Reconstitution of Early Paclitaxel Biosynthetic Network

Abstract

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. This diverts metabolic flux away from the paclitaxel precursor, taxadien-5α-ol, thus previous attempts of reconstitution have not yielded sufficient material for characterization, regardless of the heterologous host. Here, we structurally characterized four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression, levels of these proposed byproducts decrease with a concomitant increase in the accumulation of taxadien-5α-ol by four-fold. This engineered system enabled the reconstitution of a six step biosynthetic pathway to produce isolatable 5α,10β-diacetoxy-taxadien-13α-ol. Furthermore, we showed that this pathway may function as a metabolic network rather than a linear pathway. The engineering of the paclitaxel biosynthetic network demonstrates thatTaxusgenes can coordinatively function for the biosynthetic production of key early stage paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes.

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

“…The discovery also highlights that catalytic promiscuity is somewhat common in the cytochrome P450s and acyltransferases of baccatin III biosynthesis. Previous studies have demonstrated that T5aH can catalyze the formation of many unknown oxygenated products other than taxadien-5a-ol (59)(60)(61), and recently it was revealed that this enzyme even has weak cyclase activity leading to the oxetane structure (62), which indicates its functional promiscuity. T13aH could catalyze C13 hydroxylation with taxadien-5a-ol and taxadien-5a-yl acetate as substrates, thereby exhibiting a substrate flexibility similar to that of cytochrome P450s in paclitaxel biosynthesis (24).…”