 1 and 2 was determined by the PGME method, while the amino acid group in 3 was identified by advanced Marfey's method. Based on the structure of the modified side chains, the origin of 1-3 is proposed. Further experiments indicated that 1 and 3 displayed moderate histone deacetylase inhibitory activity, suggesting that not only the hydroxamate group but also the N,N-dimethyl group were essential for the inhibitory activity.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/isolamento & purificação , Humanos , Ácidos Hidroxâmicos/isolamento & purificação , Estrutura Molecular , República da Coreia , Microbiologia do Solo , Streptomyces/químicaRESUMO
A novel 17-allylamino-17-demethoxygeldanamycin (17-AAG) glucoside (1) was obtained from in vitro enzymatic glycosylation using a UDP-glycosyltransferase (YjiC). The water-solubility of compound 1 was approximately 10.5 times higher than that of the substrate, 17-AAG. Compound 1 showed potential anti-proliferative activities against five human cancer cell lines, with IC50 values ranging from 5.26 to 28.52 µM. Further studies also indicated that compound 1 could inhibit the growth of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (Akt, c-Raf, Bcl-2, and HIF-1α). In addition, compound 1 showed greater potential anti-tumor efficacy than 17-AAG in nude mice xenografted with CNE-2Z cells. Therefore, we suggest that in vitro enzymatic glycosylation is a powerful approach for the structural optimization of 17-AAG.
Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Glucosídeos/farmacologia , Glicosiltransferases/metabolismo , Lactamas Macrocíclicas/farmacologia , Difosfato de Uridina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glucosídeos/biossíntese , Glucosídeos/química , Glicosilação , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Relação Estrutura-AtividadeRESUMO
Two new macrolide metabolites of the hygrolidin family, catenulisporidins A and B (1 and 2), together with a known compound hygrolidin (3), were isolated from the culture broth of the rare actinobacterium Catenulispora sp. KCB13F192. Their structures were elucidated on the basis of HRESIMS spectrometric and NMR spectroscopic analyses. Catenulisporidins A and B are the first example of natural hygrolidin and bafilomycin derivatives featuring a modified macrolide ring, and catenulisporidin A possesses a tetrahydrofuran ring through an ether linkage between C-7 and C-10. In cell-based fluorescent imaging and immunoblot assays, the three compounds were shown to inhibit autophagic flux in HeLa cells.
Assuntos
Macrolídeos/farmacologia , Actinobacteria/química , Autofagia/efeitos dos fármacos , Células HeLa , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Estrutura MolecularRESUMO
PURPOSE: The purpose of this study is to compare clinical patient-reported outcomes and radiographic sagittal parameters between obese and non-obese patients following open posterior lumbar spine fusion (PLSF). METHODS: A retrospective cohort study was conducted for patients who underwent open PLSF from 2011 to 2018. Patients were classified as obese as per Center for Disease Control and Prevention guidelines if their body mass index (BMI) ≥ 30 kg/m2. Preoperative and final visual analog scale (VAS) back pain, VAS leg pain, and Oswestry Disability Index (ODI) were obtained for both obese and non-obese groups. Achievement of minimal clinically important difference was evaluated. Preoperative, immediate postoperative, and final lumbar plain radiographs were assessed to measure spinopelvic parameters. Additionally, postoperative complication measures were collected. RESULTS: A total of 569 patients were included; 290 (50.97%) patients with BMI < 30 (non-obese) and 279 (49.03%) patients with BMI ≥ 30 (obese). Patients classified as obese were more likely to have a diagnosis of diabetes mellitus (p < 0.001), and American Society of Anesthesiologists Physical Status Classification System of ≥ 3 (p < 0.001). Obese patients had significantly longer operative times (p < 0.001) compared to non-obese patients. There was no difference in radiographic measurements, patient-reported outcomes, postoperative complications, or reoperations between groups. CONCLUSION: Obese patients had significantly more comorbidities and longer operative time compared to non-obese patients. However, sagittal parameters, patient-reported outcomes, inpatient complications, length of hospital stay, and reoperations were similar between groups. Given these findings, open PLSF can be considered safe and effective in obese patients after thorough consideration of related comorbidities. These slides can be retrieved under Electronic Supplementary Material.
