CAREER: Epigenetic Regulation of Gene Expression in Engineered Prokaryotes

土壤含有复杂的微生物网络，以细菌物种为主，这些微生物对土壤健康，土壤稳定性和作物生产至关重要。经过工程改造的土壤细菌品种可以通过检测和清除污染物或减少农田施肥所需的氮来保护我们的土壤。为了有效地改造土壤细菌，并将对环境的风险降至最低，需要更好地了解重要基因的开启和关闭。这项职业研究的重点是表观遗传调控-基因表达是由化学修饰的DNA控制-及其在土壤细菌的天然和工程菌株的生存和性能的作用。这项工作将成为面向研究的高级本科课程项目的来源，以及向本科生教授生物工程原理的互动教育资源。原核生物中通过基因组甲基化对基因表达的表观遗传调控已经被广泛研究。在不断变化的生长条件下，如指数生长期和稳定期生长期之间，已经观察到基因组甲基化的差异，这表明生长环境显著影响表观遗传调控。然而，对合成生物学中的表观遗传调控的理解超越了E。大肠杆菌，在现实环境中，是非常有限的。恶臭假单胞菌是一种多功能的革兰氏阴性生物，其原产于土壤，并且正在被开发为许多工业和环境应用的底盘。然而，没有全面的研究表观遗传调控，也没有任何调查的表观遗传效应的合成基因电路功能，尚未报道在恶臭假单胞菌。这个CAREER计划将研究基因组甲基化如何影响基因表达变化，这些变化对于P. putida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集成合成基因电路的功能。本提案的总体目标是研究恶臭假单胞菌在其原生土壤环境中基因组甲基化对基因表达和合成基因电路功能的影响。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)通过调节DNA甲基化来控制恶臭假单胞菌中合成基因回路的表达。拟议的研究将通过教育活动来补充，这些活动将创建新的开放式高等教育资源，用于教授合成生物学的核心原则。这项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在复杂的环境中茁壮成长。该奖项反映了NSF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.