Functional Metagenomic Reprogramming of the Human Microbiome through Mobilome Eng

项目摘要 这项研究的长期目标是开发与文化无关的策略，以有效地设计 并在活体内对人类微生物组进行编程。为此，这项提案的目标是制定一项 使名为自传播复制体基因组工程(GESTR)的技术平台能够 外源基因在人类微生物区系中的有效传递、繁殖和表达，以表征 微生物组中基因繁殖的动力学，并初步展示这种技术在 改造小鼠的肠道微生物区系。这项工作的具体目标是：i)构建自我工程的 传递含有可跟踪有效载荷基因的接合转座子并鉴定其能力 动员到人类肠道微生物区系中常见的不同菌株；ii)开发有效载荷文库 用于表征转录效率和密码子适应以实现最佳有效载荷基因表达的构建 在优势肠道微生物中；以及iii)测量基因流动和外源基因的传播 小鼠肠道微生物区系中的可自我动员的遗传元件。新开发的工具包括 多重自动化基因组工程，基于从头DNA微阵列的基因合成，以及Meta-DNA 将利用转录技术促进这一项目的发展。拟议的研究将提供 关键功能的首次演示和充分的新理解的发展，以使 这一技术平台在未来的应用中得到了更广泛的应用。传播动力学中的关键问题 还将讨论人类微生物组中横向共享基因的自然选择问题。工程学 人类微生物组具有增强的报告、预防和逆转疾病状态以及 调节食物和药物的新陈代谢有望成为改变人类的关键途径- 相关微生物进入微型传感器、微型蛋白质生产工厂和适应性生物修复 系统。这项技术具有开发常见微生物的临床疗法的潜力- 相关疾病，如肠道疾病(如克罗恩病、IBD、慢性消化不良)、口腔疾病(如牙科疾病) (如龋齿)、泌尿生殖道(如感染、性病)和皮肤(如异位湿疹)。

Project Summary The long-term goal of this research is to develop culture-independent strategies to effectively engineer and program the human microbiome in vivo. To this end, the objective of this proposal is to develop an enabling technology platform, named Genome Engineering by Self-Transmissible Replicons (GESTR), to allow the efficient delivery, propagation and expression of exogenous genes in the human microbiota, to characterize the kinetics of gene propagation in the microbiome, and to initially demonstrate the utility of such techniques in engineering the gut microbiota of mice. The specific goals of this work are i) to construct engineered self- transmitting conjugative transposons containing trackable payload genes and characterize their ability to mobilize into different strains typically found in the human gut microbiota; ii) to develop libraries of payload constructs to characterize transcriptional efficiency and codon adaptation for optimal payload gene expression in dominant gut microbes; and iii) to measure gene flow and transmission of exogenous genes by engineered self-mobilizable genetic elements in the gut microbiota of a murine model. Newly developed tools including Multiplex Automated Genome Engineering, de novo DNA-microarray-based gene synthesis, and meta- transcriptomics will be utilized to facilitate the development of this project. The proposed research will provide the first demonstration of key functionality and the development of sufficient new understanding to enable the broader use of this technology platform in future applications. Key questions in the dynamics of transmission and natural selection of laterally shared genes in the human microbiome will also be addressed. Engineering the human microbiome with augmented capabilities to report, prevent and reverse disease states and to modulate the metabolism of foods and drugs promises to be a critical avenue towards transforming human- associated microbes into micro-sensors, miniature protein-production factories, and adaptive bioremediation systems. This technology holds potential for development of clinical therapeutics of common microbial- associated diseases such as those of the gut (e.g. Crohn's, IBD, chronic maldigestion), oral cavity (e.g. dental caries), urogenital tract (e.g. infections, STDs), and skin (e.g. ectopic eczema).