Assuntos
Vértebras Lombares , Fusão Vertebral , Avaliação da Deficiência , Feminino , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Fusão Vertebral/efeitos adversos , Resultado do TratamentoRESUMO
IMPORTANCE: Forehead feminization cranioplasty (FFC) is an important component of gender-affirming surgery and has become increasingly popular in recent years. Little objective evidence exists for the procedure's safety and clinical impact via patient-reported outcome measures (PROMs). OBJECTIVE: To determine what complications are observed following FFC, the relative frequency of complications by surgical technique, and what impact the procedure has on patient's quality of life. DATA SOURCES: Database searches were performed in PubMed/MEDLINE, Scopus, CINAHL, Cochrane CENTRAL, Cochrane Database of Systematic Reviews, and PsycINFO. The search terms included variations of forehead setback/FFC. Both controlled vocabularies (i.e., MeSH and CINAHL's Suggested Subject Terms) and keywords in the title or abstract fields were searched. STUDY SELECTION: Two independent reviewers screened the titles and abstracts of all articles. Two independent surgeon reviewers evaluated the full text of all included articles, and relevant data points were extracted. MAIN OUTCOMES AND MEASURES: Complications and complication rate observed following FFC. Additional outcome measures were the approach utilized, concurrent procedures performed, and the use and findings of a PROM. RESULTS: Ten articles describing FFC were included, encompassing 673 patients. The overall pooled complication rate was 1.3%. PROMs were used in half of studies, with no standardization among studies. CONCLUSIONS AND RELEVANCE: Complications following FFC are rare and infrequently require reoperation. Further studies into standardized and validated PROMs in facial feminization patients are warranted. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.
Assuntos
Testa , Qualidade de Vida , Feminização , Testa/cirurgia , Humanos , Masculino , Medidas de Resultados Relatados pelo Paciente , Satisfação do Paciente , Resultado do TratamentoRESUMO
The advances of genomic sequence analyses and genome mining tools have enabled the exploration of untapped microbial natural products. Through genome mining studies to discover cryptic natural products, we found biosynthetic genes encoding a new lasso peptide in the genome sequence of a soil bacterium, Streptomyces sp. KCB13F003 isolated from Ulleung Island (a small volcanic island), Korea. The production and purification of the encoded peptide, named ulleungdin, were achieved by optimizing the culture conditions followed by LC-MS-targeted isolation. Structure elucidation was performed by NMR spectroscopic and MS spectrometric analyses and chemical means (Marfey's and GITC derivatizations), proving ulleungdin to be a new 15-mer class II lasso peptide with a threaded structure. Biological evaluation with the cell invasion assay and time-lapse cell tracking analysis revealed that ulleungdin has significant inhibitory activities against cancer cell invasion and migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Mineração de Dados/métodos , Genômica/métodos , Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Células A549 , Fermentação , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Invasividade Neoplásica , Neoplasias/patologia , Peptídeos/química , República da Coreia , Streptomyces/químicaRESUMO
Three cyclic lipopeptides, including one known (1) and two new (2 and 3) compounds, that possess the rare enamide linkage group were discovered from Streptomyces sp. KCB14A132, an actinobacterium isolated from a soil sample collected from Jeung Island, Korea. The NMR and MS-based characterization showed that they differed in the amino acid residues in the peptide backbone. Application of Marfey's analysis, GITC derivatization, and modified Mosher's method, as well as ECD measurements provided the absolute configurations of enamidonin (1) and those of new compounds enamidonins B and C (2 and 3). The two new enamidonin analogues were shown to exhibit antibacterial activity against Gram-positive bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus. Furthermore, evaluation of the extraction conditions and a close inspection of the LC-MS chromatograms revealed that the N, N-acetonide unit of the enamidonin family was formed during the acetone extraction process. The chemically prepared deacetonide derivatives of enamidonins were found to lack antibacterial activity, demonstrating that the dimethylimidazolidinone residue is necessary for antibacterial activity.
