Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase

Abstract

In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

Comparative metabolomics reveals the metabolic variations between two endangered Taxus species (T. fuana and T. yunnanensis) in the Himalayas

“…Then, several intermediate enzymatic reactions, such as hydroxylation, acetylation and N -benzoylation, occur in the pathway [ 2 ]. For example, the acetylation of 10-deacetylbaccatin III (10-DAB) is an essential step to produce baccatin III, which is an advanced precursor for Taxol biosynthesis [ 8 , 9 ]. Thereafter, the C 13 -side chain is appended to baccatin III to form N -debenzoyl-2′-deoxytaxol [ 10 , 11 ].…”

Comparative metabolomic analysis reveals the variations in taxoids and flavonoids among three Taxus species

“…Taxol (generic name paclitaxel) is the major bioactive component of the Taxus species widely used in the treatment of various cancers, such as ovarian cancer, breast cancer and squamous cancers [1]. Since its approval for ovarian cancer treatment in 1992, the demand for paclitaxel and its derivatives has increased [2]. Several barriers, including low content of taxoids, exhausted natural resources and high loss rate of purification, limited the increasing of paclitaxel supply.…”

“…Then, a series of hydroxylation, acetylation and N -benzoylation of taxane skeleton produce a number of intermediates in the pathway towards paclitaxel. For example, acetylation of 10-deacetylbaccatin-III (10-DAB) produces baccatin III, an advanced intermediate for paclitaxel biosynthesis [2, 12]. The assembly of C13-side chain appended to baccatin III (BAC) to form N -debenzoyl-2′-deoxytaxol is considered as the final step of the paclitaxel biosynthesis pathway [13].…”

Comparative metabolomic analysis reveals the variations in taxoids and flavonoids among three Taxus species

“…Taxol (generic name paclitaxel) is the major bioactive component of the Taxus species widely used in the treatment of various cancers, such as ovarian cancer, breast cancer and squamous cancers [ 1]. Since its approval for ovarian cancer treatment prescription in 1992, the demand for paclitaxel and its derivatives have increased [ 2]. Several barriers, including low content of taxoids, exhausted natural resources and high loss rate of purification, limited the increasing of paclitaxel supply [ 3].…”

“…Then, a series of hydroxylation, acetylation and N-benzoylation of taxane skeleton produce a number of intermediates in the pathway towards paclitaxel. For example, acetylation of 10deacetylbaccatin-III (10-DAB III) produces baccatin III, an advanced intermediate for paclitaxel biosynthesis [ 2,13]. The assembly of C13-side chain appended to baccatin III (BAC) to form N-debenzoyl-2'-deoxytaxol is considered as the final step of the paclitaxel biosynthesis pathway [ 14].…”