Browsing by Author "El-Sayed, Ashraf S. A"

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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
(American Society for Microbiology, 2023-10) Rady, Amgad M; El-Sayed, Ashraf S. A; El-Baz, Ashraf F; Abdel-Fattah, Ghada G; Magdeldin, Sameh; Ahmed, Eman; Osama, Aya; Hassanein, Sameh E; Saed, Hend; Yassin, Marwa
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 com- pletely 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-hydroxy- phenyl)-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 indus- trial 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.
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.