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  • 1
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    Online Resource
    p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation
    MDPI AG ; 2020
    In:  Molecules Vol. 25, No. 18 ( 2020-09-12), p. 4186-
    Details
    In: Molecules, MDPI AG, Vol. 25, No. 18 ( 2020-09-12), p. 4186-
    Abstract: Actinosynnema species produce diverse natural products with important biological activities, which represent an important resource of antibiotic discovery. Advances in genome sequencing and bioinformatics tools have accelerated the exploration of the biosynthetic gene clusters (BGCs) encoding natural products. Herein, the completed BGCs of dnacin B1 were first discovered in two Actinosynnema pretiosum subsp. auranticum strains DSM 44131T (hereafter abbreviated as strain DSM 44131T) and X47 by comparative genome mining strategy. The BGC for dnacin B1 contains 41 ORFs and spans a 66.9 kb DNA region in strain DSM 44131T. Its involvement in dnacin B1 biosynthesis was identified through the deletion of a 9.7 kb region. Based on the functional gene analysis, we proposed the biosynthetic pathway for dnacin B1. Moreover, p-amino-phenylalanine (PAPA) unit was found to be the dnacin B1 precursor for the quinone moiety formation, and this was confirmed by heterologous expression of dinV, dinE and dinF in Escherichia coli. Furthermore, nine potential PAPA aminotransferases (APAT) from the genome of strain DSM 44131T were explored and expressed. Biochemical evaluation of their amino group transformation ability was carried out with p-amino-phenylpyruvic acid (PAPP) or PAPA as the substrate for the final product formation. Two of those, APAT4 and APAT9, displayed intriguing aminotransferase ability for the formation of PAPA. The proposed dnacin B1 biosynthetic machinery and PAPA biosynthetic investigations not only enriched the knowledge of tetrahydroisoquinoline (THIQ) biosynthesis, but also provided PAPA building blocks to generate their structurally unique homologues.
    Type of Medium: Online Resource
    ISSN: 1420-3049
    URL: Article
    DOI: 10.3390/molecules25184186
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2008644-1
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  • 2
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    Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes
    MDPI AG ; 2020
    In:  Biomolecules Vol. 10, No. 6 ( 2020-06-03), p. 851-
    Details
    In: Biomolecules, MDPI AG, Vol. 10, No. 6 ( 2020-06-03), p. 851-
    Abstract: In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.
    Type of Medium: Online Resource
    ISSN: 2218-273X
    URL: Article
    DOI: 10.3390/biom10060851
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2701262-1
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  • 3
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    Online Resource
    Insight into Structural Characteristics of Protein-Substrate Interaction in Pimaricin Thioesterase
    MDPI AG ; 2019
    In:  International Journal of Molecular Sciences Vol. 20, No. 4 ( 2019-02-18), p. 877-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 20, No. 4 ( 2019-02-18), p. 877-
    Abstract: As a polyene antibiotic of great pharmaceutical significance, pimaricin has been extensively studied to enhance its productivity and effectiveness. In our previous studies, pre-reaction state (PRS) has been validated as one of the significant conformational categories before macrocyclization, and is critical to mutual recognition and catalytic preparation in thioesterase (TE)-catalyzed systems. In our study, molecular dynamics (MD) simulations were conducted on pimaricin TE-polyketide complex and PRS, as well as pre-organization state (POS), a molecular conformation possessing a pivotal intra-molecular hydrogen bond, were detected. Conformational transition between POS and PRS was observed in one of the simulations, and POS was calculated to be energetically more stable than PRS by 4.58 kcal/mol. The structural characteristics of PRS and POS-based hydrogen-bonding, and hydrophobic interactions were uncovered, and additional simulations were carried out to rationalize the functions of several key residues (Q29, M210, and R186). Binding energies, obtained from MM/PBSA calculations, were further decomposed to residues, in order to reveal their roles in product release. Our study advanced a comprehensive understanding of pimaricin TE-catalyzed macrocyclization from the perspectives of conformational change, protein-polyketide recognition, and product release, and provided potential residues for rational modification of pimaricin TE.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms20040877
    Language: English
    Publisher: MDPI AG
    Publication Date: 2019
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    In Silico Discovery of Aminoacyl-tRNA Synthetase Inhibitors
    MDPI AG ; 2014
    In:  International Journal of Molecular Sciences Vol. 15, No. 1 ( 2014-01-20), p. 1358-1373
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 15, No. 1 ( 2014-01-20), p. 1358-1373
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms15011358
    Language: English
    Publisher: MDPI AG
    Publication Date: 2014
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 5
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    Comparative Transcriptome-Based Mining of Genes Involved in the Export of Polyether Antibiotics for Titer Improvement
    MDPI AG ; 2022
    In:  Antibiotics Vol. 11, No. 5 ( 2022-04-29), p. 600-
    Details
    In: Antibiotics, MDPI AG, Vol. 11, No. 5 ( 2022-04-29), p. 600-
    Abstract: The anti-coccidiosis agent salinomycin is a polyether antibiotic produced by Streptomyces albus BK3-25 with a remarkable titer of 18 g/L at flask scale, suggesting a highly efficient export system. It is worth identifying the involved exporter genes for further titer improvement. In this study, a titer gradient was achieved by varying soybean oil concentrations in a fermentation medium, and the corresponding transcriptomes were studied. Comparative transcriptomic analysis identified eight putative transporter genes, whose transcription increased when the oil content was increased and ranked top among up-regulated genes at higher oil concentrations. All eight genes were proved to be positively involved in salinomycin export through gene deletion and trans-complementation in the mutants, and they showed constitutive expression in the early growth stage, whose overexpression in BK3-25 led to a 7.20–69.75% titer increase in salinomycin. Furthermore, the heterologous expression of SLNHY_0929 or SLNHY_1893 rendered the host Streptomyces lividans with improved resistance to salinomycin. Interestingly, SLNHY_0929 was found to be a polyether-specific transporter because the titers of monensin, lasalocid, and nigericin were also increased by 124.6%, 60.4%, and 77.5%, respectively, through its overexpression in the corresponding producing strains. In conclusion, a transcriptome-based strategy was developed to mine genes involved in salinomycin export, which may pave the way for further salinomycin titer improvement and the identification of transporter genes involved in the biosynthesis of other antibiotics.
