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

目前人类依赖不可再生的化石资源来生产化学物质和材料，再加上能源和碳密集型的转化过程，这些挑战促使人们开发新的方法，将可再生原料转化为有用的产品。生物技术的进步使构成生物经济的方法和商业活动，包括那些着重于转化可持续原料的方法和商业活动有了大幅度的增长。然而，许多生物基产品的成本仍然没有足够的竞争力来取代化石衍生产品。同样，通过生物生产可获得的产品范围经常受到常用微生物对所希望生产的化合物的敏感性的限制。最后，由于污染是一个重要的问题，生物工艺通常以分批或进料分批模式操作，而化学工艺则受益于连续生产固有的生产力优势。该项目专注于一种特殊生物的发展，这种生物表现出一套不寻常的物理特征，以帮助解决这些挑战。该微生物能够在包括超临界CO2 （scCO2）在内的两相系统中生长，超临界CO2是许多有毒产物的首选溶剂，并且抑制大多数生物体的生长。这个第二阶段既要通过持续提取来保护微生物不受高浓度产品的影响，又要尽量减少污染的风险。该研究项目将使人们对这种生物的行为有更深入的了解，增加基因工程工具和流线型菌株，所有这些都将为综合生物工艺操作带来新的机会，将生产与提取结合起来。这项工作将有助于培养研究生和博士后研究人员，以及合作机构之间的研究交流。新内容也将引入实验实验室课程。本项目的目的是利用减少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。将构建转座子文库，用于基因敲除（即完全消除相关酶活性）和过表达，并评估结果文库，以确定基因集在不同环境条件下的必要性。转录组学（RNA测序）和翻译组学（核糖体分析）研究将在文库成员身上进行，以了解在分子水平上对培养条件变化的系统生物学反应。相关数据也将用于挖掘和设计新的生物零件，以扩大基因工程的工具箱。最后，将构建显示所需耐受性表型的减少基因组菌株并进行工程设计以产生目标化合物，并对这些菌株进行相对于野生型前身的评估。该项目将提供对有毒溶剂耐受性的更深入了解，并提供评估这些复杂表型的工作流程。这是一个长期努力的下一步，即引入新的、非模式生物，这些生物天生就有利于生物处理，最终目标是推动一个强大的、可持续的生物经济。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 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.