Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering
Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering
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DOI：
10.1016/j.ymben.2006.04.001
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发表时间：
2006-09-01
影响因子：
8.4
通讯作者：
Walker, Elsbeth L.
中科院分区：
文献类型：
--
作者：
Nims, Ezekiel;Dubois, Camille P.;Walker, Elsbeth L.
红豆杉植物悬浮细胞培养物提供了用于治疗许多癌症的紫杉醇（Taxol（R））的可持续来源。为了开发最佳的紫杉醇生物合成过程，必须更好地了解紫杉烷生物合成途径的调控。本研究通过RNA凝胶印迹分析和RT-PCR检测紫杉醇生物合成途径基因在东北红豆杉细胞系P991中的表达谱，并与紫杉烷代谢产物水平进行比较。茉莉酸甲酯（MJ）诱导（100 μ M），紫杉醇积累到3.3毫克/升和三尖杉宁碱2.2毫克/升诱导后7天，但都没有观察到在此之前的时间。10-诱导后第7天，脱乙酰巴卡亭III积累到3.3 mg/L，巴卡亭III积累到1.2 mg/L。早期途径酶基因GGPPS、TASY和T5 α H在6小时内被MJ诱导上调，并在其丰度降低之前持续24小时。这项研究揭示了在早期紫杉烷合成中对生物合成途径分支的一侧的偏好，其中编码T α H的转录本在用MJ诱导后是丰富的，但编码交替分支的两种酶（TDAT和T10 β H）的转录本在诱导后不高度表达。转录本编码的酶DBBT和DBAT的MJ诱导上调。他们的产品，10-脱乙酰浆果赤霉素III和浆果赤霉素III，分别在6小时内的转录丰度的初始增加积累。重要的是，两个末端酶转录本（BAPT和DBTNBT）的稳态水平远低于早期途径步骤的转录本。这些是靶向代谢工程途径中的潜在步骤，以增加紫杉醇在悬浮细胞培养物中的积累。(C)2006年爱思唯尔公司All rights reserved.
Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol(R)) 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 mu M), 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 T5 alpha H are upregulated 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 T alpha H are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch (TDAT and T10 beta H) 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. (C) 2006 Elsevier Inc. All rights reserved.