Browsing by Author "Sitohy, Basel"

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    Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation
    (MDPI, 6/30/2020) El-Sayed, Ashraf S.A.; El-Sayed, Manal T.; Rady, Amgad M.; Zein, Nabila; Enan, Gamal; Shindia, Ahmed; El-Hefnawy, Sara; Sitohy, Mahmoud; Sitohy, Basel
    MDPI Open Access Journals zoom_out_map search menu Journals Molecules Volume 25 Issue 13 10.3390/molecules25133000 molecules-logo Submit to this Journal Review for this Journal Edit a Special Issue ► Article Menu Open AccessReview Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation by Ashraf S.A. El-Sayed 1,*,Manal T. El-Sayed 1,Amgad M. Rady 2,Nabila Zein 3,Gamal Enan 1,Ahmed Shindia 1,Sara El-Hefnawy 1,Mahmoud Sitohy 4OrcID andBasel Sitohy 5,6,*OrcID 1 Enzymology and Fungal Biotechnology Lab (EFBL), Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt 2 Faculty of Biotechnology, October University for Modern Sciences and Arts, Cairo 12566, Egypt 3 Chemistry Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt 4 Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt 5 Department of Clinical Microbiology, Infection and Immunology, Umeå University, SE-90185 Umeå, Sweden 6 Department of Radiation Sciences, Oncology, Umeå University, SE-90185 Umeå, Sweden * Authors to whom correspondence should be addressed. Molecules 2020, 25(13), 3000; https://doi.org/10.3390/molecules25133000 Received: 24 May 2020 / Revised: 17 June 2020 / Accepted: 23 June 2020 / Published: 30 June 2020 (This article belongs to the Special Issue Drug Discovery and Development Based on Native/Engineered Microorganisms) View Full-Text Download PDF Browse Figures Cite This Paper Abstract Endophytic fungi have been considered as a repertoire for bioactive secondary metabolites with potential application in medicine, agriculture and food industry. The biosynthetic pathways by fungal endophytes raise the argument of acquisition of these machineries of such complex metabolites from the plant host. Diterpenoids “Taxol” is the most effective anticancer drug with highest annual sale, since its discovery in 1970 from the Pacific yew tree, Taxus brevifolia. However, the lower yield of Taxol from this natural source (bark of T. brevifolia), availability and vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions are the challenges. Endophytic fungi from Taxus spp. opened a new avenue for industrial Taxol production due to their fast growth, cost effectiveness, independence on climatic changes, feasibility of genetic manipulation. However, the anticipation of endophytic fungi for industrial Taxol production has been challenged by the loss of its productivity, due to the metabolic reprograming of cells, downregulating the expression of its encoding genes with subculturing and storage. Thus, the objectives of this review were to (1) Nominate the endophytic fungal isolates with the Taxol producing potency from Taxaceae and Podocarpaceae; (2) Emphasize the different approaches such as molecular manipulation, cultural optimization, co-cultivation for enhancing the Taxol productivities; (3) Accentuate the genome mining of the rate-limiting enzymes for rapid screening the Taxol biosynthetic machinery; (4) Triggering the silenced rate-limiting genes and transcriptional factors to activates the biosynthetic gene cluster of Taxol.
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    Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia
    (MDPI, 09/11/2021) El-Sayed, Ashraf S. A; Fathalla, Maher; Shindia, Ahmed A; Rady, Amgad M; El-Baz, Ashraf F; Morsy, Yara; Sitohy, Basel; Sitohy, Mahmoud
    Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett– Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel- filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 ◦C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg2+, confirming the identity of Mg2+ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis.