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1.
Microb Cell Fact ; 23(1): 13, 2024 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-38183102

RESUMEN

BACKGROUND: S. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein. RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain. CONCLUSION: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.


Asunto(s)
Proteómica , Streptomyces lividans , Streptomyces lividans/genética , Transporte de Proteínas , Transporte Biológico , Regulación hacia Arriba
2.
Mol Cell Proteomics ; 18(3): 423-436, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30498012

RESUMEN

Protein secretion is a central biological process in all organisms. Most studies dissecting bacterial secretion mechanisms have focused on Gram-negative cell envelopes such as that of Escherichia coli However, proteomics analyses in Gram negatives is hampered by their outer membrane. Here we studied protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, in which most of the secretome is released in the growth medium. We monitored changes of the secretome as a function of growth phase and medium. We determined distinct protein classes of "house-keeping" secreted proteins that do not change their appearance or abundance in the various media and growth phases. These comprise mainly enzymes involved in cell wall maintenance and basic transport. In addition, we detected significant abundance and content changes to a sub-set of the proteome, as a function of growth in the different media. These did not depend on the media being minimal or rich. Transcriptional regulation but not changes in export machinery components can explain some of these changes. However, additional downstream mechanisms must be important for selective secretome funneling. These observations lay the foundations of using S. lividans as a model organism to study how metabolism is linked to optimal secretion and help develop rational optimization of heterologous protein production.


Asunto(s)
Proteínas Bacterianas/metabolismo , Medios de Cultivo/análisis , Proteómica/métodos , Streptomyces lividans/crecimiento & desarrollo , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos/microbiología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Esenciales , Modelos Biológicos , Streptomyces lividans/metabolismo
3.
Microb Cell Fact ; 16(1): 232, 2017 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-29274637

RESUMEN

BACKGROUND: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. RESULTS: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. CONCLUSION: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.


Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Expresión Génica , Rhodothermus/enzimología , Streptomyces lividans/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Celulasa/química , Celulasa/genética , Estabilidad de Enzimas , Calor , Transporte de Proteínas , Rhodothermus/genética , Streptomyces lividans/metabolismo
4.
Nat Commun ; 15(1): 4479, 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38802343

RESUMEN

Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides , Microscopía por Crioelectrón , Proteínas de la Membrana , Presenilina-1 , Humanos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Presenilina-1/metabolismo , Presenilina-1/química , Presenilina-1/genética , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Endopeptidasas/metabolismo , Endopeptidasas/química , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Unión Proteica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/química , Enfermedad de Alzheimer/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/química , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/química , Modelos Moleculares , Proteolisis
5.
Front Microbiol ; 12: 604034, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33935985

RESUMEN

Streptomyces lividans TK24 is a relevant Gram-positive soil inhabiting bacterium and one of the model organisms of the genus Streptomyces. It is known for its potential to produce secondary metabolites, antibiotics, and other industrially relevant products. S. lividans TK24 is the plasmid-free derivative of S. lividans 66 and a close genetic relative of the strain Streptomyces coelicolor A3(2). In this study, we used transcriptome and proteome data to improve the annotation of the S. lividans TK24 genome. The RNA-seq data of primary 5'-ends of transcripts were used to determine transcription start sites (TSS) in the genome. We identified 5,424 TSS, of which 4,664 were assigned to annotated CDS and ncRNAs, 687 to antisense transcripts distributed between 606 CDS and their UTRs, 67 to tRNAs, and 108 to novel transcripts and CDS. Using the TSS data, the promoter regions and their motifs were analyzed in detail, revealing a conserved -10 (TAnnnT) and a weakly conserved -35 region (nTGACn). The analysis of the 5' untranslated region (UTRs) of S. lividans TK24 revealed 17% leaderless transcripts. Several cis-regulatory elements, like riboswitches or attenuator structures could be detected in the 5'-UTRs. The S. lividans TK24 transcriptome contains at least 929 operons. The genome harbors 27 secondary metabolite gene clusters of which 26 could be shown to be transcribed under at least one of the applied conditions. Comparison of the reannotated genome with that of the strain Streptomyces coelicolor A3(2) revealed a high degree of similarity. This study presents an extensive reannotation of the S. lividans TK24 genome based on transcriptome and proteome analyses. The analysis of TSS data revealed insights into the promoter structure, 5'-UTRs, cis-regulatory elements, attenuator structures and novel transcripts, like small RNAs. Finally, the repertoire of secondary metabolite gene clusters was examined. These data provide a basis for future studies regarding gene characterization, transcriptional regulatory networks, and usage as a secondary metabolite producing strain.

