Application of plant and bacterial enzymes in the biocatalysis of paclitaxel (110.3)

Despite robust production of paclitaxel by Phyton Biotech, the FDA reported (01/01/12 ‐ 07/31/12, www.fda.gov , May 2012) that unreliable manufacturing and patient demand created a paclitaxel (Taxol) shortage, causing delays in cancer treatment. Phyton uses Taxus plant cells to produce paclitaxel via minimally 19 intermediates. These cells also produce ~3 times more paclitaxel precursors, baccatin III and 10‐deacetylbaccatin III combined, than paclitaxel. We show preliminary data on an alternative in vitro biosynthetic method to convert the baccatins to paclitaxel. This 4 to 5‐step biocatalytic route employs only 2‐3 Taxus acyltransferases (a phenylpropanoyl‐ and benzoyltransferase) and two CoA ligases (a peptidyl synthetase adenylation module and a benzoyl CoA ligase) co‐opted from bacteria. This simplified pathway is shorter than the previously used 17‐step semisynthesis to make paclitaxel, also beginning from baccatin III. In addition, we can biosynthesize efficacious paclitaxel analogs, and the pathway intermediates are easier to purify than from complex mixtures in plant cells. The Taxus phenylpropanoyltransferase on the pathway uses( R )‐β‐phenylalanyl CoA as a substrate. A plant aminomutase converts ( S )‐α‐phenylalanine to ( R )‐β‐Phenylalanine by rotating the reaction intermediate 180°, while an isozyme from bacteria makes the ( S )‐enantiomer without rotating the intermediate.