Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

El-Sayed, Ashraf S. A.; Fathalla, Maher; Shindia, Ahmed A.; Rady, Amgad M.; El-Baz, Ashraf F.; Morsy, Yara; Sitohy, Basel; Sitohy, Mahmoud

doi:10.3390/scipharm89040048

Cited by 3 publications

(1 citation statement)

References 69 publications

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“…Aspergillus terreus , an endophyte of Ficus elastica ( 9 , 15 ), Cestrum parqui ( 15 ), Cinnamomum camphora ( 26 ), Aspergillus flavus , “endophyte of Astragalus sp.” ( 17 ), and Penicillium chrysogenum ( 20 ) have been reported as a potent CPT producers. However, the suppression of CPT productivity with fungal storage and subculturing are the challenges that limits their further implementation ( 9 , 14 – 16 , 20 , 27 , 28 ).…”

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confidence: 99%

Proteomics and metabolomics analyses of camptothecin-producing Aspergillus terreus reveal the integration of PH domain-containing proteins and peptidylprolyl cis/trans isomerase in restoring the camptothecin biosynthesis

Rady,

El-Sayed,

El-Baz

et al. 2023

Microbiol Spectr

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Attenuating the expression of fungal camptothecin biosynthetic genes with subculturing is the challenge that halts their further implementation. The camptothecin productivity of the subcultured Aspergillus terreus has been restored upon addition of Ficus elastica indigenous microbiome; however, the identity of triggering signals of A. terreus camptothecin biosynthesis remains ambiguous. In this study, differential proteomics and metabolomics analyses were implemented to unravel the differentially abundant proteins and metabolites associated with the weakening/restoration of the biosynthetic machinery of camptothecin by A. terreus . The functional proteins, namely, ribosomal proteins, ATP, metal ion, and GTP binding proteins, were abolished by the seventh culture of A. terreus ; however, the expression of these proteins was completely restored upon addition of F. elastica microbiome. Among the proteins of highly altered abundance, Pleckstrin homology (PH) domain-containing protein, peptidylprolyl cis / trans isomerase, 60S ribosomal protein, and So-Cu domain-containing proteins were significantly decreased with subculturing of A. terreus and strikingly restored upon addition of F. elastica microbiome. The metabolites 5,7-dihydroxy-2-(4-hydroxyphenyl)-3,6-dimethoxy-4H-chromen and glutaric acid of A. terreus were significantly decreased with subculturing and completely restored upon addition of F. elastica microbiome. The most differentially abundant metabolites were involved in glycolysis, TCA cycle, mevalonate pathway, terpenoids and shikimate synthesis, and ultimately with camptothecin biosynthesis. Thus, overexpression of PH domain-containing protein and peptidylprolyl cis / trans isomerase could be a new avenue for a metabolically stable camptothecin producing A. terreus . IMPORTANCE Decreasing the camptothecin productivity by fungi with storage and subculturing is the challenge that halts their further implementation to be an industrial platform for camptothecin (CPT) production. The highest differentially abundant proteins were Pleckstrin homology (PH) domain-containing proteins and Peptidyl-prolyl cis / trans isomerase that fluctuated with the subculturing of A. terreus with a remarkable relation to CPT biosynthesis and restored with addition of F. elastica microbiome.

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Section: Introductionmentioning

confidence: 99%

Proteomics and metabolomics analyses of camptothecin-producing Aspergillus terreus reveal the integration of PH domain-containing proteins and peptidylprolyl cis/trans isomerase in restoring the camptothecin biosynthesis

Rady,

El-Sayed,

El-Baz

et al. 2023

Microbiol Spectr

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Production, bioprocessing and antiproliferative activity of camptothecin from Aspergillus terreus, endophyte of Cinnamomum camphora: restoring their biosynthesis by indigenous microbiome of C. camphora

Eldeghidy,

Abdel-Fattah,

El-Sayed

et al. 2023

Microb Cell Fact

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Fungal producing potency of camptothecin (CPT) raise the hope for their usage to be a platform for industrial production of CPT, nevertheless, attenuation of their productivity of CPT with the subculturing and preservation is the challenge. So, screening for novel endophytic fungal isolates with a reliable CPT-biosynthetic stability was the objective. Among the isolated endophytic fungi from the tested medicinal plants, Aspergillus terreus OQ642314.1, endophyte of Cinnamomum camphora, exhibits the highest yield of CPT (89.4 μg/l). From the NMR, FT-IR and LC–MS/MS analyses, the extracted CPT from A. terreus gave the same structure and molecular mass fragmentation pattern of authentic CPT (349 m/z). The putative CPT had a significant activity against MCF7 (0.27 µM) and HEPG-2 (0.8 µM), with a strong affinity to inhibits the human Topoisomerase 1 activity (IC50 0.362 μg/ml) as revealed from the Gel-based DNA relaxation assay. The purified CPT displayed a strong antimicrobial activity for various bacterial (E. coli and B. cereus) and fungal (A. flavus and A. parasiticus) isolates, ensuring the unique tertiary, and stereo-structure of A. terreus for penetrating the microbial cell walls and targeting the topoisomerase I. The higher dual activity of the purified CPT as antimicrobial and antitumor, emphasize their therapeutic efficiency, especially with growth of the opportunistic microorganisms due to the suppression of human immune system with the CPT uses in vivo. The putative CPT had an obvious activity against the tumor cell (MCF7) metastasis, and migration as revealed from the wound healing assay. The overall yield of A. terreus CPT was maximized with the Blackett-Burman design by twofolds increment (164.8 μg/l). The CPT yield by A. terreus was successively diminished with the multiple fungal subculturing, otherwise, the CPT productivity of A. terreus was restored, and increased over the zero culture upon coculturing with C. camphora microbiome (1.5% w/v), ensuring the restoring of CPT biosynthetic potency of A. terreus by the plant microbiome-derived chemical signals “microbial communication”. This is the first report exploring the feasibility of A. terreus “endophyte of C. camphora” to be a preliminary platform for commercial production of CPT with a reliable sustainability upon uses of indigenous C. camphora microbiome.

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Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases

El-Hady,

ElSayed,

Wadan

et al. 2024

Microb Cell Fact

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Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 μg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC–MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 μM) and MCF7 (3.2 μM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.

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Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

Cited by 3 publications

References 69 publications

Proteomics and metabolomics analyses of camptothecin-producing Aspergillus terreus reveal the integration of PH domain-containing proteins and peptidylprolyl cis/trans isomerase in restoring the camptothecin biosynthesis

Proteomics and metabolomics analyses of camptothecin-producing Aspergillus terreus reveal the integration of PH domain-containing proteins and peptidylprolyl cis/trans isomerase in restoring the camptothecin biosynthesis

Production, bioprocessing and antiproliferative activity of camptothecin from Aspergillus terreus, endophyte of Cinnamomum camphora: restoring their biosynthesis by indigenous microbiome of C. camphora

Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases

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