Poster: Metabolic engineering

Abs # 299: Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

Presenter:	Hong, Seung-Beom , dsbhong@yahoo.com
Authors	Hong, Seung-Beom  (A)   Peebles, Christie  (B)   San, Ka-Yiu  (B)   Shanks, Jacqueline  (C)   Gibson, Susan  (D)
Affiliations:	(A): Konkuk University (B): Rice University (C): Iowa State University (D): University of Minnesota

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, tryptamine is a building block for the biosynthesis of terpenoid indole alkaloids. Previously we manipulated the indole pathway by introducing an Arabidopsis feedback insensitive anthranilate synthase (AS) subunit (trp5) cDNA and a C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots. Inducible expression of one or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In order to more successfully engineer the indole pathway, wild-type Arabidopsis AS subunit (ASB1) cDNA was constitutively expressed and trp5 and TDC were inducibly expressed in C. roseus hairy roots. As compared with the line expressing only trp5, AS from lines expressing both trp5 and ASB1 exhibited 50% inhibition of activity at more than four-fold higher concentration of tryptophan. Nevertheless, the tryptophan levels in both of the transgenic lines are similar to each other upon 3-day induction. However, lines expressing both trp5 and ASB1 yield dramatically larger increases (up to 19 fold) in tryptamine levels than lines expressing only trp5 with a 4-fold tryptamine increase. Endogenous TDC activities of both the trp5 ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, tryptamine in the induced lines accumulated up to 30-fold higher than that in the uninduced lines. In parallel with the remarkable accumulation of tryptamine upon induction, some terpenoid alkaloid levels were significantly increased.