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2.
Syst Appl Microbiol ; 44(6): 126270, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34653842

RESUMO

Exploration of unexplored habitats for novel actinobacteria with high bioactivity potential holds great promise in the search for novel entities. During the course of isolation of actinobacteria from desert soils, four actinobacteria, designated as 5K548T, 7K502T, 16K309T and 16K404T, were isolated from the Karakum Desert and their bioactivity potential as well as taxonomic provenances were revealed by comprehensive genome analyses. Pairwise sequence analyses of the 16S rRNA genes indicated that the four strains are representatives of putatively novel taxa within the prolific actinobacterial genus Saccharopolyspora. The strains have typical chemotaxonomic characteristics of the genus Saccharopolyspora by having meso-diaminopimelic acid as diagnostic diaminoacid, arabinose, galactose and ribose as whole-cell sugars. Consistent with this assignment, all of the isolates contained phosphatidylcholine in their polar lipid profiles and MK-9(H4) as the predominant menaquinone. The sizes of the genomes of the isolates ranged from 6.0 to 10.2 Mb and the associated G + C contents from 69.6 to 69.7 %. Polyphasic characterizations including determination of overall genome relatedness indices revealed that the strains are representatives of four novel species in the genus Saccharopolyspora. Consequently, isolates 5K548T, 7K502T, 16K404T and 16K309T are proposed as novel Saccharopolyspora species for which the names of Saccharopolyspora karakumensis sp. nov., Saccharopolyspora elongata sp. nov., Saccharopolyspora aridisoli sp. nov. and Saccharopolyspora terrae sp. nov. are proposed, respectively. Comprehensive genome analysis for biosynthetic gene clusters showed that the strains have high potential for novel secondary metabolites. Moreover, the strains harbour many antimicrobial resistance genes providing more evidence for their potentiality for bioactive metabolites.


Assuntos
Saccharopolyspora , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos/análise , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Saccharopolyspora/genética , Análise de Sequência de DNA , Vitamina K 2
3.
J Agric Food Chem ; 69(42): 12554-12565, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34657420

RESUMO

Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by Saccharopolyspora pogona. However, the yield of this compound is difficult to increase because the regulatory mechanism of secondary metabolism is still unknown. Here, the transcriptional regulator Sp13016 was discovered to be highly associated with butenyl-spinosyn synthesis and bacterial growth. Overexpression of sp13016 improved butenyl-spinosyn production to a level that was 2.84-fold that of the original strain, while deletion of sp13016 resulted in a significant decrease in yield and growth inhibition. Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway to regulate the supply of precursors. Our research helps to reveal the regulatory mechanism of butenyl-spinosyn biosynthesis and provides a reference for increasing the yield of natural products of Actinomycetes.


Assuntos
Proteômica , Saccharopolyspora , Proteínas de Bactérias/genética , Macrolídeos , Saccharopolyspora/genética
4.
Microb Cell Fact ; 20(1): 141, 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294095

RESUMO

BACKGROUND: Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. RESULTS: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. CONCLUSIONS: This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.


Assuntos
Proteínas de Bactérias/genética , Macrolídeos/metabolismo , Saccharopolyspora/crescimento & desenvolvimento , Saccharopolyspora/genética , Acetilação , Combinação de Medicamentos , Glucose/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Saccharopolyspora/fisiologia
5.
Sci Rep ; 11(1): 14779, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285307

RESUMO

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.


Assuntos
Proteínas de Bactérias/genética , Perfilação da Expressão Gênica/métodos , Macrolídeos/metabolismo , Saccharopolyspora/crescimento & desenvolvimento , Vias Biossintéticas , Meios de Cultura/química , Combinação de Medicamentos , Fermentação , Regulação Bacteriana da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Metabolismo dos Lipídeos , Saccharopolyspora/classificação , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Óleo de Soja/química
6.
Appl Microbiol Biotechnol ; 105(7): 2911-2924, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33760930

