CAREER: Epigenetics of Synthetic Biology

通过编辑细胞的DNA序列来构建或重新编程细胞是合成生物学的核心。驱动这一过程的工具的创造不仅在理解控制分子过程的基本机制方面有应用，而且在可持续生物经济的发展方面也有应用。本提案旨在阐明表观遗传学如何提高产品产量，以增强微生物中天然产品的发现。将在革兰氏阴性和革兰氏阳性细菌中检查表观遗传控制机制，以设计一个可编程系统。这项研究将成为伊利诺伊大学芝加哥分校一门新的合成生物学课程的基础，并将为包括高中在内的各个层次的学生提供研究机会。教育活动的一个关键目标是使不同背景的学生都能接触到合成生物学。表观遗传DNA甲基化通过限制性修饰系统参与细菌宿主防御，并控制DNA复制和基因表达。合成生物学家最近开始探索表观遗传学和限制性修饰系统来设计生物系统中的记忆。不幸的是，对这一机制的理解受到许多挑战的阻碍，包括外源基因表达的低产量。一种工业伯克氏菌菌株，其产生的肽capistruin是正常水平的580倍，将被用作异源试验宿主。初步研究表明，这种过度生产很可能是由影响质粒拷贝数和基因转录的表观遗传因素介导的。本研究试图通过限制修饰系统内的酶的扰动来确定这种调节的潜在基础。甲基组和转录组数据集将分别生成，然后整合以确定甲基化模式。此外，还将在革兰氏阳性菌和革兰氏阴性菌模型生物中进一步探讨表观遗传因素在控制产物产量中的作用。该项目预计将提供对产品产量随机变化基础的关键理解，从而有助于在不同模式生物中构建改进的合成生物学工具。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Constructing or reprogramming cells by editing their DNA sequence is central to synthetic biology. The creation of tools that drive this process have applications in not only understanding the fundamental mechanisms that govern molecular processes, but also in the development of a sustainable bioeconomy. This proposal seeks to elucidate how epigenetics improves product yields to enhance natural product discovery in microbes. The mechanisms of epigenetic control will be examined in Gram-negative and Gram-positive bacteria to engineer a programmable system. This research will form the basis of a new synthetic biology course at the University of Illinois at Chicago and will provide research opportunities for students at various levels, including high school. A key goal of the educational activities is to make synthetic biology accessible to students of diverse backgrounds.Epigenetic DNA methylation is involved in bacterial host defense as mediated by Restriction-Modification systems, and in the control of DNA replication and gene expression. Synthetic biologists have recently started to explore epigenetics and Restriction-Modification systems to engineer memory in biological systems. Unfortunately, understanding of this mechanism is hindered by numerous challenges, including the low production yield of heterologous gene expression. An industrial Burkholderia strain, which produces 580-times the normal levels of the peptide capistruin, will be employed as a heterologous test host. Preliminary studies have demonstrated that this overproduction is most likely mediated by epigenetic factors that impact plasmid copy number and gene transcription. This research seeks to identify the underlying basis for this regulation, by perturbation of enzymes within the Restriction-Modification system. Methylome and transcriptome datasets will be generated separately and then integrated to determine methylation patterns. In addition, the role of epigenetic factors in controlling product yields will be further explored in both Gram-positive and Gram-negative bacteria model organisms. The project is expected to provide critical understanding of the basis for stochastic variation in product yields and thereby to contribute to construction of improved synthetic biology tools in diverse model organisms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.