RESUMEN
Epicoccamide (EPC) is an O-d-mannosylated acyltetramic acid of Epicoccum origin and is a bolaamphiphilic fungal polyketide. EPC displays weak toxicity against Staphylococcus aureus and HeLa cell lines. The EPC biosynthetic gene cluster was previously identified in Epicoccum nigrum and knockout of the glycosyltransferase gene (epcB) abolished EPC production. EPC-aglycone was expected in the epcB knockout but was not found. This study demonstrates that extractive culture using the hydrophobic resin Diaion HP-20 resulted in the production of EPC-aglycone, which was isolated using chromatographic separation techniques, and its structural identity was substantiated by chemical analyses. EPC-aglycone displayed strong antibacterial activity against Staphylococcus aureus, with the minimal inhibitory concentration of 1 µg/mL (64 µg/mL for EPC). EPC-aglycone displayed higher levels of growth inhibition against HeLa cell line (the half inhibitory concentration, 19 µM) and WI-38 (15 µM) cell line than EPC (76 µM and 38 µM vs. HeLa and WI-38, respectively). The dose-response curve fit of growth inhibition indicated that EPC-aglycone adopted a shallow curve (low slope factor), which was different from that of EPC, suggesting that their cellular targets are distinct from each other. This study substantiates that the d-mannose attachment is the final step in EPC biosynthesis, showcasing a glycosylation-mediated modulation of the biological activity of simple acyltetramic acid. This study also highlights the usefulness of extractive cultures in mining cryptic microbial natural products.
Asunto(s)
Antibacterianos , Humanos , Células HeLa , Antibacterianos/farmacología , GlicosilaciónRESUMEN
Epipyrone (EPN)-A (syn. orevactaene) is a polyketide compound of 3-d-galactosyl-4-hydroxy-2-pyrone with a modified heptaene acyl moiety, produced from Epicoccum nigrum and was reported to have various biological activities. Genome analysis identified a hypothetical EPN biosynthetic gene cluster (BGC) composed of the four genes epnABCD, which encode a highly-reducing fungal polyketide synthase, a glycosyltransferase, a cytochrome P450, and a transporter. The individual gene inactivation of epnABC resulted in the total loss of EPN production, while the inactivation of a nearby transcription factor-encoding gene had no effect on the production of EPN, substantiating that epnABCD is the EPN BGC. mRNA expression indicated no epnA transcription in the epnB knockout mutant and the occurrence of the bicistronic transcription of epnAB. This study defined an EPN BGC, which is the first blueprint reported for glycosylated 2-pyrone polyketide biosynthesis.
Asunto(s)
Ascomicetos/química , Ascomicetos/genética , Piranos/metabolismo , Conformación Molecular , Familia de Multigenes , Piranos/químicaRESUMEN
Monascus purpureus recombinant mppC and mpp7 knockout strains were subjected to extractive fermentation in the context of azaphilone pigment production. Inclusion of Diaion HP-20 resin resulted in the selective production of unreduced azaphilone congeners, in addition to the early intermediate FK17-P2a, from ∆mppC and ∆mpp7 strains that would otherwise mainly produce reduced congeners. Structural determination of two novel unreduced azaphilones from the ∆mpp7 strain was accomplished. The unreduced azaphilone compound was converted into the cognate reduced congener in recombinant M. purpureus strains, demonstrating its intermediate role in azaphilone biosynthesis. This study demonstrates the possibility that extractive fermentation with Diaion HP-20 resin can be used to obtain cryptic azaphilone metabolites.
Asunto(s)
Benzopiranos/metabolismo , Microbiología Industrial/métodos , Monascus/genética , Monascus/metabolismo , Pigmentos Biológicos/metabolismo , Poliestirenos/metabolismo , Benzopiranos/química , Vías Biosintéticas , Fermentación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Oxidación-Reducción , Pigmentos Biológicos/químicaRESUMEN
OBJECTIVES: To characterize a biosynthetic gene that is selectively involved in the biosynthesis of yellow or orange components in the azaphilone polyketide pathway of Monascus. RESULTS: A reductive modification is predicted to control the relative levels of reduced (yellow) and oxidized (orange and red) components in the pathway of azaphilone pigment biosynthesis in Monascus. Targeted inactivation of a reductase gene mppE enhanced orange and red pigment production whereas overexpression of the gene promoted yellow pigment production. The effect of mppE overexpression was dependent on culture methods, and augmented yellow pigmentation was evident in a submerged culture employing a chemically defined medium. CONCLUSIONS: MppE controls the biosynthesis of the yellow pigments, ankaflavin and monascin, as a reductive enzyme in the azaphilone polyketide pathway.
