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.
