CAREER: Epigenetic Regulation of Gene Expression in Engineered Prokaryotes

土壤包含一个复杂的微生物网，以细菌种类为主，这些微生物对土壤健康、土壤稳定和作物生产至关重要。土壤细菌的工程品种可以通过检测和去除污染物，或减少农田施肥所需的氮来帮助保护我们的土壤。为了有效地改造土壤细菌，并将对环境的风险降到最低，我们需要更好地了解基因的开启和关闭是如何重要的。本职业研究的重点是表观遗传调控-通过对dna的化学修饰控制的基因表达-及其在天然和工程土壤细菌菌株的生存和表现中的作用。这项工作将成为研究型高级本科课程的项目来源，以及向本科生教授生物工程原理的互动教育资源。通过基因组甲基化对原核生物基因表达的表观遗传调控的研究还远远不够。基因组甲基化在变化的生长条件下已经观察到差异，例如在指数期和平稳期生长之间，这表明生长环境显著影响表观遗传调控。然而，对大肠杆菌以外的合成生物学底盘生物和现实环境中的表观遗传调控的理解是非常有限的。恶臭假单胞菌是一种多用途的革兰氏阴性菌，原产于土壤中，目前正被开发为许多工业和环境应用的基础。然而，目前还没有关于恶臭假单胞菌表观遗传调控的全面研究，也没有关于表观遗传对合成基因回路功能影响的研究报道。这个职业计划将研究基因组甲基化如何影响基因表达的变化，这些变化对于恶臭假单胞菌从实验室到土壤条件的转变至关重要。这些表观遗传变化可能同样影响综合合成基因回路的功能。本研究旨在研究土壤环境下基因组甲基化对恶臭假单胞菌基因表达和合成基因回路功能的影响。为了实现总体目标，将追求两个具体目标：1)确定基因组甲基化对土壤中恶臭假单胞菌内源基因表达的影响。2)通过DNA甲基化调控恶臭杆菌合成基因回路的表达。拟议的研究将得到教育活动的补充，这些活动将为教授合成生物学的核心原理创造新的开放获取的高等教育资源。在这个职业计划中提出的研究将揭示重要的表观遗传原理，这些原理允许工程生物体在复杂的环境中茁壮成长。这些知识将广泛地转化为一系列环境和工业应用的基因工程微生物的优化。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。

The soil contains a complex web of microbes, dominated by bacteria species, and these microbes are essential for soil health, soil stability and crop production. Engineered varieties of soil bacteria may help protect our soils by detecting and removing pollutants, or reducing the nitrogen needed to fertilize farmlands. To engineer soil bacteria efficiently and with minimal risk to the environment, a better understanding of how important genes are turned on and off is needed. This CAREER research focuses on epigenetic regulation – gene expression that is controlled by chemical modifications to the DNA-and its role in the survival and performance of native and engineered strains of soil bacteria. This work will be the source of projects for a research-oriented upper-level undergraduate course and for interactive educational resources that teach the principles of bioengineering to undergraduates. Epigenetic regulation of gene expression via genome methylation in prokaryotes has been vastly understudied. Differences in genome methylation have been observed under changing growth conditions, such as between exponential and stationary phase growth, which suggests that the growth environment significantly influences epigenetic regulation. However, understanding of epigenetic regulation in synthetic biology chassis organisms beyond E. coli, and in real-world environmental contexts, is extremely limited. Pseudomonas putida is a versatile Gram-negative organism that is native to soil, and is being developed as a chassis for numerous industrial and environmental applications. Yet, no comprehensive study of epigenetic regulation, nor any investigation of epigenetic effects on synthetic gene circuit function, has yet been reported in P. putida. This CAREER program will examine how genome methylation affects gene expression changes that are essential for the transition of P. putida from laboratory to soil conditions. These epigenetic changes may similarly affect the function of integrated synthetic gene circuits. The overall objective of this proposal is to investigate the impact of genome methylation on gene expression and synthetic gene circuit function in P. putida within its native soil environment. To achieve the overall objective, two specific objectives will be pursued: 1) Determine the effect of genome methylation on endogenous gene expression of P. putida in soil. 2) Control synthetic gene circuit expression in P. putida by modulation of DNA methylation. The proposed research will be complemented by educational activities that create new open-access higher education resources for teaching the core principles of synthetic biology. The research proposed in this CAREER program will uncover important epigenetic principles that permit engineered organisms to thrive within a complex environment. The knowledge will be broadly translatable to the optimization of genetically engineered microbes for a range of environmental and industrial applications.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.