    Type of Medium: Online Resource
    ISSN: 2079-6382
    URL: Article
    DOI: 10.3390/antibiotics11050600
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2681345-2
    SSG: 15,3
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  • 6
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    Online Resource
    Synthetic Biology Facilitates Antimicrobials Discovery
    MDPI AG ; 2023
    In:  Antibiotics Vol. 12, No. 3 ( 2023-03-15), p. 578-
    Details
    In: Antibiotics, MDPI AG, Vol. 12, No. 3 ( 2023-03-15), p. 578-
    Abstract: We are currently facing two big global challenges: antibiotics shortage and multidrug resistance [...]
    Type of Medium: Online Resource
    ISSN: 2079-6382
    URL: Article
    DOI: 10.3390/antibiotics12030578
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2681345-2
    SSG: 15,3
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  • 7
    Online Resource
    Online Resource
    Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin
    MDPI AG ; 2021
    In:  Molecules Vol. 26, No. 22 ( 2021-11-10), p. 6791-
    Details
    In: Molecules, MDPI AG, Vol. 26, No. 22 ( 2021-11-10), p. 6791-
    Abstract: Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein–protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein–protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein–protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.
    Type of Medium: Online Resource
    ISSN: 1420-3049
    URL: Article
    DOI: 10.3390/molecules26226791
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2008644-1
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  • 8
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    Online Resource
    The Antitumor Agent Ansamitocin P-3 Binds to Cell Division Protein FtsZ in Actinosynnema pretiosum
    MDPI AG ; 2020
    In:  Biomolecules Vol. 10, No. 5 ( 2020-04-30), p. 699-
    Details
    In: Biomolecules, MDPI AG, Vol. 10, No. 5 ( 2020-04-30), p. 699-
    Abstract: Ansamitocin P-3 (AP-3) is an important antitumor agent. The antitumor activity of AP-3 is a result of its affinity towards β-tubulin in eukaryotic cells. In this study, in order to improve AP-3 production, the reason for severe growth inhibition of the AP-3 producing strain Actinosynnema pretiosum WXR-24 under high concentrations of exogenous AP-3 was investigated. The cell division protein FtsZ, which is the analogue of β-tubulin in bacteria, was discovered to be the AP-3 target through structural comparison followed by a SPR biosensor assay. AP-3 was trapped into a less hydrophilic groove near the GTPase pocket on FtsZ by hydrogen bounding and hydrophobic interactions, as revealed by docking analysis. After overexpression of the APASM_5716 gene coding for FtsZ in WXR-30, the resistance to AP-3 was significantly improved. Moreover, AP-3 yield was increased from 250.66 mg/L to 327.37 mg/L. After increasing the concentration of supplemented yeast extract, the final yield of AP-3 reached 371.16 mg/L. In summary, we demonstrate that the cell division protein FtsZ is newly identified as the bacterial target of AP-3, and improving resistance is an effective strategy to enhance AP-3 production.
    Type of Medium: Online Resource
    ISSN: 2218-273X
    URL: Article
    DOI: 10.3390/biom10050699
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2701262-1
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  • 9
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    Online Resource
    Parameter Estimation of Electromechanical Oscillation Based on a Constrained EKF with C&I-PSO
    MDPI AG ; 2018
    In:  Energies Vol. 11, No. 8 ( 2018-08-08), p. 2059-
    Details
    In: Energies, MDPI AG, Vol. 11, No. 8 ( 2018-08-08), p. 2059-
    Abstract: By combining together the extended Kalman filter with a newly developed C & I particle swarm optimization algorithm (C & I-PSO), a novel estimation method is proposed for parameter estimation of electromechanical oscillation, in which critical physical constraints on the parameters are taken into account. Based on the extended Kalman filtering algorithm, the constrained parameter estimation problem is formulated via the projection method. Then, by utilizing the penalty function method, the obtained constrained optimization problem could be converted into an equivalent unconstrained optimization problem; finally, the C & I-PSO algorithm is developed to address the unconstrained optimization problem. Therefore, the parameters of electromechanical oscillation with physical constraints can be successfully estimated and better performed. Finally, the effectiveness of the obtained results has been illustrated by several test systems.
    Type of Medium: Online Resource
    ISSN: 1996-1073
    URL: Article
    DOI: 10.3390/en11082059
    Language: English
    Publisher: MDPI AG
    Publication Date: 2018
    detail.hit.zdb_id: 2437446-5
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