Functional Metagenomic Reprogramming of the Human Microbiome through Mobilome Eng

描述(由申请人提供)：这项研究的长期目标是开发与培养无关的策略，以有效地设计和编程体内的人类微生物群。为此，这项提议的目标是开发一个使能技术平台，称为自传复制子基因组工程(GESTR)，以允许在人类微生物区系中高效地传递、繁殖和表达外源基因，表征基因在微生物群中的繁殖动力学，并初步展示这种技术在工程小鼠肠道微生物区系中的应用。这项工作的具体目标是：i)构建含有可追踪的有效载荷基因的工程自传递接合转座子，并鉴定它们动员到通常在人类肠道微生物区系中发现的不同菌株的能力；ii)开发有效载荷构建库，以表征转录效率和密码子适应，以在优势肠道微生物中优化有效载荷基因的表达；以及iii)测量小鼠模型肠道微生物区系中通过工程设计的自动员遗传元件进行的外源基因的流动和传播。将利用新开发的工具，包括多重自动化基因组工程、基于从头DNA微阵列的基因合成和元转录组学来促进这一项目的发展。拟议的研究将提供关键功能的第一次演示，并形成足够的新理解，使这一技术平台能够在未来的应用中得到更广泛的使用。还将讨论人类微生物组中横向共享基因的传播和自然选择的动力学中的关键问题。设计人类微生物组，增强其报告、预防和逆转疾病状态以及调节食物和药物代谢的能力，有望成为将人类相关微生物转化为微型传感器、微型蛋白质生产工厂和适应性生物修复系统的关键途径。这项技术具有开发常见微生物相关疾病临床疗法的潜力，如肠道疾病(如克罗恩病、IBD、慢性消化不良)、口腔疾病(如龋齿)、泌尿生殖道疾病(如感染、性传播疾病)和皮肤疾病(如异位湿疹)。 公共卫生相关性：拟议的研究项目旨在开发基础技术，以实现对通常与人体有关的微生物的基因操纵。这些努力将促进制定预防措施和治疗方法，以对抗与微生物相关的人类疾病，包括肠道、口腔、鼻子、皮肤和泌尿生殖器官的疾病。

DESCRIPTION (provided by applicant): 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). PUBLIC HEALTH RELEVANCE: The proposed research project aims to develop foundational technologies to enable the genetic manipulation of microbes that are commonly associated with the human body. These endeavors will facilitate the development of preventative measures and therapeutics to combat microbial-associated human diseases including those of the gut, mouth, nose, skin, and urogenital organs.