Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering.
Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol) for the treatment of many cancers. To develop an optimal bioprocess for paclitaxel supply, taxane biosynthetic pathway regulation must be better understood. Here we examine the expression profile of paclitaxel biosynthetic pathway genes by RNA gel blot analysis and RT-PCR in the Taxus cuspidata cell line P991 and compare with taxane metabolite levels. Upon methyl jasmonate (MJ) elicitation (100 microM), paclitaxel accumulates to 3.3 mg/L and cephalomannine to 2.2 mg/L 7 days after elicitation but neither are observed before this time. 10-deacetylbaccatin III accumulates to 3.3 mg/L and baccatin III to 1.2 mg/L by day 7 after elicitation. The early pathway enzyme genes GGPPS, TASY, and T5alphaH are up-regulated by MJ elicitation within 6 h and continue through 24 h before their abundances decrease. This study reveals the preference for one side of the biosynthetic pathway branch in early taxane synthesis, where transcripts coding for TalphaH are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch (TDAT and T10betaH) are not highly expressed following elicitation. Transcripts encoding the enzymes DBBT and DBAT are up-regulated upon MJ elicitation. Their products, 10-deacetylbaccatin III and baccatin III, respectively, accumulate within 6 h of the initial increase in transcript abundance. Importantly, the steady-state levels of the two terminal enzyme transcripts (BAPT and DBTNBT) are much lower than transcripts of early pathway steps. These are potential steps in the pathway for targeted metabolic engineering to increase accumulation of paclitaxel in suspension cell culture.
Nims
Ezekiel
E
Plant Biology Graduate Program, University of Massachusetts, North Pleasant Street, Amherst, 01003, USA.
Dubois
Camille P
CP
Roberts
Susan C
SC
Walker
Elsbeth L
EL
D002478:Cells, Cultured; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods*; D017239:Paclitaxel / Q000096:biosynthesis*; D010940:Plant Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*
D010940:Plant Proteins; D020543:Proteome; D017239:Paclitaxel