Dataset Information

Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.

ABSTRACT: The cDNA clone for a 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the Taxol biosynthetic pathway, has been isolated from Taxus cuspidata. By using consensus sequences from an assembly of transacylases of plant origin and from many deduced proteins of unknown function, a homology-based PCR cloning strategy was employed to amplify initially a 911-bp gene fragment of the putative taxane C-10 hydroxyl acetyl transferase from Taxus. This amplicon was used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which the full-length 10-deacetylbaccatin III-10-O-transacetylase sequence was obtained. Expression of the ORF from pCWori(+) in Escherichia coli JM109 afforded a functional enzyme, as determined by (1)H-NMR and MS verification of the product baccatin III derived from 10-deacetylbaccatin III and acetyl CoA. The full-length cDNA has an ORF of 1,320 bp corresponding to a deduced protein of 440 residues with a calculated molecular weight of 49,052, consistent with the size of the operationally soluble, monomeric, native acetyl transferase. The recombinant acetyl transferase has a pH optimum of 7.5, has K(m) values of 10 microM and 8 microM for 10-deacetylbaccatin III and acetyl CoA, respectively, and is apparently regiospecific toward the 10-hydroxyl group of the taxane ring. Amino acid sequence comparison of 10-deacetylbaccatin III-10-O-acetyl transferase with taxadienol-5-O-acetyl transferase and with other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (80% and 64-67%, respectively).

SUBMITTER: Walker K

PROVIDER: S-EPMC15373 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.

Walker K K Croteau R R

Proceedings of the National Academy of Sciences of the United States of America 20000101 2

The cDNA clone for a 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the Taxol biosynthetic pathway, has been isolated from Taxus cuspidata. By using consensus sequences from an assembly of transacylases of plant origin and from many deduced proteins of unknown function, a homology-based PCR cloning strategy was employed to amplify initially a 911-bp gene fragment of the putative taxane C-10 hydroxyl acetyl transferase from Taxus. ...[more]

PMID: 10639122

Similar Datasets

Project description:Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinases but is dependent on XthA, a 3' to 5' exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as in vivoE. coli cloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3' ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5' overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCE Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an in vitro recombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, E. coli potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this in vivo cloning have realized a high level of usability, comparable to that by in vitro recombination reactions. However, the mechanism of in vivo cloning is highly controversial. Here, we clarified the fundamental mechanism underlying in vivo cloning and also constructed a strain that was optimized for in vivo cloning. Additionally, we streamlined the procedure of in vivo cloning by using a single microcentrifuge tube.

Dataset Information

Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.

Publications

Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets