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发挥着核心作用。仅这一物种每年固定的二氧化碳量就相当于全球农田。了解原绿球藻的不同基因之间的联系及其在海洋代谢中的核心作用是由于我们无法修改其基因来测试各种假设而受到阻碍。在最近取得的进展的基础上，该项目旨在制定两种不同但平行的方法来应对这一挑战。在第一个中，将通过利用在一些原绿球藻菌株中发现的新发现的遗传元件的系统来绕过将外源DNA引入原绿球藻的障碍。第二，将开发触发机制的方法，使细胞能够吸收新基因并将其整合到细胞机器中。一旦开发出来，这些方法将有助于更好地了解原绿球藻基因与海洋环境之间的联系，这对于描述海洋过程在调节全球碳循环方面的作用至关重要。这些进展还将为研究其他微生物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潜在基础的关键的第一步。由于它具有任何光合细胞中最小的基因组，原绿球藻已经是自然界中最基本的光合机器，使其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。该项目为研究生，博士后研究助理和两名技术人员提供支持。 在过去三十年中，以其在海洋中的数量优势和巨大的遗传多样性而闻名的蓝细菌原绿球藻已被开发为跨尺度系统生物学的模式生物。虽然每个细胞有大约2000个基因，但全球原绿球藻“集体”的独特基因含量可能超过80，000个基因，其中大部分基因的功能未知。因此，尽管原绿球藻在海洋新陈代谢中发挥着核心作用，但它们的多样遗传内容与塑造它的选择压力之间的关系才刚刚开始被理解。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，发现了一个移动的遗传元件系统，它代表了一个有前途的新工具，外源DNA整合到原绿球藻，电穿孔传递的类型座子，构建与天然遗传元件。第二种方法将开发利用一种新的转座子为基础的屏幕，以确定触发原绿球藻之间的自然能力活动的条件，并会导致在全基因组范围内有前途的整合位点的地图。这两种方法都具有利用原绿球藻本身DNA的优势，这应该更有效地绕过细胞对外源DNA的防御。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.