RESUMO

The Lrp and MarR families are two groups of transcriptional regulators widely distributed among prokaryotes. However, the hierarchical-regulatory relationship between the Lrp family and the MarR family remains unknown. Our previous study found that an Lrp (SACE_Lrp) from Saccharopolyspora erythraea indirectly repressed the biosynthesis of erythromycin. In this study, we characterized a novel MarR family protein (SACE_6745) from S. erythraea, which is controlled by SACE_Lrp and plays a direct regulatory role in erythromycin biosynthesis and export. SACE_Lrp directly regulated the expression of marR by specifically binding a precise site OM (5'-CTCCGGGAACCATT-3'). Gene disruption of marR increased the production of erythromycin by 45% in S. erythraea A226. We found that MarR has direct DNA-binding activity for the promoter regions of the erythromycin biosynthetic genes, as well as an ABC exporter SACE_2701-2702 which was genetically proved to be responsible for erythromycin efflux. Disruption of SACE_Lrp in industrial S. erythraea WB was an efficient strategy to enhance erythromycin production. Herein, we jointly engineered SACE_Lrp and its target MarR by deleting marR in WBΔSACE_Lrp, resulting in 20% increase in erythromycin yield in mutant WBΔLrpΔmarR compared to WBΔSACE_Lrp, and 39% to WB. Overall, our findings provide new insights into the hierarchical-regulatory relationship of Lrp and MarR proteins and new avenues for coordinating antibiotic biosynthesis and export by joint engineering regulators in actinomycetes. KEY POINTS: • The hierarchical-regulatory relationship between SACE_Lrp and MarR was identified. • MarR directly controlled the expression of erythromycin biosynthesis and export genes. • Joint engineering of SACE_Lrp-MarR regulatory element enhanced erythromycin production.


Assuntos
Saccharopolyspora , Proteínas de Bactérias/genética , Eritromicina , Humanos , Saccharopolyspora/genética
7.
Laeknabladid ; 107(3): 130-136, 2021 Mar.
Artigo em Islandês | MEDLINE | ID: mdl-33625379

RESUMO

Diseases connected with work in hay have been known in Iceland for a long time. In 1981 scientific studies of these diseases were started in Iceland at the request of the Farmers Union. The results of these studies are summarized in this article. In studies of hay a great amount of storage mites, moulds and thermophilic actinomycetes (microlyspora faeni) were found in addition to allergens from mice and pollen. Symptoms caused by hay dust were mainly from nose and eyes in people with positive skin tests, but cough, dyspnea and fever were equally common in those with negative skin tests. The most common causes of allergy in farming families were storage mites and cattle, but allergy to cats, dogs and grass pollen were less common rurally than in the Reykjavik area. When comparing individuals working in heavy hay dust with those working in cleaner air, the former group had a higher likelihood of having a positive precipitin test against micropolyspora faeni, fever after work in hay and airway obstruction. It was shown that Icelandic farmers were more likely to get emphysema than other people irrespective of smoking. In a large study of homes in the Reykjavik area almost no mites were found. In spite of this, positive specific IgE tests against dust mites were equally common as in Uppsala, Sweden, where dust mites were found in 16% of homes. In further studies it was found, that 57% of people in the study had been more and less exposed to hay dust. They had either been raised on a farm, been on a farm in the summer during childhood or owned horses and fed them with hay. We have argued that cross allergy to storage mites may be the cause of a rather common allergy to house dust mites. A new study of middle-aged individuals has shown that allergy to storage mites is a little more common in the Reykjavik area than in Aarhus, Bergen or Uppsala. The most likely explanation is that they have more often been exposed to hay dust.


Assuntos
Hipersensibilidade , Ácaros , Alérgenos , Animais , Gatos , Bovinos , Cães , Poeira , Cavalos , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/epidemiologia , Hipersensibilidade/etiologia , Islândia/epidemiologia , Camundongos , Pessoa de Meia-Idade , Saccharopolyspora
8.
Appl Microbiol Biotechnol ; 105(4): 1519-1533, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33484320

RESUMO

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona.


Assuntos
Policetídeo Sintases , Saccharopolyspora , Macrolídeos , Família Multigênica , Policetídeo Sintases/genética , Saccharopolyspora/genética
9.
Gene ; 766: 145130, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32911030

RESUMO

The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona.


Assuntos
Proteínas de Bactérias/genética , Biossíntese de Proteínas/genética , Saccharopolyspora/genética , Animais , Regulação para Baixo/genética , Metabolismo Energético/genética , Insetos/microbiologia , Proteômica/métodos , Regulação para Cima/genética
10.
Pest Manag Sci ; 77(8): 3637-3649, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32893433

RESUMO

Natural products (NPs) have long been a source of insecticidal crop protection products. Like many macrolide NPs, the spinosyns originated from a soil inhibiting microorganism (Saccharopolyspora spinosa). More than 20 years after initial registration, the spinosyns remain a unique class of NP-based insect control products that presently encompass two insecticidal active ingredients, spinosad, a naturally occurring mixture of spinosyns, and spinetoram, a semi-synthetic spinosyn product. The exploration and exploitation of the spinosyns has, unusually, been tied to an array of computational tools including artificial intelligence (AI)-based quantitative structure activity relationship (QSAR) and most recently computer-aided modeling and design (CAMD). The AI-based QSAR directly lead to the discovery of spinetoram, while the CAMD studies have recently resulted in the discovery and building of a series of synthetic spinosyn mimics. The most recent of these synthetic spinosyn mimics show promise as insecticides targeting lepidopteran insect pests as demonstrated by field studies wherein the efficacy has been shown to be comparable to spinosad and spinetoram. These and a range of other aspects related to the exploration of the spinosyns over the past 30 years are reviewed herein. © 2020 Society of Chemical Industry.


