Engineering Large scale pathways from organisms to Escherichia coli

该项目的目标是开发从一个细胞中转移大基因簇（30-300 kb）的技术。 与大肠杆菌有远亲关系的微生物，使得整个代谢途径可以被移植到 主持人这项技术将通过移动指定两种途径的基因来证明， 苯甲酸盐降解和固氮作用。杆菌第一路径 将苯甲酸盐转化为庚二酰辅酶A，庚二酰辅酶A是大肠杆菌中生物素合成的必需前体。杆菌的 所得菌株为从芳香族化合物生物合成生物素提供了新的途径。第二 途径允许E.大肠杆菌来固定分子氮，这是这种宿主的一种新的生活方式，并为未来铺平了道路。 固氮酶催化的氢生产。 智力优势：这项技术能够探索一个巨大的代谢能力库， 特征不明确但已测序的细菌。通过将整个路径移动到一个众所周知的快速- 生长宿主，代谢能力可以很容易地在工业规模中研究和利用。典型地， 来自远亲生物的大基因簇在E.大肠杆菌数 原因，包括大尺寸的外来DNA片段造成克隆困难，以及 启动子和控制序列的不相容性和不能被E.大肠杆菌RNA聚合酶 和转录因子。我们的策略受到两个自然过程的启发：水平基因转移 在细菌进化和噬菌体感染过程中。为了实现水平基因转移， 转移大基因簇将基于酵母重组系统和细菌 人工染色体（BAG）。此外，我们还将鉴定和克隆来自 供体菌转化为E.这样新宿主可以容易地表达供体基因簇，而不需要 个体优化（类似于细菌在感染过程中使用的策略）。之一 得到的菌株将能够在E.大肠杆菌，提供了一种新的途径，绕过 生物素生产中前体供应瓶颈。另一种所得菌株将能够利用N2作为 唯一氮源，展示了E.大肠杆菌，并建立进一步 代谢工程来生产氢气。 更广泛的影响：拟议的研究为研究代谢功能提供了一种新的方法， 为培养和教育新一代科学家奠定基础， 工程师特别是，在此提议的选定项目将作为一个讲习班的单元， 由FRT在俄亥俄州和形式的基础课程（代谢工程）在加州大学洛杉矶分校。

The goal of this project is to develop technologies for transferring large gene clusters (30-300 kb) from a distantly related microorganism to Escherichia coli such that an entire metabolic pathway can be grafted to the host. This technology will be demonstrated by moving genes specifying two pathways, anaerobic benzoate degradation and nitrogen fixation, from Rhodopseudomonas palustris to E. coli. The firstpathway converts benzoate to pimeloyl-coA, which is the essential precursor for biotin synthesis in E. coli. The resulting strain provides a novel path for biosynthesis of biotin from aromatic compounds. The second pathway allows E. coli to fix molecular nitrogen, a new life style for this host, and paves the way for future nitrogenase-catalyzed hydrogen production. Intellectual Merit: This technology enables the exploration of a vast repository of metabolic capability in poorly characterized, but sequenced bacteria. By moving the entire pathway to a well-known and fast- growing host, the metabolic capability can be readily studied and utilized in an industrial scale. Typically, large gene clusters from a distantly related organism are not expressedefficiently in E. coli for several reasons, including the large size of the foreign DNAfragment posing difficulty in cloning, as well as the incompatibility and inability of promoter and control sequences to be recognized by E. coli RNA polymerase and transcription factors. Our strategy here is inspired by two natural processes: horizontal gene transfer during bacterial evolution and phage infection. To achieve horizontal gene transfer, an efficient method for transferring large gene clusters will be developed based on a yeast recombination system and bacterial artificial chromosome (BAG). In addition, we will identify and clone the essential transcription elements from the donor organism into E. coli such that the new host can readily express the donor gene cluster without individual optimization (which resembles the strategy used by bacterial phages during infection). One of the resulting strains will be able to convert benzoate to biotin in E. coli, providing a novel route to by-pass the bottleneck of precursor supply in biotin production. The other resulting strain will be able to utilize N2 as the sole nitrogen source, demonstrating a new life-style for E. coli and establishing the potential for further metabolic engineering for hydrogen production. Broader Impact: The proposed research provides a new way for studying metabolic functions in uncharacterized microorganisms and a basis for training and educating new generations of scientists and engineers. In particular, selected projects proposed here will be used as modules in a workshop established by FRT at Ohio State and form the basis for a course (Metabolic Engineering) at UCLA.