Assuntos
Antibacterianos/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , Streptomyces/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Lipopeptídeos/isolamento & purificação , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND: Pterostilbene, a structural analog of resveratrol, has higher oral bioavailability and bioactivity than that of the parent compound; but is far less abundant in natural sources. Thus, to efficiently obtain this bioactive resveratrol analog, it is necessary to develop new bioproduction systems. RESULTS: We identified a resveratrol O-methyltransferase (ROMT) function from a multifunctional caffeic acid O-methyltransferase (COMT) originating from Arabidopsis, which catalyzes the transfer of a methyl group to resveratrol resulting in pterostilbene production. In addition, we constructed a biological platform to produce pterostilbene with this ROMT gene. Pterostilbene can be synthesized from intracellular L-tyrosine, which requires the activities of four enzymes: tyrosine ammonia lyase (TAL), p-coumarate:CoA ligase (CCL), stilbene synthase (STS) and resveratrol O-methyltransferase (ROMT). For the efficient production of pterostilbene in E. coli, we used an engineered E. coli strain to increase the intracellular pool of L-tyrosine, which is the initial precursor of pterostilbene. Next, we tried to produce pterostilbene in the engineered E. coli strain using L-methionine containing media, which is used to increase the intracellular pool of S-adenosyl-L-methionine (SAM). According to this result, pterostilbene production as high as 33.6 ± 4.1 mg/L was achieved, which was about 3.6-fold higher compared with that in the parental E. coli strain harboring a plasmid for pterostilbene biosynthesis. CONCLUSION: As a potential phytonutrient, pterostilbene was successfully produced in E. coli from a glucose medium using a single vector system, and its production titer was also significantly increased using a L-methionine containing medium in combination with a strain that had an engineered metabolic pathway for L-tyrosine. Additionally, we provide insights into the dual functions of COMT from A. thaliana which was characterized as a ROMT enzyme.
Assuntos
Arabidopsis/enzimologia , Escherichia coli/genética , Engenharia Metabólica/métodos , Metiltransferases/genética , Metiltransferases/metabolismo , Estilbenos/metabolismo , Aciltransferases/metabolismo , Amônia-Liases/metabolismo , Biocatálise , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Redes e Vias Metabólicas , Metionina/farmacologia , Resveratrol , S-Adenosilmetionina/metabolismo , Estilbenos/química , Tirosina/metabolismoRESUMO
A bioassay-guided investigation in conjunction with chemical screening led to the isolation of three new glycosides, ulleungoside (1), 2-methylaminobenzoyl 6-deoxy-α-l-talopyranoside (2), and naphthomycinoside (3), along with three known secondary metabolites (5-7) from Streptomyces sp. KCB13F030. Their structures were elucidated by detailed NMR and MS spectroscopic analyses. Absolute configurational analysis of the sugar units based on the magnitudes of the coupling constants, NOESY correlations, chemical derivatization, and optical rotation measurements revealed that compounds 1-3 and 5 incorporate the rare deoxyhexose 6-deoxy-α-l-talopyranose. The absolute configuration of a polyketide extender unit of 3 was determined by applying the J-based configuration analysis and modified Mosher's method. Ulleungoside (1) and naphthomycin A (7) showed in vitro inhibitory effects against indoleamine 2,3-dioxygenase activity. Further bioevaluation revealed that compounds 1 and 7 had moderate antiproliferative activities against several cancer cell lines, and compounds 5 and 6, which are members of the piericidin family, induced autophagosome accumulation.
Assuntos
Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/isolamento & purificação , Naftoquinonas/química , Naftoquinonas/isolamento & purificação , Policetídeos/química , Streptomyces/química , ortoaminobenzoatos/química , Bioensaio , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Naftoquinonas/farmacologia , Ressonância Magnética Nuclear Biomolecular , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , ortoaminobenzoatos/isolamento & purificação , ortoaminobenzoatos/farmacologiaRESUMO
Analysis of the genome sequence of Streptomyces sp. KCB13F003 showed the presence of a cryptic gene cluster encoding flavin-dependent halogenase and nonribosomal peptide synthetase. Pleiotropic approaches using multiple culture media followed by LC-MS-guided isolation and spectroscopic analysis enabled the identification of two new chlorinated cyclic hexapeptides, ulleungmycins A and B (1 and 2). Their structures, including absolute configurations, were determined by 1D and 2D NMR techniques, advanced Marfey's analysis, and GITC derivatization. The new peptides, featuring unusual amino acids 5-chloro-l-tryptophan and d-homoleucine, exhibited moderate antibacterial activities against Gram-positive pathogenic bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus.
