Mechanism of Taxadiene Synthase, a Diterpene Cyclase That Catalyzes the First Step of Taxol Biosynthesis in Pacific Yew†

Biochemistry ◽  
1996 ◽  
Vol 35 (9) ◽  
pp. 2968-2977 ◽  
Author(s):  
Xiaoyan Lin ◽  
Mehri Hezari ◽  
Alfred E. Koepp ◽  
Heinz G. Floss ◽  
Rodney Croteau
Keyword(s):  
Taxadiene Synthase ◽  
Taxol Biosynthesis ◽  
Diterpene Cyclase

scholarly journals Salicylic Acid-Responsive Factor TcWRKY33 Positively Regulates Taxol Biosynthesis in Taxus chinensis in Direct and Indirect Ways

2021 ◽  
Vol 12 ◽  
Author(s):  
Ying Chen ◽  
Hua Zhang ◽  
Meng Zhang ◽  
Wenli Zhang ◽  
Ziqi Ou ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Luciferase Activity ◽  
Wrky Transcription Factor ◽  
Taxus Chinensis ◽  
Taxadiene Synthase ◽  
Taxol Biosynthesis ◽  
Group I ◽  
Excellent Candidate ◽  
Direct Target ◽  
Transient Overexpression

Taxol is a rare secondary metabolite that accumulates considerably in Taxus species under salicylic acid (SA) and methyl jasmonate treatment. However, the molecular mechanism of its accumulation remains unclear. We investigated TcWRKY33, a nuclear-localized group I WRKY transcription factor, as an SA-responsive regulator of taxol biosynthesis. Overexpression and RNA interference of TcWRKY33 confirmed that TcWRKY33 regulates the expression of most taxol biosynthesis genes, especially 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) and taxadiene synthase (TASY), which were considered as key enzymes in taxol biosynthesis. Transient overexpression of TcWRKY33 in Taxus chinensis leaves resulted in increased taxol and 10-deacetylbaccatin accumulation by 1.20 and 2.16 times compared with the control, respectively. Furthermore, TcWRKY33, DBAT, and TASY were confirmed to respond positively to SA signals. These results suggested that TcWRKY33 was the missing component of taxol biosynthesis that responds to SA. The sequence analysis identified two W-box motifs in the promoter of DBAT but not in the TASY. Yeast one-hybrid and dual-luciferase activity assays confirmed that TcWRKY33 can bind to the two W-boxes in the promoter of DBAT, upregulating its expression level. Hence, DBAT is a direct target of TcWRKY33. Furthermore, TcERF15, encoding a TASY activator, also contains two W-boxes in its promoter. Yeast one-hybrid and dual-luciferase activity assays further confirmed that TcWRKY33 can upregulate TASY expression through the activation of TcERF15. In summary, TcWRKY33 transmits SA signals and positively regulates taxol biosynthesis genes in two ways: directly and through the activation of other activators. Therefore, TcWRKY33 is an excellent candidate for genetically engineering regulation of taxol biosynthesis in Taxus plants.

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

2021 ◽  
Vol 89 (4) ◽  
pp. 48
Author(s):  
Ashraf S. A. El-Sayed ◽  
Maher Fathalla ◽  
Ahmed A. Shindia ◽  
Amgad M. Rady ◽  
Ashraf F. El-Baz ◽  
...  
Keyword(s):  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Genomic Feature ◽  
Subunit Structure ◽  
Fragmentation Pattern ◽  
Intermediary Metabolites ◽  
Taxadiene Synthase ◽  
Taxol Biosynthesis ◽  
Main Challenge

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.

Transcriptome-wide analysis of AP2/ERF transcription factors involved in regulating Taxol biosynthesis in Taxus × media

2021 ◽  
Vol 171 ◽  
pp. 113972
Author(s):  
Kaikai Zhang ◽  
Luyuan Jiang ◽  
Xu Wang ◽  
Hua Han ◽  
Duanfen Chen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Taxus Media ◽  
Taxol Biosynthesis ◽  
Erf Transcription Factors

scholarly journals Taxol biosynthesis: Taxane 13 -hydroxylase is a cytochrome P450-dependent monooxygenase

2001 ◽  
Vol 98 (24) ◽  
pp. 13595-13600 ◽  
Author(s):  
S. Jennewein ◽  
C. D. Rithner ◽  
R. M. Williams ◽  
R. B. Croteau
Keyword(s):  
Cytochrome P450 ◽  
Taxol Biosynthesis

scholarly journals Cloning of taxadiene synthase gene into Arabidopsis thaliana (ecotype Columbia-0)

2010 ◽  
Vol 9 (12) ◽  
pp. 1734-1740 ◽  
Author(s):  
Khani Sajjad ◽  
Mehdi Sohani Mohammad ◽  
Mahna Nasser ◽  
Barar Jaleh ◽  
Saeed Hejazi Mohammad ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Taxadiene Synthase

scholarly journals Enhanced production of taxadiene in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Behnaz Nowrouzi ◽  
Rachel Li ◽  
Laura E. Walls ◽  
Leopold d’Espaux ◽  
Koray Malci ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Shake Flask ◽  
Scale Up ◽  
Cost Effective ◽  
Cancer Drug ◽  
List Type ◽  
Taxadiene Synthase ◽  
Enhanced Production ◽  
Anti Cancer ◽  
Positive Effect

AbstractCost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at shake flask scale. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively. Maximum taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results highlight the positive effect of coupling molecular biology tools with bioprocess variable optimisation on synthetic pathway development.HighlightsMaximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °CIntegrating fusion protein tagged-taxadiene synthase improved taxadiene titre.Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales.

scholarly journals Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

2011 ◽  
Vol 7 (7) ◽  
pp. 431-433 ◽  
Author(s):  
Mustafa Köksal ◽  
Huayou Hu ◽  
Robert M Coates ◽  
Reuben J Peters ◽  
David W Christianson
Keyword(s):  
Diterpene Cyclase ◽  
Copalyl Diphosphate Synthase
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