Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis

Abstract

Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-␣-phenylalanine to 3R-␤-phenylalanine by phenylalanine aminomutase (PAM). A gene cloning approach was based on the assumption that PAM would resemble the well known plant enzyme phenylalanine ammonia lyase. A phenylalanine ammonia lyase-like sequence acquired from a Taxus cuspidata cDNA library was expressed functionally in Escherichia coli and confirmed as the target aminomutase that is virtually identical to the recombinant enzyme and clone from Taxus The final stages of Taxol biosynthesis (see Fig. 1A) in yew species involve the assembly and attachment to C-13 of the taxane core of the N-benzoyl phenylisoserinoyl side chain, which is an important pharmacophoric descriptor of this anticancer drug (1, 2). In the current practice of Taxol production, this side chain is attached by chemical semisynthesis to baccatin III, which is derived from 10-deacetylbaccatin III, a Taxus (yew) metabolite that is much more readily available than Taxol itself (3-5). In the biosynthetic pathway, five steps are involved in the construction of the side chain. The first step is considered to be the conversion of 2S-␣-phenylalanine to 3R-␤-phenylalanine by an aminomutase that catalyzes an intramolecular migration of the amino group and a partial internal transfer of the pro-3S hydrogen (6, 7). This step is seemingly followed by the ligase-mediated activation to the corresponding CoA ester and then the transfer of ␤-phenylalanoyl to the C-13 hydroxyl of baccatin III. The resulting intermediate (designated ␤-phenylalanoyl baccatin III or N-debenzoyl-2Ј-deoxytaxol) then likely undergoes cytochrome P450-mediated hydroxylation at the side chain 2Ј-position to generate the isoserinoyl moiety and final N-benzoylation of this side chain (8) to complete the biosynthesis of Taxol. cDNAs encoding the two transferases involved in C-13 side chain assembly have been described previously (8,9).The relative abundance of ␤-phenylalanine and side chaindeficient late pathway metabolites such as baccatin III and 10-deacetylbaccatin III in vivo (10) suggests that either the CoA ligase for ␤-phenylalanine or the CoA ester-dependent ␤-phenylalanoyltransferase (9), both of which function downstream of the aminomutase, may be rate limiting in side chain assembly and, thus, in Taxol biosynthesis. Because the phenylalanine aminomutase (PAM) 1 catalyzes the first step of the side chain assembly process and shares its primary metabolite substrate, phenylalanine, with several competing, non-taxoid phenylpropanoid pathway enzymes in plants (11-13), it is therefore an important target for genetic engineering in yew or derived cell cultures to increase Taxol production yields. PAM is also of interest enzymologically because the reaction that is catalyzed is unusual, and, in addition to the adenosylcobalamin-dependent leucine 2,3-aminomutase from Andrographis paniculata and potato tubers (14 -16), it is the only other aminomutase of plant origin d...

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].…”

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).…”

New Approach To Improve Taxol Biosynthetic

“…Identification of taxol production biochemical pathways, gene over-expression and gene silencing may be able to remove a vast amount of the problems associated with its production. It seems essential to inquiry on genes involved in the biosynthesis of taxol, cloning and genetic manipulation of plants for improving the yield Taxol [10,11].…”