Characterization of Early Taxol Biosynthetic Pathway

Taxol, a microtubule stabilizer derived from conifer Taxus species, is one of the most widely used anticancer drugs approved for treating multiple cancers. Current production methods include plant cell suspension culture and semi-synthesis, but both suffer from low yield, high production cost, and non-sustainability. Heterologous production of hard-to-source medicinal plant natural products in microorganisms has been shown to be successful in many cases. However, complete elucidation of the Taxol biosynthetic pathway would be a prerequisite for heterologous production. Despite decades of study on Taxol biosynthesis, the catalytic promiscuity of early biosynthetic enzymes, specifically the cytochrome P450 taxadiene 5α-hydroxylase (T5αH) that is attributed as the second enzyme on the pathway, has become a bottleneck in elucidating Taxol biosynthesis. When heterologously expressed in different organisms, T5αH has been reported to produce multiple oxidative products instead of a single product taxadien-5α-ol as expected. To understand if this catalytic promiscuity is inherent or due to extrinsic factors, we purified major products of T5αH when expressed in either Nicotiana benthamiana or Saccharomyces cerevisiae and elucidated their structures through extensive 2D NMR. We found that T5αH can perform up to three oxidations and at unique positions on taxadiene scaffold. The novel oxidation patterns observed in both N. benthamiana and S. cerevisiae suggests that this promiscuous catalytic behavior is likely inherent of T5αH. In addition, we found further conversion of some of these scaffolds into tailored products when co-expressed with potential Taxol biosynthetic genes. Some moieties of these structures resemble other taxanes discovered in Taxus, indicating a possible involvement of these enzymes to other taxanes. The diverse oxidative product profile paves the road for rational engineering of T5αH and suggests the presence of a complex metabolic network in Taxol biosynthesis.