Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate

ACS Synth Biol. 2017 Feb 17;6(2):201-205. doi: 10.1021/acssynbio.6b00206. Epub 2016 Nov 4.

Abstract

Attempts at microbial production of the chemotherapeutic agent Taxol (paclitaxel) have met with limited success, due largely to a pathway bottleneck resulting from poor product selectivity of the first hydroxylation step, catalyzed by taxadien-5a-hydroxylase (CYP725A4). Here, we systematically investigate three methodologies, terpene cyclase engineering, P450 engineering, and hydrolase-enzyme screening to overcome this early pathway selectivity bottleneck. We demonstrate that engineering of Taxadiene Synthase, upstream of the promiscuous oxidation step, acts as a practical method for selectivity improvement. Through mutagenesis we achieve a 2.4-fold improvement in yield and selectivity for an alternative cyclization product, taxa-4(20)-11(12)-diene; and for the Taxol precursor taxadien-5α-ol, when coexpressed with CYP725A4. This works lays the foundation for the elucidation, engineering, and improved production of Taxol and early Taxol precursors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalysis
  • Cytochrome P-450 Enzyme System / metabolism
  • Hydroxylation / genetics
  • Isomerases / genetics*
  • Isomerases / metabolism*
  • Oxidation-Reduction
  • Paclitaxel / metabolism*

Substances

  • Cytochrome P-450 Enzyme System
  • Isomerases
  • taxa-4(5),11(12)-diene synthase
  • Paclitaxel