Biosynthesis of taxol, an anti-cancer drug, commences with cyclization of geranylgeranyl pyrophosphate to produce taxadiene. Taxadiene should then undergo a 5a-hydroxylation to form taxadien-5a-ol. However, the cytochrome P450 monooxygenase that catalyzes this reaction also synthesizes a by-product called oxacyclotaxane (OCT). Moreover, the two products are distributed in roughly stoichiometrically equal quantities. This suggests that taxadiene 5a-hydroxylase, and perhaps other enzymes of the taxane pathway are possibly 'error-prone' and promiscuous. OCT production irreversibly cuts the metabolic flux to taxol in half and the error propensity of the downstream enzymes could curtail taxol production even further. Improving product selectivity and reducing substrate promiscuity of these enzymes is vital to enhancing taxol production in E. coli. Accordingly, we have attempted to tune the selectivity of taxadiene 5a-hydroxylase towards taxadien-5a-ol via model-guided enzyme engineering. By reconciling experimental observations with model predictions, we hope to propose a generalized scheme for model-guided taxane enzyme engineering.

Tuning the selectivity of a Taxus cytochrome P450 monooxygenase

243rd National Meeting (2012)

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