Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/2002
Publication Date: 11/1/2002
Citation: BRINCAT, C., GIBSON, D.M., SHULER, M. ALTERATIONS IN TAXOL PRODUCTION IN PLANT CELL CULTURE VIA MANIPULATION OF THE PHENYLALANINE AMMONIA LYASE PATHWAY. BIOTECHNOLOGY PROGRESS. 2002. v. 18. p. 1149-1156. Interpretive Summary: Paclitaxel (Taxol) is a potent chemotherapeutic drug with proven utility against a range of cancers. The very limited supply of this drug from the original source, Pacific yew bark, however, prompted the development of alternative sources of production, including the use of plant cell cultures. These cultures could be an alternative source of material if production can be optimized, as well as a useful model for understanding the pathway for taxol production. In this study, we used enzyme inhibitors to block a competing pathway for one of the compounds needed in the biosynthesis of taxol. We found that the inhibitors also reduced taxol production, but increased the production of a number of other related taxanes not containing an important component necessary for activity. This information may be useful in determining what compounds might be intermediates in the biosynthetic pathway for taxol.
Technical Abstract: One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from extraneous secondary pathways. Since both the phenylisoserine side chain and the benzoyl moiety at C-2 of taxol are derived from phenylalanine, we speculated that blockage of the competing phenylpropanoid pathway might divert this intermediate into taxol biosynthesis. We targeted the critical control point for conversion of L-phenylalanine to trans-cinnamic acid by the first enzyme in the pathway, phenylalanine ammonia lyase (PAL), through the use of specific enzyme inhibitors. Cinnamic acid acted quickly in reducing PAL activity by 40 -50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.2 0 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor, -aminooxyacetic acid (AOA), almost entirely shut down Taxol production at both 0.5 and 1.5 mM when added in conjunction with 100 µM methyl jasmonate in both cell lines. L- -aminooxy- -phenylpropionic acid (AOPP) appeared to have the opposite effect of AOA, slightly enhancing Taxol production at 1 µM, but having no effect at 10 µM. The discrepancy in the effectiveness of these two compounds, AOA and AOPP, and the lack of effect with addition of phenylalanine or benzoic acid derivatives, further indicates that the impact of cinnamic acid on Taxol is unrelated to its effect on PAL, but rather to a specific effect on the taxane pathway. Based on these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.