Assuntos
Peptídeos Cíclicos/isolamento & purificação , Streptomyces/química , Sequência de Aminoácidos , Antibacterianos/química , Cromatografia Líquida , Flavinas/metabolismo , Genômica , Bactérias Gram-Positivas/efeitos dos fármacos , Hidrocarbonetos Clorados , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredutases/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/química , Infecções Estafilocócicas , Staphylococcus aureus/efeitos dos fármacos , Streptomyces/genética , Triptofano/metabolismoRESUMO
Two new phenylspirodrimane derivatives, stachybotrysin (1) and stachybotrylactone B (2), were isolated from the cultures of the marine-derived fungus Stachybotrys sp. KCB13F013. The structures were determined by analyzing the spectroscopic data (1D and 2D NMR and MS) and chemical transformation, including the modified Mosher's method and single-crystal X-ray structure analysis. Compound 1 exhibited an inhibitory effect on osteoclast differentiation in bone marrow macrophage cells via suppressing the RANKL-induced activation of p-ERK, p-JNK, p-p38, c-Fos, and NFATc1.
Assuntos
Osteoclastos/efeitos dos fármacos , Stachybotrys/química , Animais , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação para Baixo/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Biologia Marinha , Camundongos , Estrutura Molecular , NF-kappa B/antagonistas & inibidores , Osteoblastos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Salterns, one of the most extreme natural hypersaline environments, are a rich source of halophilic and halotolerant microorganisms, but they remain largely underexplored ecological niches in the discovery of bioactive secondary metabolites. In continued efforts to investigate the metabolic potential of microbial populations from chemically underexplored sites, three new lipopeptides named iturin F1, iturin F2 and iturin A9 (1-3), along with iturin A8 (4), were isolated from Bacillus sp. KCB14S006 derived from a saltern. The structures of the isolated compounds were established by 1D-, 2D-NMR and HR-ESIMS, and their absolute configurations were determined by applying advanced Marfey's method and CD spectroscopy. All isolates exhibited significant antifungal activities against various pathogenic fungi and moderate cytotoxic activities toward HeLa and src(ts)-NRK cell lines. Moreover, in an in vitro enzymatic assay, compound 4 showed a significant inhibitory activity against indoleamine 2,3-dioxygenase.
Assuntos
Bacillus/química , Bacillus/metabolismo , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Fungos/efeitos dos fármacos , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética/métodosRESUMO
Three new DOTAM (1,4,7,10-tetrakis(acetamido)-1,4,7,10-tetraazacyclododecane) complexes have been synthesized and characterized by X-ray crystallography: [Co(DOTAM)]Cl2â¢3H2O, [Ni(DOTAM)]Cl2â¢4H2O, and [Cu(DOTAM)](ClO4)2â¢H2O. Solid state and solution IR spectroscopic features for a series of [M(DOTAM)]2+ complexes (M=Mn, Co, Cu, Ni, Ca, Zn) correlate with solid state and solution coordination numbers. [Co(DOTAM)]2+, [Ni(DOTAM)]2+, and [Zn(DOTAM)]2+ are demonstrated to be six-coordinate in both the solid state and in solution, while [Mn(DOTAM)]2+ and [Ca(DOTAM)]2+ are eight-coordinate in the solid state and remain so in solution. [Cu(DOTAM)]2+, which is five-coordinate by X-ray crystallography, is shown to increase its coordination number in solution to six-coordinate.
RESUMO
BACKGROUND: Quorum sensing (QS) networks are more commonly known as acyl homoserine lactone (HSL) networks. Recently, p-coumaroyl-HSL has been found in a photosynthetic bacterium. p-coumaroyl-HSL is derived from a lignin monomer, p-coumaric acid, rather than a fatty acyl group. The p-coumaroyl-HSL may serve an ecological role in diverse QS pathways between p-coumaroyl-HSL producing bacteria and specific plants. Interference with QS has been regarded as a novel way to control bacterial infections. Heterologous production of the QS molecule, p-coumaroyl-HSL, could provide a sustainable and controlled means for its large-scale production, in contrast to the restricted feedback regulation and extremely low productivity of natural producers. RESULTS: We developed an artificial biosynthetic process for phenylacetyl-homoserine lactone analogs, including cinnamoyl-HSL, p-coumaroyl-HSL, caffeoyl-HSL, and feruloyl-HSL, using a bioconversion method via E. coli (CB1) in the co-expression of the codon-optimized LuxI-type synthase (RpaI) and p-coumaroyl-CoA ligase (4CL2nt). In addition to this, we show the de novo production of p-coumaroyl-HSL in heterologous host E. coli (DN1) and tyrosine overproducing E. coli (DN2), containing the rpaI gene in addition to p-coumaroyl-CoA biosynthetic genes. The yields for p-coumaroyl-HSL reached 93.4 ± 0.6 and 142.5 ± 1.0 mg/L in the S-adenosyl-L-methionine and L-methionine feeding culture in the DN2 strain, respectively. CONCLUSIONS: This is the first report of a de novo biosynthesis in a heterologous host yielding a QS molecule, p-coumaroyl-HSL from a glucose medium using a single vector system combining p-coumaroyl-CoA biosynthetic genes and the LuxI-type synthase gene.