Assuntos
Produtos Biológicos , Inseticidas , Inteligência Artificial , Combinação de Medicamentos , Macrolídeos , Saccharopolyspora
11.
J Agric Food Chem ; 68(49): 14660-14669, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33258371

RESUMO

Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of S. spinosa has been published, the transcriptome of S. spinosa remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of S. spinosa. Through transcriptomic analysis of different periods of S. spinosa growth, we found large numbers of differentially expressed genes and classified them according to their different functions. Based on the RNA-seq data, the CRISPR-Cas9 method was used to knock out the PEP phosphonomutase gene (orf 06952-4171). The yield of spinosyns A and D in S. spinosa-ΔPEP was 178.91 mg/L and 42.72 mg/L, which was 2.14-fold and 1.76-fold higher than that in the wild type (83.51 and 24.34 mg/L), respectively. The analysis of the mutant strains also verified the validity of the transcriptome data. The deletion of the PEP phosphonomutase gene leads to an increase in pyruvate content and affects the biosynthesis of spinosad. The replenishment of phosphoenol pyruvate in S. spinosa provides the substrate for the production of spinosad. We envision that these transcriptomic analysis results will contribute to the further study of secondary metabolites in actinomycetes.


Assuntos
Proteínas de Bactérias/metabolismo , Saccharopolyspora/enzimologia , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Macrolídeos/metabolismo , Mutação , Ácido Pirúvico/metabolismo , RNA-Seq , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Transcriptoma
12.
Chem Commun (Camb) ; 56(75): 11042-11045, 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32808942

RESUMO

Genome mining revealed the presence of two cdps-p450 operons in Saccharopolyspora antimicrobica. Heterologous expression, biochemical characterisation and structure elucidation proved that the two P450 enzymes catalyse distinct regio- and stereospecific dimerizations of cyclo-(l-Trp-l-Trp), which significantly expands the repertoire of diketopiperazine-tailoring enzymes. TtpB1 connects the monomers via C3-C3', both from the opposite side of H-11/H-11', while TtpB2 is characterised as the first P450 to mainly catalyse the unusual linkage between N1' and C3 from the H-11 side.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Peptídeos Cíclicos/química , Saccharopolyspora/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Dimerização , Conformação Molecular , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo
13.
Molecules ; 25(15)2020 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-32727097

RESUMO

Glycosyltransferases are important enzymes which are often used as tools to generate novel natural products. In this study, we describe the identification and characterization of an inverting N- and O-glycosyltransferase from Saccharopolyspora erythraea NRRL2338. When feeding experiments with 1,4-diaminoanthraquinone in Saccharopolyspora erythraea were performed, the formation of new compounds (U3G and U3DG) was observed by HPLC-MS. Structure elucidation by NMR revealed that U3G consists of two compounds, N1-α-glucosyl-1,4-diaminoanthraquinone and N1-ß-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a N1,N4-diglucosyl-1,4-diaminoanthraquinone. In order to find the responsible glycosyltransferase, gene deletion experiments were performed and we identified the glycosyltransferase Sace_3599, which belongs to the CAZy family 1. When Streptomyces albus J1074, containing the dTDP-d-glucose synthase gene oleS and the plasmid pUWL-A-sace_3599, was used as host, U3 was converted to the same compounds. Protein production in Escherichia coli and purification of Sace_3599 was carried out. The enzyme showed glycosyl hydrolase activity and was able to produce mono- and di-N-glycosylated products in vitro. When UDP-α-d-glucose was used as a sugar donor, U3 was stereoselective converted to N1-ß-glucosyl-1,4-diaminoanthraquinone and N1,N4-diglucosyl-1,4-diaminoanthraquinone. The use of 1,4-dihydroxyanthraquinone as a substrate in in vitro experiments also led to the formation of mono-glucosylated and di-glucosylated products, but in lower amounts. Overall, we identified and characterized a novel glycosyltransferase which shows glycohydrolase activity and the ability to glycosylate "drug like" structures forming N- and O-glycosidic bonds.


