Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Abstract

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.

Correlation analysis of the taxane core functional group modification, enzyme expression, and metabolite accumulation profiles under methyl jasmonate treatment

“…The poor substrate specificity among the P450 enzymes and the substrate dependence of acyl or acetyl transferases rather than a single “path” approach in the second phase suggest the complexity of the existing reaction network. In the network, each oxygenase is designed to hydroxylate the taxane core at a specific position, and the hydroxys are further promiscuously acylated (including acetylate) . Each hydroxylated and subsequently acylated product can function as a substrate of varying efficiencies for each P450 and acyltransferase, resulting in the formation of diverse taxane fluxes, many of which find their way to DAB, B‐III, taxol, and TC.…”

“…In the network, each oxygenase is designed to hydroxylate the taxane core at a specific position, and the hydroxys are further promiscuously acylated (including acetylate). 11 Each hydroxylated and subsequently acylated product can function as a substrate of varying efficiencies for each P450 and acyltransferase, resulting in the formation of diverse taxane fluxes, many of which find their way to DAB, B-III, taxol, and TC. DAB and B-III have the same core as taxol and could be used as precursors for taxol semisynthesis.…”

Transcriptome Analysis ofTaxus cuspidataNeedles Based on 454 Pyrosequencing

“…Meanwhile, Taxol biosynthesis competes with side routes produced by acyl/aroyl or the oxidation of the taxane nucleus derived from common precursors. Identification of these side-route genes could have an important implication in eventually increasing Taxol yields [26]. For this purpose, we screened our 454 datasets and discovered candidate genes of CYP450, epoxidase, CoA ligases, and N-ben- zoyltransferase ( Table 3S, Supporting Information).…”

De novo leaf and root transcriptome analysis to explore biosynthetic pathway of Celangulin V in Celastrus angulatus maxim

“…For example, two BAHD alcohol acetyltransferases were identified from L. x intermedia glandular trichome database, which convert a variety of monoterpenes to monoterpene esters using coenzyme A as a cofactor [67]. Several taxoid-O-acetyl transferases (like TAX 9 and TAX 14), obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones, could catalyze some acylation steps of Taxol biosynthesis [68, 69]. We obtained four unigenes that were predicted to be the BAHD acyltransferase from transcriptome data, probably participating in biosynthesis of Celangulin V, according to acylated groups on C5, C6, C8, C9 and C14 positions of β-dihydroagarofuran skeleton.…”