Heterologous Production of Eleutherobin Precursors

描述(申请人提供)：萜类化合物是一类不同的天然化合物，具有同样多样和强大的生物活性。由于萜类化合物的实际用途取决于提取或合成这些化合物的能力，因此只有最丰富或最容易合成的化合物才被商业化开发。这个小企业技术转移第一阶段项目的长期目标是通过以经济的方式生产原本稀缺的萜类化合物来促进新的抗癌化合物的临床测试和使用。Eleutherobin和Sarcodictyin A和B是具有细胞毒性的萜类化合物，其作用方式类似于非常成功的抗癌药物紫杉醇。然而，供应的限制阻碍了这些令人兴奋的化合物的开发。这项第一阶段的研究将使用创新的方法来鉴定、克隆和表达负责形成Eleutherobin碳骨架的萜烯环酶。通过检测环化酶的活性，我们将分离环化酶并对其进行部分测序。然后，这些部分序列将被用于设计PCR探针，以克隆全长的环化酶基因。环化酶的序列将作为发现完整的生物合成途径的起点。由于海洋动物产生的许多化合物是微生物共生体的产物，环化酶基因可以识别与我们感兴趣的化合物相关的生物合成基因簇。在细菌中发现并表达出过量产生萜类前体的生物合成途径将使大规模发酵生产有价值的萜类成为可能。这项研究是与加州大学合作进行的，将有助于扩大萜类环酶结构、功能和进化的领域，并将为研究生和本科生提供教育机会。这项技术可能导致开发有希望的新型萜类药物，在破坏自然资源和排放合成化学废水方面，消费者和环境的成本更低。

DESCRIPTION (provided by applicant): Terpenoids are a diverse class of natural compounds with equally diverse and potent bioactivities. Because the practical use of terpenoids hinges on the ability to extract or synthesize these compounds, only the most abundant or easily synthesized compounds have been commercially developed. The long-term goal of this Small Business Technology Transfer Phase I project is to facilitate the clinical testing and use of new cancer compounds through the economical production of otherwise scarce terpenes. Eleutherobin and sarcodictyin A and B are cytotoxic terpenes that act in a manner analogous to very successful anti-cancer drug Taxol. Supply limitations, however, has precluded the development of these exciting compounds. This Phase I research will use innovative methods to identify, clone and express the terpene cyclase responsible for the formation of the eleutherobin carbon backbone. By detecting the activity of the cyclase, we will isolate and partially sequence the eunicellane cyclase. These partial sequences will then be used to design PCR probes to clone the full length cyclase gene. The sequence of the cyclase will act as a starting point in the discovery of the full biosynthetic pathway. As many compound produced by marine animals are the product of microbial symbionts, the cyclase gene allow the identification of biosynthetic gene clusters associated with our compound of interest. The discovery and expression of the identified biosynthetic pathway in bacteria that over-produce terpene precursors will allow the production of valuable terpenes in large scale fermentations. This research, conducted in collaboration with the University of California, will contribute to the expanding field of terpene cyclase structure, function and evolution and will provide educational opportunities for graduate and under-graduate students. This technology could result in the development of promising new terpene-based drugs produced at a lower cost to consumers and to the environment, in terms of destruction of natural resources and the release of synthetic chemical effluents.