Assuntos
4-Butirolactona/análogos & derivados , Escherichia coli/metabolismo , 4-Butirolactona/análise , 4-Butirolactona/biossíntese , 4-Butirolactona/química , Cromatografia Líquida de Alta Pressão , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Percepção de Quorum , Nicotiana/enzimologia , Tirosina/metabolismoRESUMO
BACKGROUND: Styrene and its derivatives as monomers and petroleum-based feedstocks are valuable as raw materials in industrial processes. The chemical reaction for styrene production uses harsh reaction conditions such as high temperatures or pressures, or requires base catalysis with microwave heating. On the other hand, production of styrene and its derivatives in Escherichia coli is an environmental friendly process to produce conventional petroleum-based feedstocks. RESULTS: An artificial biosynthetic pathway was developed in E. coli that yields 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene from simple carbon sources. This artificial biosynthetic pathway has a codon-optimized phenolic acid decarboxylase (pad) gene from Bacillus and some of the phenolic acid biosynthetic genes. E. coli strains with the tal and pad genes, the tal, sam5, and pad genes, and the tal, sam5, com, and pad genes produced 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene, respectively. Furthermore, these pathways were expressed in a tyrosine overproducing E. coli. The yields for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene reached 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation. CONCLUSIONS: Our system is the first to use E. coli with artificial biosynthetic pathways for the de novo synthesis of 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene in a simple glucose medium. Similar approaches using microbial synthesis from simple sugar could be useful in the synthesis of plant-based aromatic chemicals.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Poliestirenos/metabolismo , Tirosina/metabolismoRESUMO
During the chemical investigation of marine-derived fungus, an unusual diketopiperazine (DKP) alkaloid, haenamindole (1), was isolated from a culture of the marine-derived fungus Penicillium sp. KCB12F005. The structure of 1, which possesses benzyl-hydroxypiperazindione and phenyl-pyrimidoindole rings system in the molecule, was elucidated by analysis of NMR and MS data. The stereochemistry of 1 was determined by ROESY and advanced Marfey's method.
Assuntos
Organismos Aquáticos/química , Dicetopiperazinas/química , Penicillium/química , Dicetopiperazinas/isolamento & purificação , Dicetopiperazinas/farmacologia , Espectroscopia de Ressonância Magnética , Estrutura Molecular , EstereoisomerismoRESUMO
Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been introduced for the treatment of non-small cell lung cancer (NSCLC), the emergence of secondary T790M mutation in EGFR or amplification of the Met proto-oncogene restrain the clinical success of EGFR-TKIs. Since heat shock protein-90 (Hsp90) stabilizes various oncoproteins including EGFR and c-Met, the inhibition of Hsp90 activity appears as a rational strategy to develop anticancer drugs. Despite preclinical efficacy of geldanamycin-anasamycin (GA)-derivatives containing benzoquinone moiety as Hsp90 inhibitors, the hepatotoxicity of these GA-derivatives restricts their therapeutic benefit. We have prepared WK-88 series of GA-derivatives, which lack the benzoquinone moiety. In this study, we have examined the anticancer effects of WK88-1 in Met-amplified- and gefitinib-resistant (HCC827GR) NSCLC cells and its parental HCC827 cells. Treatment with WK88-1 reduced the cell viability in both HCC827 and HCC827GR cells, which was associated with marked decrease in the constitutive expression of Hsp90 client proteins, such as EGFR, ErbB2, ErbB3, Met and Akt. Moreover, WK88-1 attenuated phosphorylation of these Hsp90 client proteins and reduced the anchorage-independent growth of HCC827GR cells. Administration of WK88-1 did not cause hepatotoxicity in animals and significantly reduced the growth of HCC827GR cells xenograft tumors in nude mice. Our study provides evidence that ErbB3 might be a client for Hsp90 in Met-amplified NSCLCs. In conclusion, we demonstrate that inhibition of Hsp90 dampens the activation of EGFR- or c-Met-mediated survival of Met-amplified NSCLCs and that WK88-1 as a Hsp90 inhibitor alleviates gefitinib resistance in HCC827GR cells.