6.
Microorganisms ; 9(3)2021 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-33805695

RESUMEN

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO's top pathogens list). Seven of them-HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14-representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 µM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9-77.8 µM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 < 65 µM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 µM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

7.
FEMS Microbiol Lett ; 365(22)2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30299471

RESUMEN

Bacteria are of tremendous importance in the pharma- and bio-industry as producers of a broad range of economically interesting metabolites and proteins. Gram-positive bacteria are valuable hosts for the production of heterologous proteins for obvious reasons. Contrary to Gram-negative bacteria, Gram-positive bacteria release their secreted proteins immediately into the spent culture broth as they are not hindered by an outer membrane. Secretory protein production also avoids the formation of inclusion bodies, hence facilitating downstream processing. Eight protein secretion pathways have been described in Gram-positive bacteria, but solely the general secretion or Sec pathway and, to a lesser extent, the Twin-arginine pathway, are used for the recombinant protein production. This process is not always successful, but might be hampered by inefficient secretion, misfolding of the recombinant protein, its degradation by proteases and metabolic burden by the host hindering proper growth and diminishing product yield. In this review, the different protein export avenues will be briefly discussed, and the potential means to optimize protein secretion and yields for the Streptomyces lividans model presented. The proposed approaches are largely applicable for other Streptomyces host systems.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biotecnología , Vías Secretoras , Streptomyces/metabolismo , Fermentación , Transporte de Proteínas , Proteínas Recombinantes , Streptomyces lividans/metabolismo
8.
Vet World ; 11(10): 1364-1370, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30532487

RESUMEN

AIM: In view of various peroxidase applications, the searching for new sources of unique peroxidase properties is highly desirable. The present study aims to evaluate the efficiency of the peroxidase of locally grown sycamore latex (POL) for conjugation with antibodies and to study the conjugate optimal conditions, storage stability, and affinity toward different substrates as compared with commercial horseradish peroxidase (HRP). MATERIALS AND METHODS: Anti-mouse antibodies were prepared in rabbits and purified by protein A sepharose affinity column chromatography. The POL and HRP conjugates were prepared by one-step glutaraldehyde coupling method. The reactivity of the prepared conjugates was examined using the enzyme-linked immunosorbent assay (ELISA). The optimal enzymatic conditions, storage stability, and affinity toward substrates were also determined for both the conjugates. RESULTS: The POL showed higher percent recovery (98%) than HRP (78%) over the initial activity after conjugation process. The POL and HRP conjugates showed ELISA titers of 1:120 and 1:80, respectively, demonstrating high binding affinity of POL-conjugate. The POL-conjugate showed high thermal stability up to 70°C compared with HRP-conjugate up to 40°C. After conjugation, POL had wide pH stability (5.0-8.0) compared with HPR (4.5-6.0). Both of the prepared conjugates had a high affinity toward the substrates used in immunoassays with lower Km values. The POL-conjugate showed high storage stability for its enzymatic activity and ELISA titer compared with HRP-conjugate after 1 year at -20°C. CONCLUSION: The POL of Ficus sycomorus latex is an efficient source for labeling antibodies and could be utilized in immunodiagnostic kits.

9.
Front Microbiol ; 9: 3019, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30581427

RESUMEN

Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model.

10.
Rev. bras. farmacogn ; 28(5): 594-601, Sept.-Oct. 2018. tab, graf
Artículo en Inglés | LILACS | ID: biblio-977737

RESUMEN

Abstract In this study, mango seed kernels extract contained a considerable amount of phenolics and flavonoids (17,400 and 3325 mg/100 g seed, respectively). The HPLC profiling revealed that hesperidin was the major phenolic compound of the mango seed kernels extract. This is the first report find hesperidin in mango extracts. The phenolic compounds of mango seed kernels extract were effective in scavenging free radicals of DPPH and ABTS with IC50 values of 47.3 and 7.9 µg/ml, respectively. The total antioxidant activity of mango seed kernels extract based on the reduction of molybdenum was also measured. The phenolic compounds of mango seed kernels extract potentially inhibited the protease, fibrinogenase, phospholipase A2, l-amino acid oxidase, hyaluronidase, and hemolytic activities of the most dangerous Cerastes cerastes and Echis coloratus viper venoms. The phenolic compounds of mango seed kernels extract could completely neutralize the hemorrhage and lethality of both venoms in experimental animals. It could be concluded that the mango seed kernels extract phenolic compounds with potential antioxidant activity are considered as a new avenue in the viper bite treatment.

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