Figure 3.

Outline of metabolic engineering in transgenic E. californica cells. (A) Overexpression of the rate-limiting enzyme, norcoclaurine 6-O-methyltransferase (6OMT), using C. japonica cDNA in California poppy cells led to an increase in the accumulation of the main alkaloid of the cells, sanguinarine, as shown by the upward arrow. (B) Introduction of a branch pathway with scoulerine 9-O-methyltransferase (SMT) also induced a marked shift in the metabolic flow, as shown by the arrows. SMT overexpression led to a significant reduction in the accumulation of sanguinarine-type alkaloids with two methylenedioxy ring structures, but induced the production of protoberberine-type alkaloids, such as berberine and columbamine, and of allocryptopine- and chelerythrine-type alkaloids with one methylenedioxy ring and two methoxy groups. (C) The shutdown of the berberine bridge enzyme (BBE) expression with an RNAi vector clearly downregulated the accumulation of sanguinarine and the intermediate scoulerine, as shown by the downward arrow. In contrast, RNAi of BBE induced a marked accumulation of reticuline, which is a substrate of BBE, as shown by the upward arrow.