Molecular Dissection of Metabolic Channels for Sterol and Sesquiterpene Metabolism in Tobacco

类异戊二烯在自然界中普遍存在，对生命至关重要， 介导生物体与它们之间的许多相互作用。 环境 此外，植物是一个丰富的来源， 药学上重要的类异戊二烯。 然而，我们对 植物中的类异戊二烯生物合成途径是有限的。 最近 研究表明，烟草细胞悬浮培养是唯一的 适合于详细的生理和生化研究， 类异戊二烯代谢中重要但推定的分支点。 当 当真菌诱导子加入细胞培养物中时， 甾醇生产并合成和分泌抗微生物 倍半萜 甾醇生物合成的下降一直是 与角鲨烯合成酶活性的抑制相关， 以及通过诱导倍半萜类化合物的生物合成， 一种倍半萜环化酶因为这两种酶 位于途径中假定的分支点， 一种酶和抑制另一种酶被解释为 一个控制碳流的重要调节机制， 从而形成最终产品。 这种解释表明， 类异戊二烯代谢发生在均质环境中 中间体自由混合， 连续或竞争酶。 最近的结果表明， 并表明独立调节的同工酶阵列 专门用于生产特定的最终产品， 沿着表面或膜沿着。这种离散的假设 代谢通道预测基因编码的选择性转录 对于激发子处理的细胞中倍半萜生物合成的酶 与控制细胞中固醇代谢的基因相比， 酶蛋白质必须具有靶向信息以插入到 正确的代谢通道或单位。 这项工作的目标是 用分子方法来检验这些预测， 克隆倍半萜环化酶和角鲨烯合成酶基因， 分析指导其表达的顺式元件，以及融合 将它们假定的靶向信号转移到报告基因上， 改变所形成的类异戊二烯的种类或量。 结果 这些研究将有助于阐明调节 类异戊二烯在植物中的代谢，并将产生重要影响， 为将来利用基因工程操纵这一途径 技术。//

Isoprenoids are ubiquitous in nature, essential for life, and mediate a number of interactions between organisms and their environment. In addition, plants are a rich source for pharmaceutically important isoprenoids. Yet, our understanding of the isoprenoid biosynthetic pathway in plants is limited. Recent studies indicate that tobacco cell suspension cultures are uniquely suited for detailed physiological and biochemical studies of one important but putative branch point in isoprenoid metabolism. When fungal elicitors are added to the cell cultures, the cultures cease sterol production and instead synthesize and secrete anti-microbial sesquiterpenoids. The decline in sterol biosynthesis has been correlated with suppression of squalene synthetase enzyme activity, and the induction of sesquiterpenoid biosynthesis wit the induction of a sesquiterpene cyclase. Because these two enzymes are positioned at a putative branch point in the pathway, the induction of one enzyme and the suppression of the other were interpreted as an important regulatory mechanism controlling carbon flow and hence, end product formation. This interpretation suggested that isoprenoid metabolism was occurring in a homogeneous environment with intermediates mixing freely and accessible to other to successive or competing enzymes. Recent results suggest otherwise and indicate that independently regulated arrays of isozymes dedicated to the production of specific end products might be aligned along a surface or membrane.. This hypothesis of discrete metabolic channels predicts selective transcription of genes coding for enzymes of sesquiterpene biosynthesis in elicitor-treated cells versus genes for sterol metabolism in control cells, and that the enzyme proteins must have targeting information for insertion into the correct metabolic channel or unit. The goals of this work are to test these predictions using a molecular approach including cloning of the sesquiterpene cyclase and squalene synthetase genes, analysis of the cis-elements directing their expression, and fusion of their putative targeting signal onto a reporter gene which will either alter the kind or amount of isoprenoid formed. Results from these studies will help elucidate the mechanisms regulating isoprenoid metabolism in plants, and will have important impact on for future manipulations of this pathway using genetic engineering technology.//