Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis

Abstract

The structural pharmacophore of Taxol, responsible for binding the N terminus of the ␤-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

“…Genes encoding the phenylalanine aminomutase [18] and the two responsible side chain transferases [19,20] have been isolated. In the case of the transfer reactions, functional screening of a family of previously acquired Taxus acyl/aroyl transferase cDNAs [13] yielded the C13 phenylpropanoid side chain-CoA acyl transferase that mediates the 13-O-esterification of baccatin III [20], and the benzoyl CoA-dependent transferase responsible for the final benzamidation step of the Taxol pathway [19].…”

“…In the case of the transfer reactions, functional screening of a family of previously acquired Taxus acyl/aroyl transferase cDNAs [13] yielded the C13 phenylpropanoid side chain-CoA acyl transferase that mediates the 13-O-esterification of baccatin III [20], and the benzoyl CoA-dependent transferase responsible for the final benzamidation step of the Taxol pathway [19]. The lack of absolute substrate specificity by the recombinant transferases led to some ambiguities with regard to the precise timing of hydroxylation and acyl transfer steps in side chain assembly [14] that were only recently resolved by demonstration that the cytochrome P450 2′-hydroxylase operates only on Ndebenzoyl-2′-deoxytaxol (5), thus implicating the construction sequence of O-esterification, followed by 2′-hydroxylation, followed by N-benzamidation [17].…”

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

“…Four of these six initial isolation studies were conducted with native Taxus plant sources providing the enzymes of interest. A concerted attempt was then made to generate cDNA libraries from Taxus cell cultures and to begin identifying and confirming subsequent pathway genes Huang et al 2001;Jennewein et al 2001Jennewein et al , 2004bLong and Croteau 2005;Schoendorf et al 2001;Walker and Croteau 2000a, b;Walker et al 2002aWalker et al , b, 2000Walker et al , 2004.…”

“…Generally, Croteau's group favored S. cerevisiae for P450 heterologous expression (Jennewein et al 2003(Jennewein et al , 2004aSchoendorf et al 2001;, most likely owing to the similarities to the native plant host. E. coli was prominently featured as an expression host with other pathway genes (Huang et al 1998;Walker and Croteau 2000a, b;Walker et al 2002aWalker et al , b, 2000Walker et al , 2004. Each of these heterologous expression attempts set the stage for heterologous biosynthetic production.…”

“…Considering that the current state-of-the-art is the production of early stage Taxol intermediates through heterologous hosts, this discussion will summarize success to date and the steps needed to realize and capitalize upon full pathway reconstitution. Hefner et al 1998), taxadiene synthase (Wildung and Croteau 1996;Koepp et al 1995;Huang et al 2001), taxadiene 5α-hydroxylase (Jennewein et al 2004a), taxane 2α-Obenzoyltransferase (TBT; Walker and Croteau 2000b), 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT; Walker and Croteau 2000a), baccatin III:3-amino-3-phenylpropanoyltransferase (BAPT; Walker et al 2002a), and phenylalanine aminomutase (PAM; Walker et al 2004). Other enzymes (taxadien-5α-ol-O-acetyl transferase, taxoid 10β-hydroxylase, taxoid 2α-hydroxylase, taxoid 9α-hydroxylase, taxane 13α-hydroxylase, taxoid 7β-hydroxylase, and 3′-N-debenzoyl-2′-deoxytaxol N-benzyoyltransferase) have been identified and/or characterized but not conclusively assigned a location in the overall pathway order (Walker et al 2000;Schoendorf et al 2001;Croteau et al 2006;Jennewein et al 2001;Walker et al 2002b).…”