Asunto(s)
Benzopiranos/metabolismo , Monascus/metabolismo , Oxidorreductasas/metabolismo , Pigmentos Biológicos/metabolismo , Policétidos/metabolismoRESUMEN
Citrinin (3) is a polyketide-derived mycotoxin, that is, produced by Monascus, Penicillium, and Aspergillus spp. and is a common contaminant in a number of agricultural products. ctPKS, a non-reducing type iterative polyketide synthase with a C-terminal reductive domain, is proposed to generate the polyketide backbone of 3. The targeted gene inactivation of ctn-orf1 or ctn-orf3 gene resulted in the accumulation of a benzaldehyde derivative 6, and the ectopic expression of ctPKS/ctnB in yeast produced 6, demonstrating that ctPKS generates 6 with the support of CtnB and suggesting that Ctn-ORF1/Ctn-ORF3 converts 6 into 3. The Δctn-orf1 mutant also produced a novel benzdialdehyde derivative 10. When either 6 or 10 was fed into a ΔctPKS mutant, 3 was readily detected, which confirms that both 6 and 10 are involved in the biosynthesis of 3. A bioconversion experiment of 6 in the ectopic expression system demonstrated that ctn-orf3 expression, but not ctn-orf1 expression, efficiently consumed 6. The resulting metabolite(s) of 6 could not be identified, however. A recombinant Ctn-ORF3 enzyme was demonstrated to convert 6 into 10 and a hypothetical carboxylic derivative 8, which substantiates that Ctn-ORF3 oxidizes the exocyclic methyl moiety of 6. Ctn-ORF1 is thus proposed to reduce 8 and the subsequent non-enzymatic reactions to complete the biosynthesis of 3. The present study delineates the biosynthetic route of 3, proposing the biochemical mechanism, that is, involved in producing the natural dihydropyranoquinone structure.
Asunto(s)
Citrinina/metabolismo , Dioxigenasas/metabolismo , Proteínas Fúngicas/metabolismo , Monascus/metabolismo , Sintasas Poliquetidas/metabolismo , Antibacterianos/metabolismo , Benzopiranos/metabolismo , Vías Biosintéticas , Ciclización , Dioxigenasas/genética , Proteínas Fúngicas/genética , Marcación de Gen , Monascus/genética , Mutación , Oxidación-Reducción , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Sintasas Poliquetidas/genéticaRESUMEN
Azaphilone polyketides are synthesized by iterative non-reducing fungal polyketide synthases (NR-fPKSs) with a C-terminus reductive domain (-R). Several azaphilone biosynthetic gene clusters contain a putative serine hydrolase gene; the Monascus azaphilone pigment (MAzP) gene cluster harbors mppD. The MAzP productivity was significantly reduced by a knockout of mppD, and the MAzP NR-fPKS-R gene (MpPKS5) generated its product in yeast only when co-expressed with mppD. Site-directed mutations of mppD for conserved Ser/Asp/His residues abolished the product formation from the MpPKS5/mppD co-expression. MppD and its homologs are thus proposed as a new protein factor involved in the product formation of NR-fPKS-R.
Asunto(s)
Monascus/enzimología , Pigmentos Biológicos/biosíntesis , Sintasas Poliquetidas/metabolismo , Benzopiranos , Técnicas de Inactivación de Genes , Genes Fúngicos , Modelos Moleculares , Estructura Molecular , Monascus/genética , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Sintasas Poliquetidas/genéticaRESUMEN
Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-(13)C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed ß-keto group modifications.
