Heterologous Expression and Characterization of a “Pseudomature” Form of Taxadiene Synthase Involved in Paclitaxel (Taxol) Biosynthesis and Evaluation of a Potential Intermediate and Inhibitors of the Multistep Diterpene Cyclization Reaction

2000 ◽  
Vol 379 (1) ◽  
pp. 137-146 ◽  
Author(s):  
David C Williams ◽  
Mark R Wildung ◽  
Alan Qingwu Jin ◽  
Dolan Dalal ◽  
John S Oliver ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Cyclization Reaction ◽  
Taxadiene Synthase ◽  
Taxol Biosynthesis

scholarly journals Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis

2004 ◽  
Vol 279 (52) ◽  
pp. 53947-53954 ◽  
Author(s):  
Kevin D. Walker ◽  
Karin Klettke ◽  
Takumi Akiyama ◽  
Rodney Croteau
Keyword(s):  
Heterologous Expression ◽  
Taxol Biosynthesis

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.

scholarly journals Cloning, heterologous expression, and characterization of a novel thioesterase from natural sample

Heliyon ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. e06542
Author(s):  
Suharti ◽  
Gita Mahardika ◽  
Raissa ◽  
Laksmi Dewi ◽  
Heni Yohandini ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Natural Sample

scholarly journals Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

2013 ◽  
Vol 97 (13) ◽  
pp. 5753-5769 ◽  
Author(s):  
Simon Carlsen ◽  
Parayil Kumaran Ajikumar ◽  
Luca Riccardo Formenti ◽  
Kang Zhou ◽  
Too Heng Phon ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression

scholarly journals Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome

3 Biotech ◽  
2017 ◽  
Vol 7 (4) ◽  
Author(s):  
Ravi K. Shah ◽  
Amrutlal K. Patel ◽  
Deepti M. Davla ◽  
Ishan K. Parikh ◽  
Ramalingam B. Subramanian ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Molecular Cloning ◽  
Functional Characterization ◽  
Cellulolytic Enzyme ◽  
Rumen Metagenome ◽  
Buffalo Rumen

Heterologous expression and characterization of l-amino acid deaminases from Proteus mirabilis in Escherichia coli

2008 ◽  
Vol 136 ◽  
pp. S300 ◽  
Author(s):  
Jin-Oh Baek ◽  
Jeong-Woo Seo ◽  
Ohsuk Kwon ◽  
Su-Il Seong ◽  
Ik-Hwan Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Proteus Mirabilis

scholarly journals Heterologous expression in Pichia pastoris and biochemical characterization of the unmodified sulfatase from Fusarium proliferatum LE1

2017 ◽  
Vol 30 (8) ◽  
pp. 571-571 ◽  
Author(s):  
Svetlana A Korban ◽  
Kirill S Bobrov ◽  
Maria A Maynskova ◽  
Stanislav N Naryzhny ◽  
Olga L Vlasova ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Heterologous Expression ◽  
Biochemical Characterization ◽  
Fusarium Proliferatum
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