| 1. |
- El-Sayed, Ashraf S.A., et al.
(author)
-
Production, bioprocess optimization and anticancer activity of Camptothecin from Aspergillus terreus and Aspergillus flavus, endophytes of Ficus elastica
- 2021
-
In: Process Biochemistry. - : Elsevier. - 1359-5113 .- 1873-3298. ; 107, s. 59-73
-
Journal article (peer-reviewed)abstract
- Attenuating the biosynthetic potency of camptothecin producing fungi with the multiple subculturing and storage is the major challenge that limits the scaling-up of this approach. Thus, screening for novel fungal isolates with reliable metabolic stability and sustainability for camptothecin production is the objective of this work. Among the recovered isolates, Aspergillus terreus (320 μg/l) and A. flavus (290 μg/l) "endophytes of Ficus elastica" were the most potent camptothecin producing isolates. The chemical identity of extracted camptothecin was resolved from the HPLC, NMR, FTIR and LC–MS analyses, with a strong antiproliferative activity against MCF7 (0.18 μM), LS174 T, HCT29 (0.29−0.43 μM), HEPG-2 (0.73 μM) cell lines. The yield of camptothecin was decreased by about > 60 % by the 7th subculturing for both fungal isolates. Upon using Blackett-Burman design to optimize their nutritional requirements by the fungal isolates, their yield of camptothecin were increased by approximately 2 folds, revealing the essentiality of some carbon, nitrogen and growth elicitors for biosynthesis of camptothecin. Interestingly, the biosynthetic machinery of camptothecin by the 7th generation fungal isolates were completely restored upon addition of 1% surface sterilized leaves of F. elastica, while all the other experimented plant extracts did not display any effect on camptothecin production. So, chemicals signals derived from the plant /or its entire microbiome "microbial communication" triggering the expression of camptothecin biosynthetic gene cluster of these fungi, could be the most conceivable hypothesis explaining the attenuation and restoration processes of camptothecin biosynthesis by target the fungal isolates. This is the first report describing the feasibility of A. terreus and A. flavus "endophytes of F. elastica" for camptothecin production with reliable metabolic and sustainable biosynthetic potency upon addition of the plant host's entire microbiome, that could be a preliminary platform for scaling-up of camptothecin production.
|
|
| 2. |
- El-Sayed, Ashraf S. A., et al.
(author)
-
Purification and biochemical characterization of taxadiene synthase from bacillus koreensis and stenotrophomonas maltophilia
- 2021
-
In: Scientia pharmaceutica. - : MDPI. - 0036-8709 .- 2218-0532. ; 89:4
-
Journal article (peer-reviewed)abstract
- 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.
|
|
