Engineering Large scale pathways from organisms to Escherichia coli

描述（由申请人提供）：本项目的目标是开发将大基因簇（30-300 kb）从远亲微生物转移到大肠杆菌的技术，以便将整个代谢途径移植到宿主中。这项技术将通过将指定两种途径的基因从沼泽红球藻转移到E.杆菌第一条途径将苯甲酸转化为庚二酰辅酶A，庚二酰辅酶A是大肠杆菌中生物素合成的必需前体。杆菌该菌株为芳香族化合物生物合成生物素提供了一条新途径。第二条途径允许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，并为未来固氮酶催化制氢铺平了道路。 智力优势：这项技术使人们能够探索一个巨大的储存库的代谢能力，在穷人的特点，但测序细菌。通过将整个途径转移到一个众所周知的和快速生长的宿主，代谢能力可以很容易地在工业规模上进行研究和利用。通常，来自远亲生物的大基因簇在大肠杆菌中不能有效表达。大肠杆菌中的启动子和控制序列的不相容性以及不能被大肠杆菌识别，这些原因包括外源DNA片段的大尺寸造成克隆的困难。coli 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：细菌进化过程中的水平基因转移和噬菌体感染。为了实现水平基因转移，将开发一种基于酵母重组系统和细菌人工染色体（BAG）的高效转移大基因簇的方法。此外，我们还将从供体生物中鉴定并克隆必需的转录元件到大肠杆菌中。这样新宿主可以很容易地表达供体基因簇而无需单独优化（这类似于细菌感染期间使用的策略）。其中一个菌株将能够在大肠杆菌中将苯甲酸盐转化为生物素。coli，为克服生物素生产中前体供应的瓶颈提供了一条新途径。另一个菌株将能够利用N2作为唯一的氮源，展示了E.大肠杆菌，并建立了进一步的代谢工程制氢的潜力。 更广泛的影响：这项研究为研究未知微生物的代谢功能提供了一种新的方法，并为培养和教育新一代科学家和工程师奠定了基础。特别是，这里提出的选定项目将被用作由FRT在俄亥俄州建立的研讨会的模块，并形成在加州大学洛杉矶分校的课程（代谢工程）的基础。

DESCRIPTION (provided by applicant): 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 first pathway 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 fastgrowing 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 expressed efficiently in E. coli for several reasons, including the large size of the foreign DNA fragment 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.