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

合作研究：压力下的平衡：开发具有最小基因组的菌株用于集成生物加工

• 批准号: 2218260
• 负责人: Jason Boock
• 金额: $ 32.83万
• 依托单位: Miami University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2218260&HistoricalAwards=false
• 关键词: Collaborative; Research; Poise; under; pressure

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.

目前的挑战是依赖不可再生的化石资源来产生人类所依赖的化学品和材料，沿着转化这些化学品和材料的能源和碳密集型过程，这促使开发新的方法来将可再生原料转化为有用的产品。 生物技术的进步导致生物经济中的方法和商业活动大幅增长，包括那些专注于可持续原料转化的方法和商业活动。 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。 通过生物生产可获得的产品的范围经常受到常用微生物对所需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操作，而化学过程受益于连续生产固有的生产率优势。 该项目的重点是开发一种特殊的生物体，这种生物体显示出一套不寻常的物理特征，以帮助应对这些挑战。 该微生物能够在包括超临界CO2（scCO 2）的两相系统中生长，超临界CO2是许多有毒产品的优先溶剂，并且抑制大多数生物体的生长。 第二阶段应通过连续提取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。 该研究项目将使人们更好地了解这种生物的行为，增加基因工程工具和简化菌株，所有这些都将为集成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。 本项目的目标是利用简化基因组方法阐明所选微生物对scCO 2耐受性的基因型-表型关系。 目标包括使用转座子文库确定基因的必要性，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。 将构建两种基因敲除的转座子文库（即，相关酶活性的完全消除）和过表达，并且将评估所得文库以建立基因组在不同环境条件下的必要性。 将对文库成员进行转录组学（RNA测序）和翻译组学（核糖体分析）研究，以了解在分子水平上对培养条件变化的系统生物学反应。 相关数据还将用于挖掘和设计新的生物部件，以扩大基因工程的工具箱。 最后，将构建并工程化显示所需耐受性表型的简化基因组菌株以产生目标化合物，并相对于野生型前体评价这些菌株。 该项目将提供对有毒溶剂耐受性的更深入理解，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，旨在引入新的、非模式生物，这些生物本质上是可持续的生物加工，最终目标是推进一个强大的、可持续的生物经济。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。