Asunto(s)
Sintasas Poliquetidas/metabolismo , Streptomyces/enzimología , Silenciador del Gen , Lactonas/metabolismo , Familia de Multigenes/genética , Sintasas Poliquetidas/química , Sintasas Poliquetidas/deficiencia , Sintasas Poliquetidas/genética , Propionatos/metabolismo , Estructura Terciaria de Proteína , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Acute esophageal necrosis is uncommon in the literature. Its etiology is unknown, although cardiovascular disease, hemodynamic compromise, gastric outlet obstruction, alcohol ingestion, hypoxemia, hypercoagulable state, infection, and trauma have all been suggested as possible causes. A 67-year-old female underwent a coronary angiography (CAG) for evaluation of chest pain. CAG findings showed coronary three-vessel disease. We planned percutaneous coronary intervention (PCI). Coronary arterial dissection during the PCI led to sudden hypotension. Six hours after the index procedure, the patient experienced a large amount of hematemesis. Emergency gastrofibroscopy was performed and showed mucosal necrosis with a huge adherent blood clot in the esophagus. After conservative treatment for 3 months, the esophageal lesion was completely improved. She was diagnosed with acute esophageal necrosis. We report herein a case of acute esophageal necrosis occurring in a patient undergoing percutaneous coronary intervention.
Asunto(s)
Estenosis Coronaria/terapia , Enfermedades del Esófago/etiología , Esófago/patología , Intervención Coronaria Percutánea/efectos adversos , Enfermedad Aguda , Anciano , Angiografía Coronaria , Estenosis Coronaria/diagnóstico , Estenosis Coronaria/fisiopatología , Enfermedades del Esófago/diagnóstico , Enfermedades del Esófago/tratamiento farmacológico , Esofagoscopía , Esófago/efectos de los fármacos , Femenino , Hemodinámica , Humanos , Necrosis , Valor Predictivo de las Pruebas , Inhibidores de la Bomba de Protones/uso terapéutico , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Ultrasonografía Intervencional , Cicatrización de HeridasRESUMEN
Monascus spp. produce several well-known polyketides such as monacolin K, citrinin, and azaphilone pigments. In this study, the azaphilone pigment biosynthetic gene cluster was identified through T-DNA random mutagenesis in Monascus purpureus. The albino mutant W13 bears a T-DNA insertion upstream of a transcriptional regulator gene (mppR1). The transcription of mppR1 and the nearby polyketide synthase gene (MpPKS5) was significantly repressed in the W13 mutant. Targeted inactivation of MpPKS5 also gave rise to an albino mutant, confirming that mppR1 and MpPKS5 belong to an azaphilone pigment biosynthetic gene cluster. This M. purpureus sequence was used to identify the whole biosynthetic gene cluster in the Monascus pilosus genome. MpPKS5 contains SAT/KS/AT/PT/ACP/MT/R domains, and this domain organization is preserved in other azaphilone polyketide synthases. This biosynthetic gene cluster also encodes fatty acid synthase (FAS), which is predicted to assist the synthesis of 3-oxooactanoyl-CoA and 3-oxodecanoyl-CoA. These 3-oxoacyl compounds are proposed to be incorporated into the azaphilone backbone to complete the pigment biosynthesis. A monooxygenase gene (an azaH and tropB homolog) that is located far downstream of the FAS gene is proposed to be involved in pyrone ring formation. A homology search on other fungal genome sequences suggests that this azaphilone pigment gene cluster also exists in the Penicillium marneffei and Talaromyces stipitatus genomes.
Asunto(s)
Proteínas Fúngicas/genética , Monascus/genética , Familia de Multigenes , Pigmentos Biológicos/biosíntesis , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Monascus/enzimología , Monascus/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismoRESUMEN
The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.
Asunto(s)
Acilcoenzima A/metabolismo , Acil-CoA Deshidrogenasas/metabolismo , Antibacterianos/química , Filipina , Streptomyces/enzimología , Acil-CoA Deshidrogenasas/química , Acil-CoA Deshidrogenasas/genética , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Filipina/biosíntesis , Familia de Multigenes , Plásmidos , Homología de Secuencia de Aminoácido , Espectrofotometría , Streptomyces/química , Streptomyces/genética , Transformación BacterianaRESUMEN
dTDP-L-rhamnose (dTDP-Rha)-synthesizing dTDP-6-deoxy-L-lyxo-4-hexulose reductase (4-KR) and dTDP-Rha 4-epimerase were characterized from Burkholderia thailandensis E264 by utilizing rmlD(Bth) (BTH_I1472) and wbiB(Bth) (BTH_I1476), respectively. Incubation of the recombinant WbiB(Bth) with RmlA/RmlB/RmlC/Tal, which has previously been shown to generate dTDP-6-deoxy-L-talose (dTDP-6dTal) from α-D-glucose-1-phosphate, dTTP, and NADPH, produced dTDP-Rha. (1)H NMR measurements confirmed that both RmlA/RmlB/RmlC/Tal/WbiB(Bth) and RmlA/RmlB/RmlC/RmlD produced dTDP-Rha. WbiB(Bth) alone produced dTDP-Rha when incubated with dTDP-6dTal. This is the first report to demonstrate epimerase activity interconverting between dTDP-Rha and dTDP-6dTal.