Assuntos
Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Saccharopolyspora/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Genoma Bacteriano , Glicosilação , Glicosiltransferases/classificação , Glicosiltransferases/genética , Saccharopolyspora/genética , Homologia de Sequência
14.
Environ Microbiol ; 22(8): 3218-3233, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32400083

RESUMO

This study provides an example in the emerging field of biocodicology showing how metagenomics can help answer relevant questions that may contribute to a better understanding of the history of ancient manuscripts. To this end, two Slavonic codices dating from the 11th century were investigated through shotgun metagenomics. Endogenous DNA enabled to infer the animal origin of the skins used in the manufacture of the two codices, while nucleic sequences recovered from viruses were investigated for the first time in this material, opening up new possibilities in the field of biocodicology. In addition, the microbiomes colonizing the surface of the parchments served to determine their conservation status and their latent risk of deterioration. The saline environment provided by the parchments selected halophilic and halotolerant microorganisms, which are known to be responsible for the biodegradation of parchment. Species of Nocardiopsis, Gracilibacillus and Saccharopolyspora, but also members of the Aspergillaceae family were detected in this study, all possessing enzymatic capabilities for the biodeterioration of this material. Finally, a relative abundance of microorganisms originating from the human skin microbiome were identified, most probably related to the intensive manipulation of the manuscripts throughout the centuries, which should be taken with caution as they can be potential pathogens.


Assuntos
DNA Antigo , Manuscritos como Assunto/história , Microbiota , Animais , Biodegradação Ambiental , Conservação dos Recursos Naturais , DNA Antigo/isolamento & purificação , Europa Oriental , História Antiga , Humanos , Saccharopolyspora , Pele/microbiologia
15.
Int J Syst Evol Microbiol ; 70(5): 3241-3246, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375933

RESUMO

A novel Gram-stain-positive, aerobic, non-motile actinobacterium, designated strain E2AT, was isolated from a coral sample and examined using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain E2AT formed a distinct phyletic lineage in the genus Saccharopolyspora and was closely related to S. cavernae CCTCC AA 2012022T (96.4 %) and S. lacisalsi CCTCC AA 2010012T (95.3 %). The isolate grew at 15-35 °C, pH 5-12 and in the presence of 1-16 % (w/v) NaCl. The cell-wall diamino acid was meso-DAP. Major fatty acids identified were iso-C15 : 0, iso-C16 : 0 and C17 : 1 ω8c. The predominant menaquinone was MK-9(H4). The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, one unidentified glycolipid, one unidentified phospholipid and one unidentified aminolipid. The genomic DNA G+C content was 68.6 mol%. Based on the data from the polyphasic taxonomic study reported here, strain E2AT represents a novel species within the genus Saccharopolyspora, for which the name Saccharopolyspora coralli sp. nov. is proposed. The type strain is E2AT=(JCM 31844T=MCCC 1A17150T).


Assuntos
Antozoários/microbiologia , Filogenia , Saccharopolyspora/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Saccharopolyspora/isolamento & purificação , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
16.
Microb Cell Fact ; 19(1): 27, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046731

RESUMO

BACKGROUND: Saccharopolyspora pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. pogona are yet to characterize. RESULTS: In the present study, the overexpression of the tetR-like gene sp1418 in S. pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. CONCLUSION: This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. pogona, and provided insights into the improvement of secondary metabolite production.


Assuntos
Proteínas de Bactérias/metabolismo , Saccharopolyspora/crescimento & desenvolvimento , Saccharopolyspora/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Redes e Vias Metabólicas , Saccharopolyspora/genética
17.
ACS Synth Biol ; 9(3): 655-670, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32078772

RESUMO

Saccharopolyspora erythraea is used for industrial erythromycin production. To explore the physiological role of intracellular energy state in metabolic regulation by S. erythraea, we initially overexpressed the F1 part of the endogenous F1F0-ATPase in the high yielding erythromycin producing strain E3. The F1-ATPase expression resulted in lower [ATP]/[ADP] ratios, which was accompanied by a strong increase in the production of a reddish pigment and a decreased erythromycin production. Subsequent transcriptional analysis revealed that the lower intracellular [ATP]/[ADP] ratios exerted a pleotropic regulation on the metabolism of S. erythraea. The lower [ATP]/[ADP] ratios induced physiological changes to restore the energy balance, mainly via pathways that tend to produce ATP or regenerate NADH. The F1-ATPase overexpression strain exhibited a state of redox stress, which was correlated to an alteration of electron transport at the branch of the terminal oxidases, and S. erythraea channeled the enhanced glycolytic flux toward a reddish pigment in order to reduce NADH formation. The production of erythromycin was decreased, which is in accordance with the net ATP requirement and the excess NADH formed through this pathway. Partial growth inhibition by apramycin increased the intracellular [ATP]/[ADP] ratios and demonstrated a positive correlation between [ATP]/[ADP] ratios and erythromycin synthesis. Finally, overexpression of the entire F1F0-ATPase complex resulted in 28% enhanced erythromycin production and markedly reduced pigment synthesis in E3. The work illustrates a feasible strategy to optimize the distribution of fluxes in secondary metabolism.


