EDGE FGT: Genetic Tools for Picocyanobacteria that Dominate the Oceans

作为地球上最丰富的光合生物，海洋蓝藻原绿球藻在为海洋食物网提供能量和营养方面起着核心作用。仅这个物种每年固定的二氧化碳就相当于全球农田固定的二氧化碳量。我们无法修改原绿球藻的基因来测试各种假设，这阻碍了我们理解原绿球藻的不同基因与其在海洋代谢中的核心作用之间的联系。在最近进展的基础上，该项目旨在开发两种不同但平行的方法来应对这一挑战。首先，利用在某些原绿球藻菌株中发现的新发现的遗传元素系统，可以绕过将外源DNA引入原绿球藻的障碍。第二，将开发触发机制的方法，使细胞能够吸收并将新基因整合到它们的细胞机制中。一旦发展起来，这些方法将使人们更好地了解原绿球藻基因与海洋环境之间的联系，这对于表征海洋过程在调节全球碳循环中所起的作用至关重要。这些进展也将为研究其他微生物有机体的类似方法的发展提供蓝图。最后，开发这些技术是使原绿球藻成为人工光合作用的潜在基础的关键的第一步。由于原绿球藻的基因组是所有光合细胞中最小的，它已经是自然界中最基本的光合机器，这使它成为开发生物工程工具的理想候选者。该项目旨在开发的工具将为新的调查和生物技术应用提供巨大的潜力。本项目资助研究生1名，博士后1名，技术人员2名。在过去的三十年中，蓝藻原绿球藻以其在海洋中的数量优势和巨大的遗传多样性而闻名，已被发展成为跨尺度系统生物学的模式生物。虽然每个细胞大约有2000个基因，但全球原绿球藻“集体”的独特基因含量可能超过8万个基因，其中大多数都有未知的功能。因此，尽管原绿球藻在海洋代谢中起着核心作用，但人们对原绿球藻多样化的基因含量与形成它的选择压力之间的关系才刚刚开始了解。由于缺乏遗传系统，这一挑战的进展一直停滞不前。在EDGE计划支持的先前工作中，研究人员开发了一种将外源DNA引入原绿球藻细胞的方法，这代表了基因组操作的重要一步。在此过程中，发现了一种可移动遗传元件系统，它代表了一种将外源DNA整合到原绿球藻中的新工具，即电穿孔传递由天然遗传元件构建的tycheposons。第二种方法是利用一种新的基于转座子的筛选来确定触发原绿球藻自然能力活性的条件，并将产生有希望的整合位点的全基因组图谱。这两种方法的优点都是利用原绿球藻本身的DNA，可以更有效地绕过细胞对外源DNA的防御。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。

As the most abundant photosynthetic organism on the planet, the marine cyanobacterium Prochlorococcus plays a central role in providing energy and nutrients to ocean food webs. This species alone fixes approximately as much carbon dioxide annually as global croplands. Understanding the link between the diverse genes of Prochlorococcus and its central role in ocean metabolism is hindered by our inability to modify its genes to test various hypotheses. Building on recent progress, this project aims to develop two distinct but parallel approaches to the challenge. In the first, barriers to the introduction of foreign DNA to Prochlorococcus will be bypassed by exploiting a system of newly discovered genetic elements that are found in some Prochlorococcus strains. In the second, approaches to trigger mechanisms that allow cells to take up and integrate new genes into their cellular machinery will be developed. Once developed, these approaches would allow better understanding of the link between Prochlorococcus genes and the ocean environment, which is critical for characterizing the role ocean processes play in regulating the global carbon cycle. These advances will also provide a blueprint for the development of similar methodologies for studying other microbial organisms. Finally, developing these technologies is a critical first step in enabling Prochlorococcus as a potential chassis for artificial photosynthesis. Since it has the smallest genome of any photosynthetic cell, Prochlorococcus is already the most basic photosynthetic machine occurring in nature, making it the ideal candidate for developing the tools that would enable bioengineering. The tools this project aims to develop would provide immense potential for new inquiry and biotechnology applications. This project provides support for graduate students, a postdoctoral research associate, and two technicians. Over the past three decades, the cyanobacterium Prochlorococcus, notable for its numerical dominance of the oceans and enormous genetic diversity, has been developed as a model organism for cross-scale systems biology. While each cell has around 2000 genes, the total unique gene content of the global Prochlorococcus ‘collective’ likely exceeds 80,000 genes, most of which have unknown function. Thus, despite their central role in ocean metabolism, the relationship between Prochlorococcus’ diverse genetic content, and the selective pressures that have shaped it, is only beginning to be understood. Progress on this challenge has been stalled by the lack of a genetic system. In previous work supported by the EDGE program, the researchers developed a method of introducing exogenous DNA into Prochlorococcus cells, which represented a significant step forward in genomic manipulation. In the process, a system of mobile genetic elements was discovered, which represent a promising new tool for integration of exogenous DNA into the Prochlorococcus, electroporation delivered tycheposons that are constructed with native genetic elements. A second approach to be developed utilizes a novel transposon-based screen to identify conditions for triggering natural competence activity among Prochlorococcus and will result in a genome-wide map of promising integration sites. Both approaches have the advantage of utilizing DNA from Prochlorococcus itself, which should more effectively bypass cell defenses against exogenous DNA.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.