Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III

Abstract

Paclitaxel is a well-known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. Here, we identified a bifunctional cytochrome P450 enzyme (Taxane oxetanase, TOT) that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, representing a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9-position (T9αH). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Unraveling the Catalytic Mechanism of Taxadiene-5α-hydroxylase from Crystallography and Computational Analyses

“…Interestingly, recent studies suggested that the T5OH intermediate may undergo further oxidation by CYP725A4, leading to the formation of an oxetane intermediate . However, another recent work demonstrated that the generation of the oxetane intermediate is mediated by another novel P450 enzyme . The mechanism of the oxetane formation is worthwhile to gauge with QM/MM in our future work.…”

Regulatory microRNAs and phasiRNAs of paclitaxel biosynthesis in Taxus chinensis

“…The full names of abbreviations are listed as: GGPPS, geranylgeranyl diphosphate synthase; TXS, Taxadiene synthase; T5αOH, taxane 5α-hydroxylase; TAT, taxadien-5α-ol-O-acetyl-transferase; T13αOH, taxane 13α-hydroxylase; T10βOH, taxane 10β-hydroxylase; T14βOH, taxane 14β-hydroxylase; T2αOH, taxane 2α-hydroxylase; T7βOH, taxane 7β-hydroxylase; TBT, taxane 2α-O-benzoyl transferase; DBAT, 10-deacetyl-baccatin III-10-Oacetyltransferase; PAM, phenylalanine aminomutase; BAPT, 13-O-(3-amino-3-phenylpropanoyl) transferase; DBTNBT, 30-N-debenzoyl-20-deoxytaxol-Nbenzoyltransferase. The metabolic processes were adapted from previous reports ( Croteau et al., 2006 ; Cheng et al., 2021 ; Zhang et al., 2023 ; Jiang et al., 2024 ).…”

“…Six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes ( Liu et al., 2024 ). The screening strategy for the biosynthesis pathway of paclitaxel is constantly being updated, new enzyme genes like TOT1 and T9αH1 were innovatively proposed and precisely located in the synthesis pathway ( Jiang et al., 2024 ). In our study, most of the known genes involved in paclitaxel biosynthesis are located on an 80.46-Mb region and a 141.69-Mb region on chromosome 9, which were consistent with previous studies, and the number of identified enzyme genes in the clusters also increased ( Supplementary Table 3 ; Xiong et al., 2021 ).…”

“…Subsequently, the tricyclic taxane backbone undergoes extremely complex modifications mediated by many oxygenases, acyltransferases, and benzoyltransferases, including the 2α-, 5α-, 7β-, 9α-, 10β-, and 13α-hydroxylases, and TAT (taxadienol 5α- O -acetyl transferase) and DBAT (10-deacetylbaccatin III 10- O -acetyltransferase) ( Walker and Croteau, 2000 ; Walker et al, 2000 ; Jennewein and Croteau, 2001 ; Jennewein et al, 2001 ; Walker et al, 2002 ; Chau and Croteau, 2004 ; Kaspera and Croteau, 2006 ; Long et al, 2008 ). Recently, two key enzymes have been identified for artificial construction of the baccatin III biosynthetic pathway, including Taxane oxetanase 1 (TOT1) representing a previously unknown enzyme mechanism for oxetane ring formation and T9αH for the taxane oxidation of the C9- position ( Jiang et al., 2024 ).…”

Enzyme and Pathway Engineering for Improved Betanin Production in Saccharomyces cerevisiae

“…Subsequent enzymatic actions include the conversion of l -dopa to betalamic acid by dopa-4,5-dioxygenase enzyme (DOD) and the glycosylation of cyclo -dopa at the 5- O position by the cyclo -dopa 5- O -glucosyltransferase (CDGT), ultimately resulting in the synthesis of betanin through the spontaneous condensation of the cyclo -dopa-5- O -glucoside with betalamic acid. The burgeoning field of synthetic biology has provided platforms for the production of complex natural products, with successful examples including the precursors of paclitaxel, artemisinin, and vincristine, − which offers a viable solution to overcome the constraints associated with plant extraction method by genetically engineering biosynthetic pathways in microbes. Betanin bioproduction in Saccharomyces cerevisiae with an initial titer of 17 mg/L was achieved by introducing betanin biosynthetic pathway from plants .…”