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

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.A cDNA clone for (S)-tetrahydroberberine oxidase was isolated from an EST library prepared from high berberineproducing 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.

Identification and Characterization of Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in Coptis Species

“…Thus, several genes encoding proteins associated with berberine biosynthesis have been identified and characterized. These include the berberine bridge enzyme (BBE; Bird et al, 2003 ; Minami et al, 2008 ), which converts ( S )-reticuline to berberine, SOMT ( Takeshita et al, 1995 ), ( S )-canadine synthase (CAS; Ikezawa et al, 2003 ), and ( S )-tetrahydroprotoberberine oxidase (STOX; Matsushima et al, 2012 ; Sato, 2013 ). However, the pathway of epiberberine, which is closely related to berberine in structure, has not been determined.…”

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…In particular, berberine is formed from (S)-canadine by the FAD-linked enzyme (S)-tetrahydroprotoberberine oxidase (STOX) (Fig. 1) (Amann et al 1984;Gesell et al 2011;Matsushima et al 2012). However, berberine and other modified protoberberines have generally not been reported in opium poppy (Shamma and Moniot 1978;Shulgin and Perry 2002).…”

Microbial production of isoquinoline alkaloids as plant secondary metabolites based on metabolic engineering research

“…As a consequence of the difficulties encountered during the attempted production of active recombinant CjTHBO in E. coli and Saccharomyces cerevisiae cells, recombinant CjTHBO was heterologously expressed in E. californica cells and its metabolic activities subsequently characterized. 26) …”