Asunto(s)
Burkholderia/enzimología , Carbohidrato Epimerasas/química , Desoxiazúcares/química , Hexosas/química , Azúcares de Nucleósido Difosfato/química , Nucleótidos de Timina/química , Burkholderia/genética , Cromatografía Líquida de Alta Presión , Estructura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Galbonolides A and B are antifungal compounds, which are produced by Streptomyces galbus. A multimodular polyketide synthase (PKS) was predicted to catalyze their biosynthesis, and a methoxymalonyl-acyl carrier protein (methoxymalonyl-ACP) was expected to be involved in the biosynthesis of galbonolide A. Cloning of a methoxymalonyl-ACP biosynthesis locus (galGHIJK) and the flanking regions has revealed that the locus is colocalized with beta-ketoacyl synthase (KAS)-related genes (orf3, 4, and 5), but separated from any multimodular PKS gene cluster in S. galbus. A galI-disruption mutant (SK-galI-5) is unable to produce galbonolide A, but can synthesize galbonolide B, indicating that galGHIJK is involved in the biosynthesis of galbonolide A. A disruption mutant of orf4 is severely impaired in the production of both galbonolides A and B. These results indicate that galGHIJK and the KAS genes are involved in the biosynthesis of galbonolides, although they are not colocalized with a multimodular PKS gene cluster. We further propose that a single galbonolide PKS generates two discrete structures, galbonolides A and B, by alternatively incorporating methoxymalonate and methylmalonate, respectively.
Asunto(s)
Proteína Transportadora de Acilo/genética , Vías Biosintéticas/genética , Familia de Multigenes , Sintasas Poliquetidas/genética , Streptomyces/enzimología , Antifúngicos/metabolismo , Clonación Molecular , Eliminación de Gen , Orden Génico , Lactonas/metabolismo , Modelos Biológicos , Mutagénesis Insercional , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-l-talose (dTDP-6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlA(Kkf), RmlB(Kkf), RmlC(Kkf), and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, alpha-d-glucose-1-phosphate, and NAD(P)H. The identity of dTDP-6dTal was validated using (1)H and (13)C NMR spectroscopy. K. kifunensistal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family, providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.
Asunto(s)
Lactonas/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , Streptomycetaceae/genética , Streptomycetaceae/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Clonación Molecular , Variación Genética , Datos de Secuencia Molecular , Streptomycetaceae/enzimología , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificaciónRESUMEN
The fact that adpA promoter activity is enhanced by S-adenosylmethionine without the involvement of the A-factor/ArpA regulatory cascade suggests the existence of additional transcriptional regulators for adpA expression in Streptomyces griseus. In this study, an additional adpA promoter regulatory protein, named ArfA, that is conserved among many bacteria was identified using DNA affinity purification from the cell extracts of Streptomyces lividans. The interactions of ArfA with the adpA promoter from S. griseus and with the bldH promoter from S. lividans were specific and both adpA and bldH promoters required ArfA for the wild-type level of their expressions in S. lividans. bldH of S. lividans is a homolog of adpA of S. lividans. ArfA-deletion mutant had only 70% of the normal undecylprodigiosin production. This result was confirmed by reduced redD promoter activity in the ArfA-deletion mutant. These results suggest that ArfA is a new type of DNA-binding regulator.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Streptomyces lividans/metabolismo , Cromatografía de Afinidad , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Eliminación de Gen , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Regiones Promotoras Genéticas , Streptomyces lividans/genéticaRESUMEN
A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.
Asunto(s)
Proteínas Bacterianas/fisiología , Gentamicinas/biosíntesis , Micromonospora/metabolismo , Trisacáridos/metabolismo , Proteínas Bacterianas/genética , Silenciador del Gen , Ingeniería Genética , Micromonospora/genéticaRESUMEN
Because ATP is an extracellular effector in animal and plant systems and derivatives of ATP, such as S-adenosylmethionine and cAMP, can control antibiotic production and morphological differentiation in Streptomyces, we hypothesized that extracellular ATP (exATP) can also affect physiologies of Streptomyces. We found that the addition of 10 microM exATP to Streptomyces coelicolor A3(2) cultures resulted in enhanced actinorhodin and undecylprodigiosin production and morphological differentiation on solid medium. However, these phenotypes were reduced by the addition of a 10-fold higher concentration of exATP (100 microM). Intracellular ATP concentrations were also modulated in response to changes in exATP. ATP analogs, added at a 100-fold lower concentration, affected Streptomyces similarly to that seen for 10 microM exATP. The enhanced promoter activity of actII-orf4 indicated that 10 microM exATP affect the transcriptional level for actinorhodin production. Results from this study suggest that exATP is an effector for the physiology of S. coelicolor and careful manipulation of exATP may significantly enhance the high-yield production of antibiotics by S. coelicolor.
Asunto(s)
Adenosina Trifosfato/metabolismo , Espacio Extracelular/metabolismo , Streptomyces coelicolor/metabolismo , Adenosina Trifosfato/análogos & derivados , Antraquinonas/metabolismo , Antibacterianos/metabolismo , Medios de Cultivo/metabolismo , AMP Cíclico/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/metabolismo , Regiones Promotoras Genéticas , Streptomyces coelicolor/citología , Streptomyces coelicolor/genética , Transcripción GenéticaRESUMEN
In the present study, a mechanism for S-adenosylmethionine (SAM) to promote secondary metabolism was characterized in terms of bldH sl) expression in Streptomyces lividans. A previous study demonstrated that SAM, on application at 2 microM, induces the transcription of the strR promoter (strRp), which originates from Streptomyces griseus, in S. lividans. An inactivation study verified that bldH sl is essential to strRp transcription in S. lividans and it was demonstrated that the effects of SAM on the induction of strRp activity, on the transcription of redZ and actII-orf4, and on antibiotic production were compromised when the unique leucine TTA-codon of bldH sl was changed to TTG. Western blot analysis revealed that SAM supplementation enhances the expression of bldH sl when the TTA-codon was intact but not when the TTG replacement was provided. This study validates the involvement of BldH sl in the potentiating effect of SAM on the antibiotic production and substantiates that SAM controls the expression of bldH sl through the TTA-codon control in translating bldH mRNA. It is argued here that the intracellular SAM-level modulates the maturation of bldA, which encodes the UUA-codon tRNA and controls secondary metabolism in S. lividans.
Asunto(s)
Proteínas Bacterianas/metabolismo , Codón/genética , Regulación Bacteriana de la Expresión Génica , S-Adenosilmetionina/metabolismo , Streptomyces lividans/metabolismo , Proteínas Bacterianas/genética , Codón/metabolismo , Codón de Terminación , Leucina/metabolismo , Streptomyces lividans/genética , Estreptomicina/biosíntesis , Transcripción GenéticaRESUMEN
S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (ASCO5260). ASCO5260 displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in ASCO5260. Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , S-Adenosilmetionina/farmacología , Streptomyces coelicolor/fisiología , Esporas Bacterianas/fisiologíaRESUMEN
AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (DeltaafsK-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S. avermitilis, yet not in the mutant harboring T168D or T165D, demonstrating that tandem phosphorylation on Thr-165 and Thr-168 in AfsKav is the mechanism modulating avermectin production in response to S-adenosylmethione accumulation in S. avermitilis.
Asunto(s)
Ivermectina/análogos & derivados , Morfogénesis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Streptomyces/genética , Treonina/fisiología , Regulación Bacteriana de la Expresión Génica , Ivermectina/metabolismo , Mutación/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/citología , Streptomyces/enzimología , Streptomyces/fisiologíaRESUMEN
The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the A. kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-l-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase, a conserved set of enzymes involved in formation, attachment, and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar d-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-d-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitrosugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.