Pathway engineering of benzylisoquinoline alkaloid biosynthesis in transgenic California poppy cells with ectopic expression of tetrahydroberberine oxidase from <i>Coptis japonica</i>

• Matsushima Yasutaka

  Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University

• Minami Hiromichi

  Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University

• Hori Kentaro

  Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University

• Sato Fumihiko

  Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University

Bibliographic Information

Other Title

• Pathway engineering of benzylisoquinoline alkaloid biosynthesis in transgenic California poppy cells with ectopic expression of tetrahydroberberine oxidase from Coptis japonica
• Pathway engineering of benzylisoquinoline alkaloid biosynthesis in transgenic California ppopy cells with ectopic expression of tetrahydroberberine oxidase from Coptis japonica.

Abstract

(S)-Tetrahydroberberine oxidase is the enzyme in the last step of berberine biosynthesis. While a previous report described the isolation of cDNA of tetrahydroberberine oxidase (THBO) from cultured Coptis japonica cells, we later found that purified THBO was heavily contaminated by triosephosphate isomerase. Here, we report the re-isolation of THBO cDNA from cultured C. japonica cells and its functional characterization in transgenic California poppy cells.<br>A cDNA clone for (S)-tetrahydroberberine oxidase was isolated from an EST library prepared from high berberine-producing cultured C. japonica cells based on the partial amino acid sequence of the purified enzyme. Analyses of the nucleotide sequences of the cloned cDNA inserts of 1728 base pairs revealed an open reading frame that encoded a 540-amino acid polypeptide with putative 28-amino acid signal peptides and a mature polypeptide with a molecular mass of 57,748. A protein blast search also shows that CjTHBO belongs to the FAD-containing berberine bridge enzyme oxidoreductase family. Since all attempts to produce active recombinant CjTHBO in Escherichia coli and Saccharomyces cerevisiae cells failed, we tried to express CjTHBO in California poppy (Eschscholzia californica) cells. When transgenic California poppy cells that ectopically expressed CjTHBO under the control of Cauliflower mosaic virus 35S promoter were established, the transgenic cells showed small but evident new alkaloid peaks, which were scarcely detected in control cells that did not express CjTHBO. LC-MS analyses showed that these peaks were coptisine and dehydrocheilanthifoline, which were expected to be generated by the reaction of CjTHBO from pathway intermediates, i.e., cheilanthifoline and stylopine. The usefulness of CjTHBO for metabolic engineering in transgenic California poppy cells is discussed.

Journal

• Plant Biotechnology

  Plant Biotechnology 29 (5), 473-481, 2012

  Japanese Society for Plant Biotechnology

Keywords

• Benzophananthridine alkaloid biosynthesis
• California poppy
• <i>Coptis japonica</i>
• (<i>S</i>)-tetrahydroberberine oxidase.

Details

• CRID: 1390282679305893888
• NII Article ID: 130002500899
• NII Article ID: 40019545301
• NII Book ID: AA11250821
• DOI: 10.5511/plantbiotechnology.12.1101a
• ISSN: 13476114
• ISSN: 13424580
• NDL BIB ID: 024195583
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I024195583
• Web Site: https://www.jstage.jst.go.jp/article/plantbiotechnology/29/5/29_12.1101a/_pdf
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed