Collaborative Research: Poise under pressure: developing strains with minimal genomes for integrated bioprocessing

目前的挑战是依赖不可再生的化石资源来生产人类所依赖的化学品和材料，以及转化它们的能源和碳密集型过程，这促使开发新的方法，将可再生原料转化为有用的产品。生物技术的进步导致了构成生物经济的方法和商业活动的大幅增长，包括那些侧重于可持续原料转换的方法和商业活动。然而，事实仍然是，许多生物产品的成本没有足够的竞争力来取代化石衍生产品。通过生物生产获得的产品范围往往受到常用微生物对希望生产的化合物的敏感性的限制，这也是事实。最后，由于污染是一个重要的问题，生物过程通常以分批或补料分批模式运行，而化学过程则受益于连续生产所固有的生产率优势。这个项目的重点是开发一种特殊的有机体，这种生物体表现出一组不同寻常的物理特征，以帮助应对这些挑战。该微生物能够在包括超临界二氧化碳(ScCO2)的两相系统中生长，超临界二氧化碳是许多有毒产品的优先溶剂，并抑制大多数生物的生长。这一第二阶段应通过连续提取来保护微生物免受高浓度产品的影响，并将污染风险降至最低。这项研究项目将使人们更好地了解这种生物的行为，增加基因工程工具，以及简化菌株，所有这些都将为将生产和提取结合在一起的综合生物过程操作带来新的机会。这项工作将使研究生和博士后研究人员的培训以及合作机构之间的研究交流成为可能。新的内容也将被引入到实验实验室课程中。这个项目的目标是用简化基因组的方法阐明所选微生物对scCO2耐受性的基因型-表型关系。目标包括利用转座子文库确定基因的重要性，开发用于生物体基因组规模工程的强大工具，以及构建用于生物生产的最小基因组菌株。将构建用于基因敲除(即完全消除相关酶活性)和过度表达的转座子文库，并将对所得文库进行评估，以确定不同环境条件下基因集的重要性。将对文库成员进行转录学(RNA测序)和翻译组学(核糖体图谱)研究，以在分子水平上了解系统对培养条件变化的生物学反应。相关数据还将被用于挖掘和设计新的生物艺术，以扩大基因工程的工具箱。最后，将构建一个显示所需耐受性表型的简化基因组菌株，并对其进行工程设计，以产生目标化合物，并将这些菌株与野生型前身进行评估。该项目将提供对有毒溶剂耐受性的更深层次的了解，并提供评估这些复杂表型的工作流程。这是一项长期努力的下一步，目的是引入新的、非模式生物，这些生物天生就有利于生物加工，最终目标是推进强大、可持续的生物经济。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The current challenges of dependence on non-renewable fossil sources to generate the chemicals and materials that humans depend upon, along with the energy- and carbon-intensive processes that transform them, motivates the development of new approaches to convert renewable feedstocks to useful products. Advances in biotechnology have resulted in substantial growth within the methodologies and commercial activities that comprise the Bioeconomy, including those with a focus on the conversion of sustainable feedstocks. However, it remains the case that the costs of many bio-based products are not sufficiently competitive to displace fossil-derived counterparts. It is also true that the scope of products accessible through biological production is frequently constrained by the sensitivity of commonly-used microbes to the compounds that are desired to be produced. Finally, because contamination is a significant concern, bioprocesses are usually operated in batch or fed-batch mode, while chemical processes benefit from the productivity advantages inherent to continuous production. This project focuses on the development of a particular organism that displays an unusual set of physical characteristics to help addresses these challenges. The microbe is capable of growth in a two-phase system that includes supercritical CO2 (scCO2), which is a preferential solvent for many toxic products as well as inhibits growth of most organisms. This secondary phase should both protect the microbe from high concentrations of the product through continuous withdrawal, and minimize the risks of contamination. This research project will result in greater understanding of the behavior of this organism, an increase in genetic engineering tools, and a streamlined strain, all of which will lead to new opportunities for integrated bioprocess operations, which couple production with extraction. The work will enable the training of graduate students and postdoctoral researchers as well as research exchanges between the collaborating institutions. New content will also be introduced into an experimental laboratory curriculum.The goal of this project is to elucidate the genotype-phenotype relationship of tolerance to scCO2 of the chosen microorganism using a reduced genome approach. The objectives include the determination of gene essentiality using transposon libraries, development of robust tools for genome-scale engineering of the organism, and construction of a minimal genome strain for use in bioproduction. Transposon libraries will be constructed for both gene knockouts (i.e., complete elimination of associated enzyme activities) and overexpression, and the resulting libraries will be assessed to establish essentiality of gene sets under different environmental conditions. Transcriptomic (RNA sequencing) and translatomics (ribosome profiling) studies will be performed on library members to understand systematic biological responses to changes in culture conditions at the molecular level. The associated data will also be used to mine and design new bioparts to expand a toolbox for genetic engineering. Lastly, a reduced genome strain displaying the desired tolerance phenotype will be constructed and engineered to produce target compounds, with these strains evaluated relative to the wild-type predecessor. This project will provide deeper understanding of tolerance to toxic solvents and provide a workflow for assessing these complex phenotypes. It is the next step in a long-term effort to introduce new, non-model organisms that are inherently advantaged for bioprocessing, with the ultimate goal of advancing a robust, sustainable Bioeconomy.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.