Assuntos
Coenzimas/genética , Eritromicina/biossíntese , Engenharia Metabólica/métodos , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Acetilcoenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Coenzimas/metabolismo , Transporte de Elétrons , Regulação Bacteriana da Expressão Gênica , Microrganismos Geneticamente Modificados , NAD/genética , NAD/metabolismo , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Metabolismo Secundário
18.
Am J Physiol Lung Cell Mol Physiol ; 318(4): L631-L643, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31940220

RESUMO

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that may progress to fibrosis and significant risk of death. HP develops following repeated exposures to inhaled environmental antigens; however, only a fraction of the exposed population develops the disease, suggesting that host genetics contribute to disease susceptibility. We used the BXD family of mice with the Saccharopolyspora rectivirgula (SR) model of HP to investigate the role of genetics in susceptibility to HP. The BXD family is derived from a B6 mother and a D2 father and has been used to map susceptibility loci to numerous diseases. B6, D2, and BXD progeny strains were exposed to SR for 3 wk, and the development of HP was monitored. The B6 and D2 strains developed alveolitis; however, the cellular composition was neutrophilic in the D2 strain and more lymphocytic in the B6 strain. Hematoxylin-eosin staining of lung sections revealed lymphoid aggregates in B6 lungs, whereas D2 lungs exhibited a neutrophilic infiltration. Twenty-eight BXD strains of mice were tested, and the results reveal significant heritable variation for numbers of CD4+ or CD8+ T cells in the air spaces. There was significant genetic variability for lymphoid aggregates and alveolar wall thickening. We mapped a significant quantitative trait locus (QTL) on chromosome 18 for CD8+CD69+ T cells that includes cadherin 2 (Cdh2), an excellent candidate gene associated with epithelial-mesenchymal transition, which is upregulated in lungs of strains with HP. These results demonstrate that the BXD family is a valuable and translationally relevant model to identify genes contributing to HP and to devise early and effective interventions.


Assuntos
Alveolite Alérgica Extrínseca/genética , Alveolite Alérgica Extrínseca/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Variação Genética/genética , Alveolite Alérgica Extrínseca/microbiologia , Animais , Transição Epitelial-Mesenquimal/genética , Feminino , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Neutrófilos/imunologia , Saccharopolyspora/imunologia , Regulação para Cima/genética
19.
Appl Environ Microbiol ; 86(3)2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31732571

RESUMO

Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.


Assuntos
Antibacterianos/biossíntese , Bacteriocinas/biossíntese , Genes Bacterianos , Saccharopolyspora/química , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Formigas/microbiologia , Bacteriocinas/isolamento & purificação , Bacteriocinas/metabolismo , Fabaceae , Família Multigênica , Saccharopolyspora/genética
20.
J Appl Microbiol ; 128(2): 314-329, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31230389

RESUMO

Actinomycetes are a rich source for secondary metabolites with a diverse array of biological activities. Among the various genera of actinomycetes, the genus Saccharopolyspora has long been recognized as a potential source for antibiotics and other therapeutic leads that belong to diverse classes of natural products. Members of the genus Saccharopolyspora have been widely reported from several natural sources including both terrestrial and marine environments. A plethora of this genus has been chemically investigated for the production of novel natural products with interesting pharmacological effects. Therefore, Saccharopolyspora is considered one of the pharmaceutical important genera that could provide further chemical diversity with potential lead compounds. In this review, the literature from 1976 until December 2018 was covered, providing a comprehensive survey of all natural products derived from this genus and their semi-synthetic derivatives along with their biological activities, whenever applicable. Moreover, the biological diversity of Saccharopolyspora species and their habitats were also discussed.


Assuntos
Produtos Biológicos/metabolismo , Saccharopolyspora/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Ecossistema , Humanos , Saccharopolyspora/química , Saccharopolyspora/classificação , Saccharopolyspora/genética
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