Engineering Large scale pathways from organisms to Escherichia coli

描述(由申请人提供)：该项目的目标是开发将大基因簇(30-300kb)从远亲微生物转移到大肠杆菌的技术，以便可以将整个代谢途径嫁接到宿主身上。这项技术将通过将指定两条途径的基因从沼泽红假单胞菌转移到大肠杆菌来展示，这两条途径是厌氧苯甲酸盐降解和固氮。第一个途径是将苯甲酸转化为苯甲酰辅酶A，这是在大肠杆菌中合成生物素的必要前体。该菌株为从芳香化合物中生物合成生物素提供了一条新的途径。第二条途径允许大肠杆菌固定分子氮，这是这种宿主的一种新的生活方式，并为未来固氮酶催化的氢气生产铺平了道路。 智力价值：这项技术使人们能够在特征不佳但已测序的细菌中探索巨大的新陈代谢能力储存库。通过将整个途径转移到众所周知的快速生长的宿主，代谢能力可以很容易地在工业规模上进行研究和利用。通常情况下，来自远缘生物的大基因簇不能在大肠杆菌中有效表达，原因包括外源DNA片段较大，导致克隆困难，以及启动子和控制序列不兼容，无法被大肠杆菌RNA聚合酶和转录因子识别。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，基于酵母重组系统和细菌人工染色体(BAG)，将开发一种高效的大基因簇转移方法。此外，我们将从供体有机体中鉴定并将必要的转录元件克隆到大肠杆菌中，以便新宿主可以在不进行个体优化的情况下容易地表达供体基因簇(这类似于细菌噬菌体在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，为绕过生物素生产中前体供应的瓶颈提供了一条新的途径。另一株产生的菌株将能够利用氮气作为唯一的氮源，展示了大肠杆菌的一种新的生活方式，并为进一步的代谢工程生产氢气奠定了潜力。 更广泛的影响：拟议的研究为研究未知微生物的代谢功能提供了一种新的方法，并为培训和教育新一代科学家和工程师提供了基础。特别是，这里提出的选定项目将作为俄亥俄州立大学FRT建立的研讨会的模块，并构成加州大学洛杉矶分校(UCLA)课程(新陈代谢工程)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.