Reprogramming Escherichia coli with synthetic protein glycosylation pathways

1159581/去天冬酰胺连接(N-连接)的蛋白质糖基化是真核细胞中对蛋白质最常见的修饰。已知在革兰氏阴性细菌中也存在N-连接的糖基化途径。细菌糖基化系统与真核过程在功能上有许多相似之处，但也有几个主要的区别。最值得注意的是，细菌N-连接的多糖的结构与真核生物中产生的结构有很大的不同。因此，来自当前细菌糖基化系统的糖蛋白很可能对人类具有免疫原性，因此治疗价值有限。这个项目寻求通过自下而上的工程将细菌的糖基化提高到以前从未探索的水平，在大肠杆菌中进行真核N-连接糖基化反应的合成途径。拟议的研究和研究培训活动预计将对社会产生广泛影响，从糖生物学科学到人类糖疗法的发展。该项目还将培养下一代训练有素的研究生，他们将被介绍给生物技术研究的广泛、跨学科性质。此外，该计划将积极和积极地扩大对科学和工程的参与。这将通过与康奈尔大学工程学多样性项目和康奈尔·路易斯·斯托克斯少数群体参与联盟(LSAMP)项目合作来实现，该项目将开发一个题为《多么甜蜜》的实践学习体验，向来自未被充分代表的群体的本科生介绍分子生物技术以及糖在生命系统中发挥的重要作用。最后，将通过与当地生物技术公司的合作，将细菌糖基化用于生物技术应用的开发带到更大的研究社区。总体而言，这些研究将为与细菌糖工程相关的新的学术追求和工业企业提供相当大的燃料。

1159581/ DeLisaAsparagine-linked (N-linked) protein glycosylation is the most common modification made to proteins in eukaryotic cells. N-linked glycosylation pathways are also known to exist in gram-negative bacteria. The bacterial glycosylation system bears many functional similarities with the eukaryotic process, but there are also several major differences between the systems. Most notably, the structures of bacterial N-linked glycans are significantly different from those produced in eukaryotes. Hence, glycoproteins derived from current bacterial glycosylation systems are likely to be immunogenic in humans and thus of limited therapeutic value. This project seeks to take bacterial glycosylation to a previously unexplored level by bottom-up engineering of a synthetic pathway for performing eukaryotic N-linked glycosylation reactions in Escherichia coli. The proposed studies and research training activities are expected to have a broad impact on society, ranging from the science of glycobiology to the development of human glycotherapeutics. This project will also cultivate the next generation of highly trained graduate students who will be introduced to the broad, interdisciplinary nature of biotechnology research. Moreover, this program will actively and aggressively broaden participation in science and engineering. This will be accomplished by collaborating with Cornell's Diversity Programs in Engineering and with the Cornell Louis Stokes Alliance for Minority Participation (LSAMP) Program to develop a hands-on learning experience entitled "How Sweet It Is" that will introduce undergraduate students from underrepresented groups to molecular biotechnology and the important role played by sugars in living systems. Finally, the development of bacterial glycosylation for biotechnological applications will be brought to a larger research community through partnership with local biotechnology companies. Overall, these studies will provide considerable fuel for new academic pursuits and industrial enterprises related to bacterial glycoengineering.